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1
Sgambato, Antonella, Valentina Pastori, Laura Russo, Simone Vesentini, Marzia Lecchi, and Laura Cipolla. "Neoglycosylated Collagen: Effect on Neuroblastoma F-11 Cell Lines." Molecules 25, no. 19 (September 23, 2020): 4361. http://dx.doi.org/10.3390/molecules25194361.
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The regeneration of the nervous system is a challenging task. Currently, regenerative medicine approaches that exploit nature-inspired cues are being studied and hold great promise. The possibility to use protein-based matrices functionalized with small oligo- and monosaccharides is of interest since these can be finely tuned to better mimic the native environment. Collagen has been selected as a promising material that has the potential to be further tailored to incorporate carbohydrates in order to drive cell behavior towards neuroregeneration. Indeed, the grafting of carbohydrates to collagen 2D matrices is proved to enhance its biological significance. In the present study, collagen 2D matrices were grafted with different carbohydrate epitopes, and their potential to drive F-11 neuroblastoma cells towards neuronal differentiation was evaluated. Collagen functionalized with α-glucosides was able to differentiate neuroblastoma cells into functional neurons, while sialyl α-(2→6)-galactosides stimulated cell proliferation.
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Breen, Shawn M., Nebojsa Andric, Tai Ping, Fang Xie, Stefan Offermans, Jan A. Gossen, and Mario Ascoli. "Ovulation Involves the Luteinizing Hormone-Dependent Activation of Gq/11 in Granulosa Cells." Molecular Endocrinology 27, no. 9 (September 1, 2013): 1483–91. http://dx.doi.org/10.1210/me.2013-1130.
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The LH receptor (LHR) activates several families of heterotrimeric G proteins, but only the activation of Gs and subsequent generation of cAMP are universally accepted as important mediators of LH actions. To examine the involvement of the Gq/11 family on the actions of LH, we crossed Cyp19Cre and Gαqf/f;Gα11−/− mice to generate mice with a granulosa cell-specific deletion of Gαq in the context of a global deletion of Gα11. Granulosa cells from Gαqf/f;Gα11−/−;Cre+ mice have barely detectable levels of Gαq/11, have a normal complement of LHR, and respond to LHR activation with a transient increase in cAMP accumulation, but they fail to respond with increased inositol phosphate accumulation, an index of the activation of Gαq/11. The LHR-provoked resumption of meiosis, cumulus expansion, and luteinization are normal. However, the Gαqf/f;Gα11−/−;Cre+ mice display severe subfertility because many of the oocytes destined for ovulation become entrapped in preovulatory follicles or corpora lutea. Because follicular rupture is known to be dependent on the expression of the progesterone receptor (Pgr), we examined the LHR-induced expression of Pgr and 4 of its target genes (Adamts-1, Ctsl1, Edn2, and Prkg2). These actions of the LHR were impaired in the ovaries of the Gαqf/f;Gα11−/−;Cre+ mice. We conclude that the defect in follicular rupture is secondary to the failure of the LHR to fully induce the expression of the Pgr. This is the first conclusive evidence for the physiological importance of the activation of Gq/11 by the LHR and for the involvement of Gαq/11 in ovulation.
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Pastori, Valentina, Alessia D’Aloia, Stefania Blasa, and Marzia Lecchi. "Serum-deprived differentiated neuroblastoma F-11 cells express functional dorsal root ganglion neuron properties." PeerJ 7 (October 30, 2019): e7951. http://dx.doi.org/10.7717/peerj.7951.
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The isolation and culture of dorsal root ganglion (DRG) neurons cause adaptive changes in the expression and regulation of ion channels, with consequences on neuronal excitability. Considering that not all neurons survive the isolation and that DRG neurons are heterogeneous, it is difficult to find the cellular subtype of interest. For this reason, researchers opt for DRG-derived immortal cell lines to investigate endogenous properties. The F-11 cell line is a hybridoma of embryonic rat DRG neurons fused with the mouse neuroblastoma line N18TG2. In the proliferative condition, F-11 cells do not display a gene expression profile correspondent with specific subclasses of sensory neurons, but the most significant differences when compared with DRGs are the reduction of voltage-gated sodium, potassium and calcium channels, and the small amounts of TRPV1 transcripts. To investigate if functional properties of mature F-11 cells showed more similarities with those of isolated DRG neurons, we differentiated them by serum deprivation. Potassium and sodium currents significantly increased with differentiation, and biophysical properties of tetrodotoxin (TTX)-sensitive currents were similar to those characterized in small DRG neurons. The analysis of the voltage-dependence of calcium currents demonstrated the lack of low threshold activated components. The exclusive expression of high threshold activated Ca2+ currents and of TTX-sensitive Na+ currents correlated with the generation of a regular tonic electrical activity, which was recorded in the majority of the cells (80%) and was closely related to the activity of afferent TTX-sensitive A fibers of the proximal urethra and the bladder. Responses to capsaicin and substance P were also recorded in ~20% and ~80% of cells, respectively. The percentage of cells responsive to acetylcholine was consistent with the percentage referred for rat DRG primary neurons and cell electrical activity was modified by activation of non-NMDA receptors as for embryonic DRG neurons. These properties and the algesic profile (responses to pH5 and sensitivity to both ATP and capsaicin), proposed in literature to define a sub-classification of acutely dissociated rat DRG neurons, suggest that differentiated F-11 cells express receptors and ion channels that are also present in sensory neurons.
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Powell, Geoffrey M. L. "Attaching cells to finite complexes, with an application to elliptic spaces." Mathematical Proceedings of the Cambridge Philosophical Society 119, no. 3 (April 1996): 483–91. http://dx.doi.org/10.1017/s0305004100074351.
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Suppose that f; Sn → E is a continuous map from the n-sphere to a 1-connected CW complex E, with n ≥ 2. One may suppose that f is a cofibration, so that there is a cofibration sequence , with f the attaching map of the cell en+1. Consider the homotopy fibre F of the inclusion E ↪ B, so that there is a homotopy fibration let δ; ΩB → F be the connectant of this fibration. The following definition is given by Félix and Lemaire in [11]: Definition 1·1. Suppose that k is a field of characteristic p ≥ 0. The attaching map f:Sn → E is: 1. p-inert if is surjective; 2. p-lazy if is zero; where H˜ denotes reduced homology and coefficients are taken in the field k.
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Chen, Xu, Ying Qin, Xinru Song, He Li, Yue Yang, Jiazhuang Guo, Tingting Cui, Jiafei Yu, Cai-Feng Wang, and Su Chen. "Green Synthesis of Carbon Dots and Their Integration into Nylon-11 Nanofibers for Enhanced Mechanical Strength and Biocompatibility." Nanomaterials 12, no. 19 (September 26, 2022): 3347. http://dx.doi.org/10.3390/nano12193347.
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Carbon dots (CDs) have been extensively explored to show good optical features, low toxicity, and good biocompatibility. Herein, we report the new synthesis of forsythia-derived CDs (F-CDs) and their incorporation into Nylon-11 nanofibers for improved mechanical properties and biocompatibility. F-CDs are prepared from a Chinese herb forsythia via a magnetic hyperthermia method in 90 s without the use of any organic solvents. The as-prepared F-CDs with rich surface functional groups can be well embedded into Nylon-11 nanofibers via electrospinning, providing Nylon-11/F-CD nanofiber mats with remarkably enhanced mechanical properties. With the incorporation of F-CDs at 10 wt% into the Nylon-11 nanofiber mats, the tensile strength increases from 7.5 to 16.6 MPa, and the elongation ratio at break increases from 39% to 125%. Moreover, the Nylon-11/F-CD nanofiber mats exhibit excellent cytocompatibility towards L929 fibroblast cells with cell viability of 96%. These findings may guide the development of various CD-embedded nanofiber mats with good mechanical properties and biocompatibility potentially useful for biomedical applications, such as tissue engineering scaffolds or wound dressing.
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Zhang, Hongyu, Peter J. Wickley, Sayantani Sinha, Ian N. Bratz, and Derek S. Damron. "Propofol Restores Transient Receptor Potential Vanilloid Receptor Subtype-1 Sensitivity via Activation of Transient Receptor Potential Ankyrin Receptor Subtype-1 in Sensory Neurons." Anesthesiology 114, no. 5 (May 1, 2011): 1169–79. http://dx.doi.org/10.1097/aln.0b013e31820dee67.
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Background Cross talk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has been demonstrated recently. Moreover, the intravenous anesthetic propofol has directly activates TRPA1 receptors and indirectly restores sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity. Methods Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. The intracellular Ca concentration was measured in individual cells via fluorescence microscopy. After TRPV1 desensitization with capsaicin (100 nM), cells were treated with propofol (1, 5, and 10 μM) alone or with propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 μM), or the TRPA1 agonist, allyl isothiocyanate (AITC; 100 μM); capsaicin was then reapplied. Results In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC after desensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC was capable of restoring TRPV1 sensitivity. Conclusions These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol's effects on sensory neurons may be clinically important and may contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue.
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Khandros, Eugene, Peng Huang, Scott A. Peslak, Belinda Giardine, Zhe Zhang, Cheryl A. Keller, Ross C. Hardison, and Gerd A. Blobel. "Understanding Heterogeneity of Fetal Hemoglobin Induction through Comparative Analysis of Stage-Matched F- and a-Cells." Blood 134, Supplement_1 (November 13, 2019): 981. http://dx.doi.org/10.1182/blood-2019-124099.
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Reversing the developmental switch from fetal (HbF, α2γ2) to adult (HbA, α2β2) hemoglobin is an important therapeutic approach in sickle cell disease (SCD) and β-thalassemia. Elevated HbF levels due to genetic variation or through therapeutic induction by hydroxyurea (HU) attenuate the severity of both disorders. HbF in healthy individuals, SCD patients, and patients treated with HU is present in a heterocellular fashion in a subset of red blood cells known as F-cells. Despite over 50 years of observations of F-cells, it is not known why only some cells in a genetically identical population are able to express HbF or respond to pharmacological inducers. Adult F-cells can potentially represent a reversion to a fetal-like epigenetic and transcriptional program, or alternatively isolated transcriptional or posttranscriptional events at the γ-globin genes. Here we set out to understand the heterogeneity of HbF activation and gain insights into whether the mechanisms underlying the heterocellular response are similar or distinct in response to different HbF inducers. To this end we developed techniques to purify differentiation stage-matched late erythroblast F-cells and non-F cells (A-cells) from the human HUDEP2 erythroid cell line and primary CD34 cell erythroid cultures using a reversible fixation protocol enabling extraction of high-quality RNA and protein. Purified F-cells from both sources were enriched for γ-globin transcripts by 200-500 fold by RT-PCR, validating the purification scheme. We profiled these cells by RNA-seq using a modified method that depletes globin mRNAs and ribosomal RNAs and is capable of detecting low abundance transcripts, as well as by mass spectrometry using size fractionation to increase the number of detected proteins. In differentiated clonal HUDEP2 cells, differences between F-cells and A-cells were remarkably small, with only 62 differentially expressed transcripts and 20 differentially expressed proteins. Top differentially expressed transcripts were γ-globin and the non-coding β-globin locus transcripts BGLT3 and HBBP1. Interestingly, there were no significant changes in known HbF regulators BCL11A, LRF, and HRI at the RNA or protein level. Gene set enrichment analysis (GSEA) using a previously generated set of differentially expressed transcripts from adult and fetal-derived CD34 erythroid cultures showed enrichment of fetal transcripts in F-cells and adult transcripts in A-cells. We also carried out transcriptome analysis of sorted matched late erythroblast F-cells and A-cells from human CD34+ cell erythroid cultures at different time points. Similar to HUDEP2 cells, only small numbers of transcripts were differentially expressed (33 at 8 days, 17 at 11 days, and 261 at 14 days). BCL11A, LRF, and HRI were not differentially expressed at the earlier timepoints, and BCL11A and HRI were at most decreased by about 20% at the 14-day mark. GSEA analysis did not show fetal transcript enrichment in day 8. At days 11 and 14, there was some enrichment of fetal transcripts in F-cells but not to the degree of HUDEP2 cells. Finally, we analyzed sorted F- and A-cells from day 11 CD34+ erythroid cultures treated with hydroxyurea and pomalidomide. Again, differences between F- and A-cells were small with hydroxyurea treatment (53 transcripts) and more significant with pomalidomide treatment (400 transcripts). We have successfully established an approach to analyze stage-matched γ-globin containing cells from a genetically identical starting population, with high degree of enrichment. Our preliminary data indicate that these cells are overall highly similar to non-γ-containing cells, but do show some enrichment of fetal-specific transcripts, more so in HUDEP2 cells. The differences between F- and A- cells are overall smaller than those observed by us and others in profiling of fetal and adult-derived erythroblasts. This suggests that F-cells are not formed by reversion to a fetal-like state but rather through specific changes at the β-globin locus. Importantly, we do not find differential levels of any known γ-globin regulators, suggesting an alternative mechanism for the heterocellular expression pattern. Studies are currently ongoing to carry out epigenetic profiling of F-cells. Disclosures Blobel: Bioverativ: Research Funding; Pfizer: Research Funding.
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Dover, GJ, and SH Boyer. "Fetal hemoglobin-containing cells have the same mean corpuscular hemoglobin as cells without fetal hemoglobin: a reciprocal relationship between gamma- and beta-globin gene expression in normal subjects and in those with high fetal hemoglobin production." Blood 69, no. 4 (April 1, 1987): 1109–13. http://dx.doi.org/10.1182/blood.v69.4.1109.1109.
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Abstract We have developed methodology that allows comparison of the mean corpuscular hemoglobin (MCH) of fetal hemoglobin (HbF)-containing red cells (F cells) with the MCH of non-F cells from the same individual. To do this, suspensions of peripheral blood erythrocytes and their internal contents are fixed with an imidodiester, dimethyl-3,3′- dithiobispropionimidate dihydrochloride (DTBP). Thereafter fixed cells are made permeable to antisera by treatment with Triton X-100 and isopropanol, reacted with a mouse monoclonal antibody (MoAb) against HbF, and then with fluorescein-conjugated antimouse IgG. No appreciable hemoglobin is lost during such manipulation. Red cells from a diversity of subjects were thus treated and examined microscopically, first by transmitted light and then by epifluorescence. A direct correlation between Coulter-derived MCH and mean absorbance of 415 nm transmitted light was found for 100 unfixed (r = 0.96) and for 100 antibody-treated fixed-permeabilized red cells (r = 0.99) among individuals selected so as to provide a range of Coulter MCH values between 20 and 35. Comparisons of microscopically derived MCH of F cells and non-F cells were statistically nondistinguishable (P greater than 0.05) in all subjects. Such comparisons included normal individuals (less than 1% F cells), SS patients (7% to 48% F cells), subjects with congenital anemia (22% to 65% F cells), individuals with heterocellular hereditary persistence of HbF (HPFH) (12% to 21% F cells), and heterozygotes for beta + thalassemia (11% to 31% F cells). We conclude that gamma- and beta-globin production within F cells is regulated in a reciprocal fashion both among normal individuals and among individuals with elevated HbF production.
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Dover, GJ, and SH Boyer. "Fetal hemoglobin-containing cells have the same mean corpuscular hemoglobin as cells without fetal hemoglobin: a reciprocal relationship between gamma- and beta-globin gene expression in normal subjects and in those with high fetal hemoglobin production." Blood 69, no. 4 (April 1, 1987): 1109–13. http://dx.doi.org/10.1182/blood.v69.4.1109.bloodjournal6941109.
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We have developed methodology that allows comparison of the mean corpuscular hemoglobin (MCH) of fetal hemoglobin (HbF)-containing red cells (F cells) with the MCH of non-F cells from the same individual. To do this, suspensions of peripheral blood erythrocytes and their internal contents are fixed with an imidodiester, dimethyl-3,3′- dithiobispropionimidate dihydrochloride (DTBP). Thereafter fixed cells are made permeable to antisera by treatment with Triton X-100 and isopropanol, reacted with a mouse monoclonal antibody (MoAb) against HbF, and then with fluorescein-conjugated antimouse IgG. No appreciable hemoglobin is lost during such manipulation. Red cells from a diversity of subjects were thus treated and examined microscopically, first by transmitted light and then by epifluorescence. A direct correlation between Coulter-derived MCH and mean absorbance of 415 nm transmitted light was found for 100 unfixed (r = 0.96) and for 100 antibody-treated fixed-permeabilized red cells (r = 0.99) among individuals selected so as to provide a range of Coulter MCH values between 20 and 35. Comparisons of microscopically derived MCH of F cells and non-F cells were statistically nondistinguishable (P greater than 0.05) in all subjects. Such comparisons included normal individuals (less than 1% F cells), SS patients (7% to 48% F cells), subjects with congenital anemia (22% to 65% F cells), individuals with heterocellular hereditary persistence of HbF (HPFH) (12% to 21% F cells), and heterozygotes for beta + thalassemia (11% to 31% F cells). We conclude that gamma- and beta-globin production within F cells is regulated in a reciprocal fashion both among normal individuals and among individuals with elevated HbF production.
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Skyberg, Jerod A., and Carolyn A. Lacey. "Hematopoietic MyD88 mediates protection against virulent Francisella tularensis infection via IFN-γ, but does not require myeloid or dendritic cell MyD88 signaling." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 148.24. http://dx.doi.org/10.4049/jimmunol.198.supp.148.24.
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Abstract Francisella tularensis is a highly infectious intracellular bacterium that causes the potentially fatal disease tularemia. MyD88 has previously been shown to confer protection against both attenuated and virulent strains of F. tularensis, however the underlying mechanisms of protection are largely unexplored. Here we investigated cell-specific mechanisms of protection against virulent F. tularensis infection using mice with conditional MyD88 deficiencies. MyD88 deficiency in myeloid or dendritic cells did not enhance susceptibility to F. tularensis, regardless of the route of infection. In addition, myeloid or dendritic cell MyD88 deficiency did not markedly hinder the production of inflammatory cytokines. In contrast, MyD88−/− mice, or mice with hematopoietic MyD88 deficiency display elevated bacterial burdens, and markedly reduced cytokine levels. While IL-12 levels were not diminished in MyD88−/− or hematopoietic MyD88-deficient mice infected with F. tularensis, IFN-γ production was abolished in these animals. In particular, IFN-γ production by splenic NK cells was abated in mice lacking hematopoietic MyD88. Neutralization of IFN-γ from wild-type, but not hematopoietic MyD88-deficient mice, resulted in elevated tissue F. tularensis burdens. Caspase-1/11−/− mice also displayed enhanced bacterial burdens, diminished serum IL-18 levels, and reduced IFN-γ production. Collectively, our data shows that hematopoietic MyD88 is required for IFN-γ production that is protective against F. tularensis infection. As IL-18 requires MyD88 for signaling to induce IFN-γ production, MyD88 signaling in hematopoietic cells, such as NK cells, may result in the production of protective IFN-γ via caspase1/11 dependent IL-18.
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Naruse, K., D. S. McGehee, and G. S. Oxford. "Differential responses of Ca-activated K channels to bradykinin in sensory neurons and F-11 cells." American Journal of Physiology-Cell Physiology 262, no. 2 (February 1, 1992): C453—C460. http://dx.doi.org/10.1152/ajpcell.1992.262.2.c453.
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The nonapeptide bradykinin (BK) excites a subset of dorsal root ganglion (DRG) neurons with putative nociceptive functions by stimulating an inward cation current. In addition, BK stimulates various intracellular signaling pathways including an elevation of intracellular Ca2+. In a DRG neuron x neuroblastoma hybrid cell (F-11), BK stimulates similar increases in intracellular [Ca2+] and inward current but also elicits a large transient outward current through Ca(2+)-activated K channels. We have investigated the mechanisms underlying differential expression of outward current responses in the two cell types at the single channel level. Although K(Ca) channel activity appears in inside-out patches from both cells exposed to Ca2+, BK applied to the extrapatch membrane of cell-attached patches activates K(Ca) channels in F-11 but not DRG neurons. Whereas single K(Ca) channels are quantitatively similar in terms of conductance, voltage-dependence, and sensitivity to tetraethylammonium, they differ in sensitivity to intracellular Ca2+. Channel activation in both cells requires at least four Ca2+ ions, but half-maximal activation occurs at slightly higher [Ca2+] for DRG neurons. The shift in the Ca2+ dose-response curve combined with the steep [Ca2+] dependence of channel open probability makes it less likely that a BK-induced rise in internal [Ca2+] induced will trigger a transient outward current and resultant hyperpolarization in a DRG neuron.
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Schmalzl, Jonas, Piet Plumhoff, Fabian Gilbert, Frank Gohlke, Christian Konrads, Ulrich Brunner, Franz Jakob, Regina Ebert, and Andre F. Steinert. "Tendon-derived stem cells from the long head of the biceps tendon." Bone & Joint Research 8, no. 9 (September 2019): 414–24. http://dx.doi.org/10.1302/2046-3758.89.bjr-2018-0214.r2.
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Objectives The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration. Methods In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions. Results Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis. Conclusion These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery. Cite this article: J. Schmalzl, P. Plumhoff, F. Gilbert, F. Gohlke, C. Konrads, U. Brunner, F. Jakob, R. Ebert, A. F. Steinert. Tendon-derived stem cells from the long head of the biceps tendon: Inflammation does not affect the regenerative potential. Bone Joint Res 2019;8:414–424. DOI: 10.1302/2046-3758.89.BJR-2018-0214.R2.
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Garetto, L. P., E. R. Morey, G. N. Durnova, A. S. Kaplansky, and W. E. Roberts. "Preosteoblast production in COSMOS 2044 rats: short-term recovery of osteogenic potential." Journal of Applied Physiology 73, no. 2 (August 1, 1992): S14—S18. http://dx.doi.org/10.1152/jappl.1992.73.2.s14.
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The influence of a 13.8-day spaceflight and approximately 8.5–11 h of recovery at 1 g on fibroblast-like osteoblast precursor cells was assessed in the periodontal ligament of rat maxillary first molars. Preosteoblasts (C + D cells), less differentiated progenitor cells (A + A′ cells), and nonosteogenic fibroblast-like cells (B cells) were identified by nuclear volume analysis (i.e., A + A′ = 40–79 microns 3; B = 80–119 microns 3; C + D greater than or equal to 120 microns 3). No differences were observed among flight (F), synchronous (SC), vivarium, and basal control groups in the A + A′ (F: 28.0 +/- 3.7 vs. SC: 27.4 +/- 2.2), B (F: 33.1 +/- 1.4 vs. SC: 32.4 +/- 2.4), or C + D (F: 38.4 +/- 4.5 vs. SC: 39.2 +/- 1.6) cell compartments (mean +/- SE, n = 5). Compared with previous spaceflight experiments, the present data are consistent with a postflight response to replenish preosteoblasts and restore periodontal ligament osteogenic potential. These data emphasize the need to 1) unequivocally determine the flight effect by killing the animals in-flight and 2) further assess the postflight recovery phenomenon.
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Kemppainen, R. J., and T. P. Clark. "Evidence for a single glucocorticoid regulated pool of adrenocorticotropin in sheep anterior pituitary." American Journal of Physiology-Endocrinology and Metabolism 268, no. 1 (January 1, 1995): E85—E91. http://dx.doi.org/10.1152/ajpendo.1995.268.1.e85.
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The goal of this study was to determine whether separate glucocorticoid-sensitive releasable pools of adrenocorticotropic hormone (ACTH) could be distinguished in sheep anterior pituitary cells. Isolated cells were cultured in serum-free medium containing 0–10 nM cortisol (F) for 7–11 days to determine whether variation in the glucocorticoid environment selectively affected ACTH release stimulated by corticotropin-releasing hormone (CRH) or arginine vasopressin (AVP). Secretion was studied using a microperifusion system. The results indicated that while the concentration of F in the medium bathing the cells profoundly influenced the magnitude of ACTH released in response to either peptide, the fractional release of total ACTH was unchanged. F concentration in culture medium similarly did not alter the negative-feedback effectiveness of a larger dose of F applied to cells 45 min before treatment with CRH or AVP. These results support the existence of a single glucocorticoid-sensitive pool of ACTH in corticotrophs.
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Frecha, Cecilia, Caroline Costa, Didier Nègre, Emmanuel Gauthier, Stephen J. Russell, François-Loïc Cosset, and Els Verhoeyen. "Stable transduction of quiescent T cells without induction of cycle progression by a novel lentiviral vector pseudotyped with measles virus glycoproteins." Blood 112, no. 13 (December 15, 2008): 4843–52. http://dx.doi.org/10.1182/blood-2008-05-155945.
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AbstractA major limitation of current lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent cells such as primary T cells, which hampers their application for gene therapy. Here we generated high-titer LVs incorporating Edmonston measles virus (MV) glycoproteins H and F on their surface. They allowed efficient transduction through the MV receptors, SLAM and CD46, both present on blood T cells. Indeed, these H/F-displaying vectors outperformed by far VSV-G-LVs for the transduction of IL-7–prestimulated T cells. More importantly, a single exposure to these H/F-LVs allowed efficient gene transfer in quiescent T cells, which are not permissive for VSV-G-LVs that need cell-cycle entry into the G1b phase for efficient transduction. High-level transduction of resting memory (50%) and naive (11%) T cells with H/F-LVs, which seemed to occur mainly through SLAM, was not at cost of cell-cycle entry or of target T-cell activation. Finally, the naive or memory phenotypes of transduced resting T cells were maintained and no changes in cytokine profiles were detected, suggesting that T-cell populations were not skewed. Thus, H/F-LV transduction of resting T cells overcomes the limitation of current lentiviral vectors and may improve the efficacy of T cell–based gene therapy.
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Kiener, Hans P., Christopher S. Stipp, Philip G. Allen, Jonathan M. G. Higgins, and Michael B. Brenner. "The Cadherin-11 Cytoplasmic Juxtamembrane Domain Promotes α-Catenin Turnover at Adherens Junctions and Intercellular Motility." Molecular Biology of the Cell 17, no. 5 (May 2006): 2366–76. http://dx.doi.org/10.1091/mbc.e05-08-0745.
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Cadherins mediate homophilic cell adhesion and contribute to tissue morphogenesis and architecture. Cadherin cell adhesion contacts are actively remodeled and impact cell movement and migration over other cells. We found that expression of a mutant cadherin-11 lacking the cytoplasmic juxtamembrane domain (JMD) diminished the turnover of α-catenin at adherens junctions as measured by fluorescence recovery after photobleaching. This resulted in markedly diminished cell intercalation into monolayers reflecting reduced cadherin-11-dependent cell motility on other cells. Furthermore, the actin cytoskeleton in cadherin-11 ΔJMD cells revealed a more extensive cortical F-actin ring that correlated with significantly higher levels of activated Rac1. Together, these data implicate the cadherin-11 cytoplasmic JMD as a regulator of α-catenin turnover at adherens junctions and actin-cytoskeletal organization that is critical for intercellular motility and rearrangement in multicellular clusters.
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Zhang, Lian, Ying Zhang, Yunlong Lei, Zhili Wei, Yi Li, Yingxiong Wang, Youquan Bu, and Chundong Zhang. "Proline-rich 11 (PRR11) drives F-actin assembly by recruiting the actin-related protein 2/3 complex in human non-small cell lung carcinoma." Journal of Biological Chemistry 295, no. 16 (March 13, 2020): 5335–49. http://dx.doi.org/10.1074/jbc.ra119.012260.
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The actin cytoskeleton is extremely dynamic and supports diverse cellular functions in many physiological and pathological processes, including tumorigenesis. However, the mechanisms that regulate the actin-related protein 2/3 (ARP2/3) complex and thereby promote actin polymerization and organization in cancer cells are not well-understood. We previously implicated the proline-rich 11 (PRR11) protein in lung cancer development. In this study, using immunofluorescence staining, actin polymerization assays, and siRNA-mediated gene silencing, we uncovered that cytoplasmic PRR11 is involved in F-actin polymerization and organization. We found that dysregulation of PRR11 expression results in F-actin rearrangement and nuclear instability in non-small cell lung cancer cells. Results from molecular mechanistic experiments indicated that PRR11 associates with and recruits the ARP2/3 complex, facilitates F-actin polymerization, and thereby disrupts the F-actin cytoskeleton, leading to abnormal nuclear lamina assembly and chromatin reorganization. Inhibition of the ARP2/3 complex activity abolished irregular F-actin polymerization, lamina assembly, and chromatin reorganization due to PRR11 overexpression. Notably, experiments with truncated PRR11 variants revealed that PRR11 regulates F-actin through different regions. We found that deletion of either the N or C terminus of PRR11 abrogates its effects on F-actin polymerization and nuclear instability and that deletion of amino acid residues 100–184 or 100–200 strongly induces an F-actin structure called the actin comet tail, not observed with WT PRR11. Our findings indicate that cytoplasmic PRR11 plays an essential role in regulating F-actin assembly and nuclear stability by recruiting the ARP2/3 complex in human non-small cell lung carcinoma cells.
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Kocsis, B., and R. P. Vertes. "Dorsal raphe neurons: synchronous discharge with the theta rhythm of the hippocampus in the freely behaving rat." Journal of Neurophysiology 68, no. 4 (October 1, 1992): 1463–67. http://dx.doi.org/10.1152/jn.1992.68.4.1463.
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1. Single-unit activity of 30 dorsal raphe (DR) neurons was recorded along with the cortical and hippocampal electroencephalogram and neck muscle electromyogram in freely behaving rats during sleep-waking states. 2. On the basis of firing rates, DR cells were divided into slow-firing (S-cells), fast firing (F-cells), and very fast firing (FF-cells) units. The S-cells (8 units) fired at rates of < 10 Hz, the F-cells (11 units) at 10-40 Hz, and the FF-cells (11 units) at 55-70 Hz. 3. The activity of 17 of 30 DR units was correlated with the theta rhythm of the hippocampus. They included both slow and rapidly firing DR neurons. These cells typically fired irregularly (single spikes or short-duration bursts of activity) during non-theta states of quiet waking and slow-wave sleep. With the change of behavioral state to awake-moving or rapid eye movement sleep, the activity of these units switched to a regular bursting pattern synchronous with the hippocampal theta rhythm. Seven of these 17 units were classified as theta-rhythmic cells on the basis of the tight phase-locking of their discharge to the hippocampal theta rhythm. The remaining 10 units were classified as theta-modulated cells on the basis of a smaller but significant coherence between unit discharge and the theta rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)
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Valent, P., J. Besemer, K. Kishi, F. Di Padova, K. Geissler, K. Lechner, and P. Bettelheim. "Human basophils express interleukin-4 receptors." Blood 76, no. 9 (November 1, 1990): 1734–38. http://dx.doi.org/10.1182/blood.v76.9.1734.1734.
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Abstract Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.
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Valent, P., J. Besemer, K. Kishi, F. Di Padova, K. Geissler, K. Lechner, and P. Bettelheim. "Human basophils express interleukin-4 receptors." Blood 76, no. 9 (November 1, 1990): 1734–38. http://dx.doi.org/10.1182/blood.v76.9.1734.bloodjournal7691734.
Full textAbstract:
Interleukin-4 (IL-4), a multipotential lymphokine reputed to play an important role in the regulation of immune responses, interacts with a variety of hemopoietic target cells through specific cell surface membrane receptors. The present study was designed to investigate whether human basophils express IL-4 binding sites. For this purpose, basophils were enriched to homogeneity (93% and 98% purity, respectively) from the peripheral blood of two chronic granulocytic leukemia (CGL) donors using a cocktail of monoclonal antibodies (MoAbs) and complement. Purified basophils bound 125I-radiolabeled recombinant human (rh) IL-4 in a specific manner. Quantitative binding studies and Scatchard plot analysis revealed the presence of a single class of high affinity IL-4 binding sites (280 +/- 40 sites per cell in donor 1 and 640 +/- 45 sites per cell in donor 2) with an apparent dissociation constant, kd, of 7.12 x 10(-11) +/- 2.29 x 10(-11) and 9.55 +/- 3.5 x 10(-11) mol/L, respectively. KU812-F, a human basophil precursor cell line, was found to express a single class of 810 to 1,500 high affinity IL-4 binding sites with a kd of 2.63 to 5.54 x 10(-10) mol/L. No change in the numbers or binding constants of IL-4 receptors was found after exposure of KU812-F cells to rhIL-3 (a potent activator of basophils) for 60 minutes. No effect of rhIL-4 on 3H-thymidine uptake, release or synthesis of histamine, or expression of basophil differentiation antigens (Bsp-1, CD11b, CD25, CD40, CD54) on primary human CGL basophils or KU812-F cells was observed.
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Dou, Keke, Xunchang Wang, Zurong Du, Huanxiang Jiang, Feng Li, Mingliang Sun, and Renqiang Yang. "Synergistic effect of side-chain and backbone engineering in thieno[2,3-f]benzofuran-based conjugated polymers for high performance non-fullerene organic solar cells." Journal of Materials Chemistry A 7, no. 3 (2019): 958–64. http://dx.doi.org/10.1039/c8ta07544a.
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A series of copolymers containing thieno[2,3-f]benzofuran unit with different alkyl side chains are synthesized. The best photovoltaic performance with power conversion efficiency over 11% have been realized.
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Franco, Robert Suez, Peter Ciraolo, Mary B. Palascak, Clinton H. Joiner, Zahida Yasin, and Donald L. Rucknagel. "Sickle Cell Patietns with a High Percentage of HBF-Containing RBC (F Cells) Have Shorter Survival of RBC that Lack HBF." Blood 104, no. 11 (November 16, 2004): 3588. http://dx.doi.org/10.1182/blood.v104.11.3588.3588.
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Abstract Percent fetal hemoglobin (HbF) is an important determinant of clinical severity in sickle cells disease (SCD). There is a dichotomous distribution of HbF in sickle cells, with one population containing 20–25% HbF (F cells) and another in which HbF is not detectable (nonF cells). Increased HbF in SCD is due to two factors: 1) Increased HbF synthesis, which also occurs in other conditions having markedly increased erythropoiesis; 2) Longer survival of F cells in the circulation compared to nonF cells, which appears to be of great importance in SCD. We previously showed the lifespan of biotin-labeled F cells in the circulation to be about three times longer than nonF cells. We now examine these differences in greater detail, focusing on two issues. The first is whether the range of HbF content (HbF per F cell, pg) that is presumably present in F cells influences cell survival. The second is whether the survival of F and/or nonF cells is dependent upon the fraction of F cells in the circulation. To address these questions, we used the previously described biotin label for RBC. Up to 10 ml of autologous RBC were labeled and reinfused, and overall RBC survival was defined by the time-dependent disappearance of labeled RBC from the circulation. At selected time points after reinfusion, HbF was evaluated in two ways: 1) The percentage of biotin-labeled cells that were F cells was determined by flow cytometry; 2) The biotin-labeled cells were isolated with streptavidin-coated magnetic beads and the percent HbF determined by HPLC. These two assays can be used to determine the individual survival of F and nonF cells and to calculate the HbF per F cell of labeled RBC as a function of time after reinfusion. There were 12 studies in 10 patients, including 2 who were studied before and after hydroxyurea (HU). A total of 4 patients were taking HU at the time of study. F cells ranged from 4 to 90%, including 2 patients with HU and one without who had greater than 88%. There was a time-dependent linear increase in HbF per F cell with a slope of 0.09 ± 0.07 pg/day (n = 11). This is consistent with longer survival of the F cells with higher HbF content. NonF cell survival was lower in patients with a higher percentage of F cells. Subjects with < 50% F cells had an S30 (time until 30% of the labeled RBC remain in the circulation, days) for nonF cells of 14.6 ± 2.9 days (1 SD, n = 5), whereas subjects with > 50% F cells had an S30 for nonF cells of 7.7 ± 2.9 days (P < 0.005). The range of S30 values was from 5 to 17 days, and there was a linear correlation between the S30 of nonF cells and % F cells with R2 = 0.65. For a given % F cells, there appeared to be no dependence on HU. These data indicate that the survival of nonF cells (but not F cells) is dependent on the percentage of F cells. Possible reasons for this include 1) the presumed higher oxygen affinity of F cells, leading to lower venous PO2 and thus increased sickling and decreased survival of the nonF cells, and 2) a more sensitive detection of damaged RBC by the RES when RBC turnover is lower due to a high percentage of F cells, again leading to decreased survival of the nonF cells. This could have important implications in chronic transfusion therapy, in which overall RBC turnover is decreased by the presence of donor cells with relatively high oxygen affinity.
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Zhou, Ya-nan, Zi-xing Chen, Koike Kenichi, Sakashita Kazuo, Wei Wang, and Jiang-nong Cen. "Detection the Expression of Notch and Its Related Molecules in Bone Marrow Cells of Acute Leukemia Patients by Quantitative Real-Time RT-PCR." Blood 106, no. 11 (November 16, 2005): 4369. http://dx.doi.org/10.1182/blood.v106.11.4369.4369.
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Abstract The notch family of transmembrane receptors play crucial roles in cell fate determination through interaction with the Delta/Serrate /Lag-2 family of ligands. The transcription of the hairyl enhancer of split (HES-1) gene is one of the known target of notch signaling. Notch1 is involved in the pathogenesis of T-acute lymphoblastic leukemia (T-ALL) carrying the very rare translocation t (7;9)(q34;q34.3). However, the function of Notch signaling in leukemogenesis is currently still unclear. To investigate the possible role of notch signaling in human acute leukemia,A quantitative real-time reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method has been established for detecting expression levels of Notch1~2, Jagged1, Delta1, Hes1 and internal reference GAPDH in primary BM samples of 34 acute leukemia patients and 11 normal controls by LightCycler using TaqMan probe; 34 pretreated acute leukemia patients includes 16 acute lymphoblastic leukemia (7 T-ALL, 9 B-ALL) and 18 acute myeloblastic leukemia (10 M1,3 M2,3 M3 and 2 M5) In all of these cases the leukemia blast cells are more than 80%. The results demonstrated as the follows: The average expression levels of Notch1 in 16 ALL were statistically higher than that in 11 normal and 18 AML [ 13225.0±3306.3 (16) vs 1725.6±1237.8 (11) vs 2354.8±2985.6(18), F=7.85, P<0.01, ] There was no statistically difference between normal and AML group( P>0.05); the average expression levels of Notch2 did not have statistically difference among ALL, normal and AML.[143671.7±372814.9 (16) vs 52178.2±36747.5 (11) vs 66520.2±62643.2 (18) F=0.15, P>0.05 ]. The average expression levels of Jagged1 in 16 ALL were statistically less than that in 18 AML and 11 normal [129.9±353.8(16) vs 375.4±697.5(18) vs 2212.5±3371.7(11), F=20.23, P<0.01] while the jagged1 level was significantly lower in AML than in normal. The average levels of Delta1 in 16 ALL were statistically higher than that in normal and AML[30884.6±104097.1(16) vs 2299.8±4301.4(11) vs 579.7±641.7(18), F=5.78, P<0.01], and no statistically difference between AML and normal has been found. The average levels of Hes1 in AML were statistically less than normal and ALL[20.1±20.1(18) vs 1013.2±2507.0 (11) vs 244.5±347.3(16), F=6.62, P<0.01, there were no statistically difference between normal and ALL,(P.>0.05)]. In addition, the average levels of Delta1 in 9 B-ALL were statistically higher than those in 7 T-ALL[60316.6±145713.4(9) vs 378.0±238.6 (7), t=4.356, p=0.001]., the average expression levels of Notch1~2, Jagged1 and Hes1 in B-ALL were no statistically difference compare to T-ALL. In conclusion, Notch1 and Delta1 expression levels in ALL were statistically higher than that in normal and AML group, there were no statistically difference between normal and AML. Jagged1 expression levels in ALL were statistically less than that in AML and least in normal. Hes1 expression levels in AML were statistically less than that in normal and ALL, while no statistically difference could be found between normal and ALL groups. Taking altogether, in AL, the expression of Hes1 had not been found upregulated, instead was downregulated in AML. No statistically difference in Notch2 expression has been found among AML, ALL and normal group. In addition, Delta1 expression levels in B-ALL were statistically higher than that in T-ALL.
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Rein, Lindsay A. M., Minyong Chen, Barbara S. Theriot, James W. Wisler, Laura M. Wingler, Richard T. Premont, Wei Chen, Julia K. L. Walker, and Robert J. Lefkowitz. "Targeting β-arrestin2 Enhances Survival in a Murine Model of Chronic Myeloid Leukemia." Blood 122, no. 21 (November 15, 2013): 857. http://dx.doi.org/10.1182/blood.v122.21.857.857.
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Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm of hematopoietic stem cells characterized by presence of a dysregulated BCR-ABL fusion protein which leads to constitutive activation of tyrosine kinase activity. Classically, CML is treated with tyrosine kinase inhibitors (TKIs). However, TKI therapy is non-curative, and persistence of quiescent leukemia stem cells likely accounts for the inability of these agents to cure CML. β-arrestins are multifunctional adapter proteins which regulate G protein-coupled receptor (GPCR) signaling and trafficking and have recently been identified as mediators of distinct cellular signaling cascades independent of G proteins. β-arrestins also play a role in the Smoothened/Hedgehog pathway, as well as in the Wingless/Frizzled (Wnt/Fz) signaling axis, both of which have been associated with the development of CML. We, therefore, hypothesize that β-arrestin2 (βarr2) is necessary for the development and propagation of CML and may function as a therapeutic target. Aim Demonstrate that loss of βarr2, using an inducible conditional knockout mouse model, slows progression of CML. Methods We used a standard murine retroviral transduction system to model chronic phase BCR-ABL positive CML. KLS cells (Lin-, Sca-1+, c-kit+) were harvested from bone marrow of donor C57BL6/J βarr2F/F-CreERT2+/- (Cre positive, CD45.2) and age matched C57BL6/J βarr2F/F-CreERT2-/- (Cre negative, CD54.2) male mice. KLS cells were retrovirally transduced with MSCV-BCR-ABL-IRES-GFP and were subsequently injected retro-orbitally into sublethally irradiated congenic wild type recipient male mice (CD45.1). Donor mice were engineered using global Cre-ER/loxP technology in order to induce site-specific recombination and loss of βarr2 when treated with tamoxifen. Mice who received Cre positive cells lost βarr2 only in hematopoietic cells. Recipient mice were treated with tamoxifen 75mg/kg daily via intraperitoneal injection for 5 days starting day 3 after transplant and were monitored for signs of leukemia development. Weekly blood analysis included WBC count, number of donor cells by flow cytometry, blood film, and qPCR for BCR-ABL expression. Survival was compared between animals receiving Cre positive (βarr2F/F-CreERT2+/-) and Cre negative (βarr2F/F-CreERT2-/-) donor cells as well as both tamoxifen treated and untreated conditions. Results Treatment of donor C57BL6/J βarr2F/F-CreERT2+/- mice with tamoxifen resulted in decreased βarr2 expression within 10 days in multiple tissues including spleen and bone marrow. By day 10, βarr2 expression was 9.5 ± 3.0% in Cre positive mice relative to pretreatment expression levels (Figure 1). In total, 8 mice received Cre positive cells and 11 mice received Cre negative cells. Ten of 11 (90.9%) Cre negative mice developed CML as evidenced by splenomegaly, leukocytosis, increased BCR-ABL expression measured by qPCR and increased number of donor cells detectable by flow cytometry. Median survival was 15 days. Six of 8 (75%) Cre positive mice developed disease with median survival of 27 days (HR 3.2, 95% CI; 1.525-12.2, p=0.013)(Figure 2). At day 11, flow cytometry for donor CD45.2 cells present in peripheral blood of recipient mice was 38 ± 2.05% in Cre negative versus 16.5± 3.9% in Cre positive mice (p<0.0001). Spleen size at death and WBC count at day 11 were not significantly different between groups. Conclusions These data demonstrate that targeting βarr2 prolongs the course of disease in chronic phase CML and raise the possibility that loss of βarr2 after disease onset may lead to disease regression. βarr2 may therefore represent an alternative therapeutic target for CML independent of tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.
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Turek, John J., Christopher P. Leamon, and Philip S. Low. "Endocytosis of folate labeled proteins: Ultrastructural localization in kb cells." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 718–19. http://dx.doi.org/10.1017/s0424820100123994.
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Recently, methods for the delivery of macromolecules into cells via covalent coupling to vitamins have been described for plant and animal cells. This method utilizing vitamin receptor endocytosis concentrates macromolecules inside the cell in an active form that is nondegradative. The purpose of this study was to determine the specific location of bovine serum albumin-folic acid-colloidal gold (BSA-F-CG) conjugates within KB cells. Bovine serum albumin was covalently coupled to folic acid (BSA-F) and used to stabilize 15 nm colloidal gold (CG) particles. KB cells were incubated with BSA-F-CG or BSA-CG at either 11°C for 2 hours or 37°C for 15 minutes to allow binding, and the cells washed to remove unbound material. Some cells were fixed immediately, and others were incubated for additional time periods. BSA-CG particles nonspecifically pinocytosed by control cells were only found in large dense endosomes after 6 hours incubation. In cells incubated with BSA-F-CG, CG particles decorated the plasma membrane, and were found in uncoated pits. At 30 minutes CG particles could be found in multivesicular bodies (MVB’s), small vesicles, and dense endosomes. At 6 hours, CG particles were found in various dense endosomes, MVB’s, dense endosomes associated with the Golgi apparatus, and free in the cytoplasm. Cells that were pulsed with BSA-F-CG for 15 minutes at 37°C had CG particles on the surface and uncoated pits, small vesicles and MVB’s. After 60 and 360 minutes, BSA-F-CG was located in MVB’s, clear and dense endosomes, and MVB’s associated with the Golgi apparatus. Control cells incubated with BSA-CG had a few CG particles located in small clear endosomes at 15 minutes and large dense endosomes at 6 hours. For comparison, 5nm colloidal gold was stabilized with transferrin (TF) and incubated alone with KB cells or coincubated with 15 nm BSA-F-CG. The 5 nm TF-CG was localized in coated pits and on the rim of vesicular structures resembling CURL (Compartment of Uncoupling of Receptor and Ligand) at 15 minutes. There was no colocalization of TF-CG and BSA-F-CG at 15 minutes. TF-CG and BSA-F-CG were found separately and together in MVB’s at 1 hour. At six hours TF-CG and BSA-F-CG frequently colocalized in large dense endosomes. Folate labeled proteins endocytosed in this study shared some common compartments with transferrin, but there were several points of divergence. Transferrin-CG is taken up via coated pits whereas BSA-F-CG enters the cell via uncoated pits. At 15 minutes, internalized TF-CG is associated primarily with vesicles resembling CURL, and BSA-F-CG is found in MVB’s. At one hour, both TF-CG and BSA-F-CG may be found separately and together in MVB’s, and at 6 hours both may be found separately and together in dense endosomes. The BSA-F-CG particles free in the cytoplasm may be released during transport of the ligand, possibly by MVB’s that associate with the Golgi apparatus. The BSA-F-CG particles localized in this study may represent the normal location of endocytosed folate or their location could be an aberration of the normal pathway.
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Hall, V., D. Compton, P. Stojkovic, M. Nesbitt, M. Herbert, A. Murdoch, and M. Stojkovic. "36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER." Reproduction, Fertility and Development 18, no. 2 (2006): 126. http://dx.doi.org/10.1071/rdv18n2ab36.
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The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.
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Hinshaw, D. B., J. M. Burger, M. T. Miller, J. A. Adams, T. F. Beals, and G. M. Omann. "ATP depletion induces an increase in the assembly of a labile pool of polymerized actin in endothelial cells." American Journal of Physiology-Cell Physiology 264, no. 5 (May 1, 1993): C1171—C1179. http://dx.doi.org/10.1152/ajpcell.1993.264.5.c1171.
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Depletion of cellular ATP is associated with profound effects on the cytoskeleton, particularly disruption of microfilaments. We examined this process in bovine pulmonary artery endothelial cells by inducing differential reductions of cellular ATP using mitochondrial inhibition and variable amounts of glucose. Reduction of cellular ATP to levels < 40% of control produced discrete stages in the visible disruption of microfilaments. Using the deoxyribonuclease I assay, a reversible 11% decrease in monomeric (G) actin occurred in conjunction with microfilament disruption. Polyacrylamide gel electrophoretic (PAGE) analysis of the detergent-insoluble cytoskeleton did not reveal any differences in actin content between normal or ATP-depleted cells. Image analysis of adherent endothelial cells that had been fixed and stained with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin revealed that an increase of F-actin of approximately 20% occurred in cells depleted of ATP. If the cells were lysed with detergent before fixation, the increase in F-actin was lost. PAGE analysis and electron microscopy of detergent-soluble material from the cells obtained by ultracentrifugation directly demonstrated the presence of a labile pool of F-actin within the cells, which increased with ATP loss. These observations suggest that ATP may play an important role in the organization and remodeling of microfilaments within cells.
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Graham, Mark, Natanya Fleming, Susan Blumenthal, Brittani Boehlke, and Eric Schuur. "Circulating tumor cells (CTCs) and molecular subtypes (MST) in a treatment algorithm for metastatic breast cancer (MBC)." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e21022-e21022. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e21022.
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e21022 Background: We measured CTC s and MST of MBC [ER+, HER2- (HR+); HER2+ (H2+); or Triple Negative (TN)] in all MBC pts in our practice and used these in an algorithm to treat MBC. Methods: CTC’s were measured in 156 pts whether beginning Rx with 1st MBC (97), or under way with Rx (59). Pts had CTC s measured over 61 mo. from 11/20/06 thr. 12/31/11; 66 of the pts had died as of 12/31/11. Pts were Rx’d with an algorithm using CTC’s and MST: Pts with ≥ 5 CTC s were Rx’d with chemoRx, usually doublet ChemoRx, but accounting for pt preferences. When CTC’s improved to < 5 for a sustained 3-4 mo. period, or if initially < 5m we sought less morbid therapies: hormonal if ER+, or single agent ChemoRx +/- biologics if ER- or hormone refractory. In a detailed statistical analysis of variables affecting Disease Specific Survival (DSS) in the first 140 pts thr. 6/30/11, with median f/u of 24.0 mo., highest CTC level (max CTC) observed during f/u was used for analysis: CTC Low (max <5, n=60); CTC Moderate (max 5-99; n=55); and CTC High (max ≥ 100; n=25). Results: We reported the distribution of pts and death rates for the 3 MST and the 3 max CTC groupings earlier (Graham, ASCO 2010); the conclusions are similar with 156 pts and longer f/u. Each MST has 40% of pts with 0-4 CTC’s at all time points. Each MST has a smaller group with CTC’s ≥ 100, 16% of all pts. 33% of deaths (22 of 66) occurred in the ≥ 100 CTC pts. 88% with ≥ 100 CTC’s died; these are early deaths. Only 13 of 66 (20%) died if CTC’s < 5, and 31 of 65 (48%) ifh 5-99 CTC’s. In the first 140 pt analyzed thr. 6/30/11, the median DSS from date of highest CTC is not reached for the CTC Low (max. f/u 54 mo.), vs. 35.8 mo. in the CTC Moderate and 3.3 mo. in the CTC High. DSS was signif. longer for the CTC Low and CTC Moderate vs. the CTC High (P<0.001 for both comparisons). DSS was also signif. greater in the CTC Low vs. the CTC Moderate (P = 0.04). Cox multivariate analysis showed that the Max CTC group (HR 4.8, 95% CI 3.1 - 7.6, p < 0.001), age ≥ 55 at time of max CTC (HR 4.0, 95% CI 2.7 - 7.9, p < 0.001), and MST (HR 2.1, 95% CI 1.4 – 3.1, p < 0.001) were predictive of DSS. Conclusions: a. A treatment algorithm of CTC’s and MST in MBC finds subgroups of MBC with long, intermediate and very short DSS; b. Effective treatments are short-lived in pts with CTC max ≥ 100.
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Taus, Naomi S., and William J. Mitchell. "The Transgenic ICP4 Promoter Is Activated in Schwann Cells in Trigeminal Ganglia of Mice Latently Infected with Herpes Simplex Virus Type 1." Journal of Virology 75, no. 21 (November 1, 2001): 10401–8. http://dx.doi.org/10.1128/jvi.75.21.10401-10408.2001.
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ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes a latent infection in neurons of sensory ganglia, including those of the trigeminal ganglia. Latent viral infection has been hypothesized to be regulated by restriction of viral immediate-early gene expression in neurons. Numerous in situ hybridization studies in mice and in humans have shown that transcription from the HSV-1 genome in latently infected neurons is limited to the latency-associated transcripts. In other studies, immediate-early gene (ICP4) transcripts have been detected by reverse transcription-PCR (RT-PCR) in homogenates of latently infected trigeminal ganglia of mice. We used reporter transgenic mice containing the HSV-1(F) ICP4 promoter fused to the coding sequence of the β-galactosidase gene to determine whether neurons in latently infected trigeminal ganglia activated the ICP4 promoter. Mice were inoculated via the corneal route with HSV-1(F). At 5, 11, 23, and 37 days postinfection (dpi), trigeminal ganglia were examined for β-galactosidase-positive cells. The numbers of β-galactosidase-positive neurons and nonneuronal cells were similar at 5 dpi. The number of positive neurons decreased at 11 dpi and returned to the level of mock-inoculated transgenic controls at 23 and 37 dpi. The number of positive nonneuronal cells increased at 11 and 23 dpi and remained elevated at 37 dpi. Viral proteins were detected in neurons and nonneuronal cells in acutely infected ganglia, but were not detected in latently infected ganglia. Colabeling experiments confirmed that the transgenic ICP4 promoter was activated in Schwann cells during latent infection. These findings suggest that the cells that express the HSV-1 ICP4 gene in latently infected ganglia are not neurons.
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Smolock, Elaine M., Ryan M. Burke, Chenjing Wang, Tamlyn Thomas, Sri N. Batchu, Xing Qiu, Martha Zettel, Keigi Fujiwara, Bradford C. Berk, and Vyacheslav A. Korshunov. "Intima modifier locus 2 controls endothelial cell activation and vascular permeability." Physiological Genomics 46, no. 17 (September 1, 2014): 624–33. http://dx.doi.org/10.1152/physiolgenomics.00048.2014.
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Carotid intima formation is a significant risk factor for cardiovascular disease. C3H/FeJ (C3H/F) and SJL/J (SJL) inbred mouse strains differ in susceptibility to immune and vascular traits. Using a congenic approach we demonstrated that the Intima modifier 2 ( Im2) locus on chromosome 11 regulates leukocyte infiltration. We sought to determine whether inflammation was due to changes in circulating immune cells or activation of vascular wall cells in genetically pure Im2 (C3H/F.SJL.11.1) mice. Complete blood counts showed no differences in circulating monocytes between C3H/F and C3H/F.SJL.11.1 compared with SJL mice. Aortic vascular cell adhesion molecule-1 (VCAM-1) total protein levels were dramatically increased in SJL and C3H/F.SJL.11.1 compared with C3H/F mice. Immunostaining of aortic endothelial cells (EC) showed a significant increase in VCAM-1 expression in SJL and C3H/F.SJL.11.1 compared with C3H/F under steady flow conditions. Immunostaining of EC membranes revealed a significant decrease in EC size in SJL and C3H/F.SJL.11.1 vs. C3H/F in regions of disturbed flow. Vascular permeability was significantly higher in C3H/F.SJL.11.1 compared with C3H/F. Our results indicate that Im2 regulation of leukocyte infiltration is mediated by EC inflammation and permeability. RNA sequencing and pathway analyses comparing genes in the Im2 locus to C3H/F provide insight into candidate genes that regulate vascular wall inflammation and permeability highlighting important genetic mechanisms that control vascular intima in response to injury.
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Gao, Fang, Chunxue Peng, Yue Zhang, Hao Wang, Hailong Shen, and Ling Yang. "Glutathione Plays a Positive Role in the Proliferation of Pinus koraiensis Embryogenic Cells." International Journal of Molecular Sciences 23, no. 23 (November 24, 2022): 14679. http://dx.doi.org/10.3390/ijms232314679.
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In the large-scale breeding of conifers, cultivating embryogenic cells with good proliferative capacity is crucial in the process of somatic embryogenesis. In the same cultural environment, the proliferative capacity of different cell lines is significantly different. To reveal the regulatory mechanism of proliferation in woody plant cell lines with different proliferative potential, we used Korean pine cell lines with high proliferative potential 001#–001 (Fast) and low proliferative potential 001#–010 (Slow) for analysis. A total of 17 glutathione-related differentially expressed genes was identified between F and S cell lines. A total of 893 metabolites was obtained from the two cell lines in the metabolomic studies. A total of nine metabolites related to glutathione was significantly upregulated in the F cell line compared with the S cell line. The combined analyses revealed that intracellular glutathione might be the key positive regulator mediating the difference in proliferative capacity between F and S cell lines. The qRT-PCR assay validated 11 differentially expressed genes related to glutathione metabolism. Exogenous glutathione and its synthase inhibitor L-buthionine-sulfoximine treatment assay demonstrated the positive role of glutathione in the proliferation of Korean pine embryogenic cells.
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Yuksek, Kamile, Wen-ling Chen, David Chien, and Jing-hsiung James Ou. "Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein." Journal of Virology 83, no. 2 (October 29, 2008): 612–21. http://dx.doi.org/10.1128/jvi.00832-08.
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ABSTRACT Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein α3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the α3-binding domain. This α3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit α3. The ability of the F protein to bind to α3 raises the possibility that the HCV F protein may regulate protein degradation in cells.
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Wang, Yadan, Jiangong Yan, Zhongmou Zhang, Minghui Chen, Xianfu Wu, and Shuangcheng Ma. "Immunosuppressive Sesquiterpene Pyridine Alkaloids from Tripterygium wilfordii Hook. f." Molecules 27, no. 21 (October 26, 2022): 7274. http://dx.doi.org/10.3390/molecules27217274.
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Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A–J (1–10), were isolated from the roots of T. wilfordii, along with ten known analogues (11–20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9–16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC50 values of 7.25 μg/mL, 8.75 μM, 0.74 μM, and 15.66 μM, respectively, and no influence on the cell viability at a concentration of 100 μg/mL (TA) or 100 μM (5, 11, and 16). Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents.
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Bessonov, Ivan, Anastasia Moysenovich, Anastasia Arkhipova, Mariam Ezernitskaya, Yuri Efremov, Vitaliy Solodilov, Peter Timashev, Konstantin Shaytan, Alexander Shtil, and Mikhail Moisenovich. "The Mechanical Properties, Secondary Structure, and Osteogenic Activity of Photopolymerized Fibroin." Polymers 12, no. 3 (March 12, 2020): 646. http://dx.doi.org/10.3390/polym12030646.
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Previously, we have described the preparation of a novel fibroin methacrylamide (FbMA), a polymer network with improved functionality, capable of photocrosslinking into Fb hydrogels with elevated stiffness. However, it was unclear how this new functionality affects the structure of the material and its beta-sheet-associated crystallinity. Here, we show that the proposed method of Fb methacrylation does not disturb the protein’s ability to self-aggregate into the stable beta-sheet-based crystalline domains. Fourier transform infrared spectroscopy (FTIR) shows that, although the precursor ethanol-untreated Fb films exhibited a slightly higher degree of beta-sheet content than the FbMA films (46.9% for Fb-F-aq and 41.5% for FbMA-F-aq), both materials could equally achieve the highest possible beta-sheet content after ethanol treatment (49.8% for Fb-F-et and 49.0% for FbMA-F-et). The elasticity modulus for the FbMA-F-et films was twofold higher than that of the Fb-F-et as measured by the uniaxial tension (130 ± 1 MPa vs. 64 ± 6 MPa), and 1.4 times higher (51 ± 11 MPa vs. 36 ± 4 MPa) as measured by atomic force microscopy. The culturing of human MG63 osteoblast-like cells on Fb-F-et, FbMA-F-et-w/oUV, and FbMA-F-et substrates revealed that the photocrosslinking-induced increment of stiffness increases the area covered by the cells, rearrangement of actin cytoskeleton, and vinculin distribution in focal contacts, altogether enhancing the osteoinductive activity of the substrate.
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Tormin, Ariane, Jan Claas Brune, Stuart Walsh, Johan Richter, Xiaolong Fan, and Stefan Scheding. "Human Primary Mensenchymal Stromal Progenitor Cells Are Highly Enriched in Both, the CD271+/CD146+ and CD271+/CD146− Bone Marrow Population with the Latter Acquiring CD146 Expression upon Culture in-Vitro." Blood 112, no. 11 (November 16, 2008): 2422. http://dx.doi.org/10.1182/blood.v112.11.2422.2422.
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Abstract Culture-derived mesenchymal stromal cells (MSC), which are attractive candidates for clinical cell therapy approaches, arise from primary MSC progenitor/stem cells in the bone marrow. Recently, several groups have reported markers (CD271, CD146, GD2, SSEA4, etc.) that allowed for an enrichment of CFU-F, i.e. primary MSC progenitors. However, the exact phenotype of the bona fide mesenchymal stem/progenitor cells has not yet not been sufficiently defined. We therefore aimed to investigate primary MSC in bone marrow subpopulations defined by the expression of CD271 and CD146, as both markers have been reported to contain all assayable CFU-F and stromal stem cells, respectively (Quirici et al., Exp. Hematol. 2002; Sacchetti et al, Cell, 2007). Utilizing multi-color flow cytometry, unfractionated human bone marrow mononuclear cells (BM-MNC) were found to contain 0.05 ± 0.05% CD271+/CD146+ cells, whereas 0.82 ± 0.43% and 0.71 ± 0.23% were single-positive for CD271 and CD146, respectively. CD271/CD146 subpopulations were FACS sorted from lineage-depleted BM-MNC (RosetteSep) and assayed for CFU-F content (n=3). CFU-F could not be detected in the CD271−/CD146− fraction. In contrast, CFU-F initiating cells were highly enriched in the CD271+/CD146+/CD45−/low fraction (1.1 ± 0.2 CFU-F per 10 plated cells), which corresponds to a ca. 400-fold enrichment compared to the entire lineage-depleted fraction (2.7 ± 3.4 CFU-F per 1 × 104 plated cells). Of note, CFU-F could also be assayed at high frequency from CD271+/CD146− cells (20.4 ± 22.6 CFU-F per 1 × 104 plated cells). Generally, CFU-F were not found in the CD271+/CD146+/CD45+ and the CD271−/CD146+ fractions, and were also not detectable within the whole CD271+/CD45+ population of unfractionated BM-MNC (n=4), which, however, gave rise to erythropoietic colonies. The two CFU-F enriched populations, i.e. CD271+/CD146+/CD45−/low and CD271+/CD146− cells, were then cultured under standard MSC growth conditions. MSC derived from both populations exhibited a typical MSC surface marker profile (CD105+, CD90+, CD73+, HLA-class I+, CD45−, CD34−, CD19−, CD14−, HLA-DR−) and typical MSC differentiation (adipocytes, osteoblasts, chondrocytes). Interestingly, MSC generated from CD271+/CD146− cells became positive for CD146 in culture and stable CD146 expression over time was observed for MSC from both populations (up to the 5th passage, average 82 ± 11%). In contrast, over the same culture period CD271 expression decreased with passage number and an average of only 10 ± 4% of the cultured cells remained positive for CD271. To further characterize the CFU-F enriched subpopulations, single cells from CD271+/CD146+/CD45−/low and CD271+/CD146−/CD45−/low cells were sorted into fibronectin-coated 96-well plates to investigate colony growth and differentiation potential. CFU-F frequencies in this assay were 4 per 96 seeded cells for both populations and all but one of the CD271+/CD146+/CD45−/low clones could be further expanded in culture. Subpopulation-derived clones were capable of typical MSC differentiation and MSC derived from CD271+/CD146−/CD45−/low clones–similar to the bulk cultures–became CD146 positive (89 ± 12%) after 2 passages, whereas here CD271 expression was not lost. Taken together, CD271+/CD146+/CD45−/low and CD271+/CD146−/CD45−/low bone marrow cells are highly enriched for primary MSC progenitor cells. The difference in CD146 expression, which disappears in culture, might relate different localizations of the primary cells in the marrow but might possibly also reflect functional differences, e.g. in stemness. Accordingly, experiments addressing in-situ location, in vivo differentiation potential, gene expression and surface-marker expression profiling of primary MSC are currently under way.
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Winkler, Dirk, Thorsten Zenz, Daniel Mertens, Annett Habermann, Hartmut Döhner, and Stephan Stilgenbauer. "In-Vitro Treatment with the AKT-Inhibitor GSK 690693 Induces Cell Death in Lymphoma Cell Lines and in Primary CLL Cells and Is Followed by Caspase-3 Activation and Cytochrome C Release." Blood 112, no. 11 (November 16, 2008): 1589. http://dx.doi.org/10.1182/blood.v112.11.1589.1589.
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Abstract The PI3K/AKT pathway acts as a critical regulator of cell survival by stimulating cell proliferation and inhibiting apoptosis and has been implicated in the pathogenesis of lymphoproliferative disorders. Therefore, inhibition of AKT seems to be a highly attractive new approach for the treatment of lymphoma. We treated 9 cell lines with AKT-nhibitor (1, 10, 20 μM) over 24h and 48h respectively: EHEB (B-CLL), GRANTA-519 (MCL), JURKAT (T-ALL) BL-60, NAMALWA and BJAB (all Burkitt’s lymphoma), L363, OPM-2 and RPMI-8226 (all multiple myeloma). To determine the rates and type of AKT-inhibitor induced cell death, FACS analyses for CD19, 7AAD, active caspase-3, cytochrome c were performed. The phosphorylation status of AKT and its downstream proteins GSK3β, p70S6k and S6 was studied by Western blotting after 5–120 minutes. In addition, 11 primary CLL samples with either del 13q (n=3), del 11q (n=2), del 17p (n=3) or a normal karyotype (n=3) were treated with AKT inhibitor (10 μM; 2.5μM; 0.625 μM; 0.156 μM). CLL samples were cultured in both standard medium as well as in HS5-(human stromal cells) conditioned medium to reduce spontaneous apoptosis of CLL in-vitro. 6 out of 11 patients had unmutated VH genes. 8 Patients were untreated, 3 were previously treated. Fludarabine (0.1 μM) was added to AKT-inhibitor in 11 cases to test for synergistic effects. CLL cells were harvested after 48 hours and 5 days to measure cell viability using Celltiter-GLO-Assay. Treatment of cell lines lead to significant rates of AKT-inhibitor induced cell death (table 1), to hyperphosphorylation of AKT and to inhibition of phosphorylation of GSK3β (after 5 min) and S6 (after 20 min) in all cell lines and of p70S6k (after 120 min) in GRANTA, JURKAT, NAMALWA and BJAB. Cell death did not depend on functional p53 gene. Treatment of primary CLL samples with AKT-inhibitor alone was followed by a decrease of cell viability in a time and concentration dependent manner regardless of the medium used (table 2). Only with the lowest concentration and when cultured in HS5-conditioned medium, no further reduction of viable cells was seen between 48h and 5d. Treatment with AKT-inhibitor as a single agent seemed to be at least as effective as treatment with fludarabine. Response was independent of the genetic subgroup, VH mutation status or prior treatment. High risk cases with del 17p responded worse to fludarabine alone when compared to cases without del 17p (i.e. 75% of viable cells after 5d at 10000 μM in cases with del 17p vs. 25% in cases without del 17p). The same fludarabine resistant cases showed good responses to treatment with AKT-inhibitor (9% of viable cells after 5d at 10000 μM in cases with del 17p). A synergistic effect was not achieved by combining AKT-inhibitor and fludarabine. Culture of CLL cells in HS5-conditioned medium resulted in lower rates of spontaneous apoptosis, but also in lower rates of AKT-inhibitor induced cell death. In conclusion, in-vitro treatment with AKT-inhibitor resulted in significant rates of cell death in cell lines and primary CLL cells, even in patients with del 17p or resistance to fludarabine. In cell lines, treatment with AKT-inhibitor was followed by typical features of apoptosis such as activation of caspase-3 and cytochrome c release. In CLL samples, prior treatment did not affect in-vitro response rates. These data underline the involvement of the PI3K/Akt pathway in the pathogenesis of lymphoma and point to an efficacy of the AKT-inhibitor in the treatment of lymphoma, multiple myeloma and CLL in-vivo. Concerning CLL, the AKT-inhibitor seems to be an attractive new treatment option even for cases with high risk cytogenetics. Using HS5-conditioned medium seems to be a well functioning method to reduce spontaneous apoptosis of CLL cells in-vitro. Table 1: rates of cell death, caspase-3 activation and cytochrome c release after treatment of cell lines with AKT inhibitor (1μM, 48h) 7AAD-positive cells active caspase-3 cytochrome c release EHEB 15% − + GRANTA-519 15% + + JURKAT 17% + + BL60 24% + + NAMALWA 25% − (+) BJAB 30% + (+) L363 15% + − OPM-2 41% + + RPMI-8226 32% + (+) Table 2: mean percentage of viable cells after treatment with AKT-Inhibitor (A), fludarabine (F; 0,1μM) and their combination (A + F) measured by Celltiter-GLO-Assay 10000 nM 2500 nM 625 nM 156,25 nM 48h 5d 48h 5d 48h 5d 48h 5d A F A + F A F A + F A A F A+ F A F A+ F A HS5 + (n=8) 94 84 (n=5) 75 (n=5) 45 22 (n=5) 25 (n=5) 88 52 91 84 80 69 22 18 76 85 HS5 − (n=11) 60 79 59 8 39 21 77 27 80 79 76 28 39 34 82 (n=10) 21
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Segerman, Anna, John P. Atkinson, Marko Marttila, Veronica Dennerquist, Göran Wadell, and Niklas Arnberg. "Adenovirus Type 11 Uses CD46 as a Cellular Receptor." Journal of Virology 77, no. 17 (September 1, 2003): 9183–91. http://dx.doi.org/10.1128/jvi.77.17.9183-9191.2003.
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ABSTRACT The 51 human adenovirus serotypes are divided into six species (A to F). Many adenoviruses use the coxsackie-adenovirus receptor (CAR) for attachment to host cells in vitro. Species B adenoviruses do not compete with CAR-binding serotypes for binding to host cells, and it has been suggested that species B adenoviruses use a receptor other than CAR. Species B adenoviruses mainly cause disease in the respiratory tract, the eyes, and in the urinary tract. Here we demonstrate that adenovirus type 11 (Ad11; of species B) binds to Chinese hamster ovary (CHO) cells transfected with CD46 (membrane cofactor protein)-cDNA at least 10 times more strongly than to CHO cells transfected with cDNAs encoding CAR or CD55 (decay accelerating factor). Nonpermissive CHO cells were rendered permissive to Ad11 infection upon transfection with CD46-cDNA. Soluble Ad11 fiber knob but not Ad7 or Ad5 knob inhibited binding of Ad11 virions to CD46-transfected cells, and anti-CD46 antibodies inhibited both binding of and infection by Ad11. From these results we conclude that CD46 is a cellular receptor for Ad11.
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Brown, Ronald L., J. Zhang, L. Qiu, A. Nett, G. Almeida-Porada, R. Herzig, and E. D. Zanjani. "Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells." Blood 104, no. 11 (November 16, 2004): 4954. http://dx.doi.org/10.1182/blood.v104.11.4954.4954.
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Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.
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Dover, GJ, KD Smith, YC Chang, S. Purvis, A. Mays, DA Meyers, C. Sheils, and G. Serjeant. "Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an X-linked gene located at Xp22.2." Blood 80, no. 3 (August 1, 1992): 816–24. http://dx.doi.org/10.1182/blood.v80.3.816.816.
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Abstract Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.
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Dover, GJ, KD Smith, YC Chang, S. Purvis, A. Mays, DA Meyers, C. Sheils, and G. Serjeant. "Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an X-linked gene located at Xp22.2." Blood 80, no. 3 (August 1, 1992): 816–24. http://dx.doi.org/10.1182/blood.v80.3.816.bloodjournal803816.
Full textAbstract:
Fetal hemoglobin (Hb F) production in sickle cell (SS) disease and in normal individuals varies over a 20-fold range and is under genetic control. Previous studies suggested that variant Hb F levels might be controlled by genetic loci separate from the beta-globin complex on chromosome 11. Using microscopic radial immunodiffusion and flow cytometric immunofluorescent assays to determine the percentage of F reticulocytes and F cells in SS and nonanemic individuals, we observed that F-cell levels were significantly higher in nonanemic females than males (mean +/- SD, 3.8% +/- 3.2% v 2.7% +/- 2.3%). F-cell production as determined by F reticulocyte levels in SS females was also higher than in SS males (17% +/- 10% v 13% +/- 8%). We tested the hypothesis that F-cell production in both normal and anemic SS individuals was controlled by an X-linked locus with two alleles, high (H) and low (L). Using an algorithm to determine the 99.8% confidence interval of a normal distribution in nonanemic individuals, we estimated that males and females with at least one H allele had greater than 3.3% F cells. Comparisons of male-male or female-female SS sib pairs with discordant F reticulocyte levels distinguished two phenotypes in SS males (L, less than 12%; H, greater than 12%) and three phenotypes in SS females (LL, less than 12%; HL, 12% to 24%, HH greater than 24%). Linkage analysis using polymorphic restriction sites along the X chromosome in eight SS and one AA family localized the F-cell production (FCP) locus to Xp22.2, with a maximum lod score (logarithm of odds of linkage v independent assortment) of 4.6 at a recombination fraction of 0.04.
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Tanaka, K., W. W. Hancock, H. Osawa, K. G. Stunkel, T. V. Alberghini, T. Diamantstein, N. L. Tilney, and J. W. Kupiec-Weglinski. "Mechanism of action of anti-IL-2R monoclonal antibodies. ART-18 prolongs cardiac allograft survival in rats by elimination of IL-2R+ mononuclear cells." Journal of Immunology 143, no. 9 (November 1, 1989): 2873–79. http://dx.doi.org/10.4049/jimmunol.143.9.2873.
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Abstract ART-18, a mouse IgG1 mAb recognizing the IL-2 binding domain of the rat p55 subunit IL-2R molecule, prevents graft rejection in various experimental models, although its mechanism of action in vivo, like that of anti-IL-2R mAb generally, remains elusive. These studies were designed to define whether IL-2R+ T effector cells were actually eliminated or their function merely inhibited by comparing directly the in vitro and in vivo efficacy of intact ART-18 and its F(ab)/F(ab')2 fragments. Addition of each mAb preparation profoundly suppressed MLR set up between naive LEW responders and x-radiated BN stimulators, suggesting that mAb fragments retained Ag binding functions in vitro. However, both ART-18 F(ab) and F(ab')2 were ineffectual in vivo as judged by their inability to affect acute (8 days) rejection of (LEW X BN)F1 cardiac allografts in LEW recipients (graft survival ca. 11 and 9 days, respectively, compared to ca. 21 days after therapy with intact ART-18, p less than 0.001). The sera levels of ART-18 and ART-18 F(ab')2 were 4 to 5 micrograms/ml, but only less than 0.5 micrograms/ml of F(ab) could be detected. The therapeutic failure of ART-18 fragments was unrelated to potential host sensitization, as rat antimouse F(ab) or F(ab')2 serum IgG titers remained in the same range as those against intact ART-18. The role of the Fc portion of Ig in the mode of action of ART-18 was then tested further by flow microfluorimetry analysis of host mononuclear spleen cells and immunoperoxidase stains of the graft infiltrate. IL-2R+ cells were abundant in rats treated with ART-18 fragments, comparable to acutely rejecting controls. In contrast, IL-2R expression was abolished in animals undergoing ART-18 therapy. The elimination of IL-2R+ cells is required to prolong cardiac allograft survival in rats after IL-2R targeted treatment with ART-18 mAb.
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White, C. A., E. Dimitriadis, A. Sharkey, C. J. Stoikos, and L. A. Salamonsen. "254.Interleukin-11 enhances endometrial stromal cell decidualisation via activation and inhibition of target genes." Reproduction, Fertility and Development 16, no. 9 (2004): 254. http://dx.doi.org/10.1071/srb04abs254.
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Differentiation of endometrial stromal cells into decidual cells is essential for successful embryo implantation. Interleukin (IL)-11 signalling is required for decidualisation in the mouse (1,2) and the expression pattern of IL-11 and its receptors during the menstrual cycle suggests a role for IL-11 in human decidualisation (3). Exogenous IL-11 has been shown to enhance hormone-induced decidualisation of human endometrial stromal cells in culture (4). This study aimed to determine the effects of IL-11 on downstream gene expression in endometrial stromal cells following 12 days of progesterone-induced decidualisation, and to examine the expression and functional significance of IL-11 target genes during this process. Stromal cells isolated from endometrial biopsies (n = 6) were decidualised with 17β-oestradiol and medroxyprogesterone acetate (EP) or EP with 100 ng/mL recombinant human IL-11. Medium was changed every 48 h, and total RNA extracted on Day 12 for gene expression analysis using custom-made 15K cDNA microarrays. Quantitative real-time RT-PCR was performed on the same samples to confirm gene expression levels. In subsequent experiments (n = 2), cells were cytocentrifuged onto glass slides for immunocytochemistry using specific antibodies. Microarray analysis revealed 16 upregulated and 11 downregulated cDNAs in EP + IL-11 compared to EP treated cells. Among these were IL-1β (6.1-fold upregulated) and insulin-like growth factor binding protein (IGFBP)-5 (3.6-fold downregulated). Using real-time RT-PCR, IL-11 was confirmed to increase IL-1β (fold change 1.3–107.1) and decrease IGFBP-5 (fold change 2.8–469.0) transcript abundance in 6 patients. Immunolocalisation of IL-1β in EP and EP + IL-11 treated cells revealed more intense vesicular cytoplasmic staining with IL-11 treatment, while staining intensity for IGFBP-5 was not affected. Interactions between IL-11 and its downstream targets IL-1β and IGFBP-5 are likely to have functional importance in early pregnancy, and may provide novel targets for the manipulation of human fertility. (1) Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG (1998) Nat. Med. 4, 303–308. (2) Bilinski P, Roopenian D, Gossler A (1998) Gene Dev. 12, 2234–2243. (3) Dimitriadis E, Salamonsen LA, Robb L (2000) Mol. Hum. Reprod. 6, 907–914. (4) Dimitriadis E, Robb L, Salamonsen LA (2002) Mol. Hum. Reprod. 8, 636–643.
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Zhou, P., G. D. Anderson, S. Savarirayan, H. Inoko, and C. S. David. "Thymic deletion of V beta 11+, V beta 5+ T cells in H-2E negative, HLA-DQ beta+ single transgenic mice." Journal of Immunology 146, no. 3 (February 1, 1991): 854–59. http://dx.doi.org/10.4049/jimmunol.146.3.854.
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Abstract DQw6b transgenic mice have been generated by microinjecting a linearized cosmid clone containing 34-kb DQb genomic DNA, isolated from HLA-homozygous B cell line AKIBA (DR2, Dw12, DQw6), into embryos of (CBA x B10.M)F2 or (SWR x SJL)F2. Among 85 mice screened, eight mice were transgene-positive. The transgene in seven of eight founders was germline-transmitted. FACS analysis and immunohistochemical studies with DQ beta-specific mAb demonstrated that DQ beta molecules in association with mouse A alpha f molecules are expressed on peripheral mononuclear cells, spleen cells, and in thymic medulla. More interestingly, V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells, but not V beta 8.2-bearing T cells, were clonally deleted in the H-2E-negative but DQ beta+ progeny of two selected founders (260-23 and 258-10). The deletion was found to take place intrathymically during the transition stage from immature to mature thymocyte development. We postulate that although human DQ genes are more homologous to mouse H-2A genes, A alpha f/DQ beta hybrid molecules may possess the same self-peptide- (or superantigen)-presenting epitope as E alpha/E beta molecules critical for deletion of V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells in thymus. Our results also confirm the previous findings that accessory molecules on thymocytes such as CD4 may be involved in thymic selection, and further suggest that an interaction of mousE CD4 and mouse A alpha chain is required for the clonal deletion.
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Maier-Redelsperger, Micheline, Mariane de Montalembert, Antoine Flahault, Maria Grazia Neonato, Rolande Ducrocq, Marie-Pierre Masson, Robert Girot, Jacques Elion, and the French Study Group on Sickle Cell Disease. "Fetal Hemoglobin and F-Cell Responses to Long-Term Hydroxyurea Treatment in Young Sickle Cell Patients." Blood 91, no. 12 (June 15, 1998): 4472–79. http://dx.doi.org/10.1182/blood.v91.12.4472.
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Abstract We have studied the cellular and molecular responses to long-term hydroxyurea (HU) treatment in 29 severely affected young patients with sickle cell disease (mean age, 10.9 ± 4.1 years). Patients received HU at 20 mg/kg/d on 4 consecutive days per week initially, with a monthly escalated dose avoiding marrow-toxicity (mean steady-state dose, 34.2 ± 4.6 mg/kg/d) for 12 to 36 months (mean duration, 22 months). The studied parameters were hemoglobin F (HbF), F reticulocytes (F retics), F cells, the amount of HbF per F cell (F/F cell), polymer tendency at 40% and 70% oxygen saturation, and hemolysis. Initial HbF (Fi) was dispersed (from 0.85% to 13.9%). HbF increased in all patients but 1. HbF at maximal response (Fmax) reached a sustained level varying from a 1.5-fold to a 16-fold Fi after a variable delay (6 to 24 months). Fmax was not related to HU dosage, but ▵F (Fmax − Fi) was strongly correlated to ▵MCV (MCVmax − MCVi). HbF increase resulted from the increase of both F cells and F/F cell. In this rather short series, Fi and Fmax were not significantly associated with age, gender, or β-globin haplotype. Neither Fmax nor ▵F was related to bone marrow reserve, as measured by baseline reticulocyte or neutrophil counts. However, Fmax was highly dependent on Fi. When patients are individualized into three groups according to Fmax (group 1, Fmax >20% [12 patients]; group 2, 10% < Fmax < 20% [11 patients]; group 3, Fmax <10% [5 patients]), Fi is significantly different between groups, being the highest in group 1. In addition, the best responders (group 1) were significantly different from patients in the two other groups with higher levels of total hemoglobin, decreased bilirubin, and decreased polymer tendency.
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Maier-Redelsperger, Micheline, Mariane de Montalembert, Antoine Flahault, Maria Grazia Neonato, Rolande Ducrocq, Marie-Pierre Masson, Robert Girot, Jacques Elion, and the French Study Group on Sickle Cell Disease. "Fetal Hemoglobin and F-Cell Responses to Long-Term Hydroxyurea Treatment in Young Sickle Cell Patients." Blood 91, no. 12 (June 15, 1998): 4472–79. http://dx.doi.org/10.1182/blood.v91.12.4472.412k16_4472_4479.
Full textAbstract:
We have studied the cellular and molecular responses to long-term hydroxyurea (HU) treatment in 29 severely affected young patients with sickle cell disease (mean age, 10.9 ± 4.1 years). Patients received HU at 20 mg/kg/d on 4 consecutive days per week initially, with a monthly escalated dose avoiding marrow-toxicity (mean steady-state dose, 34.2 ± 4.6 mg/kg/d) for 12 to 36 months (mean duration, 22 months). The studied parameters were hemoglobin F (HbF), F reticulocytes (F retics), F cells, the amount of HbF per F cell (F/F cell), polymer tendency at 40% and 70% oxygen saturation, and hemolysis. Initial HbF (Fi) was dispersed (from 0.85% to 13.9%). HbF increased in all patients but 1. HbF at maximal response (Fmax) reached a sustained level varying from a 1.5-fold to a 16-fold Fi after a variable delay (6 to 24 months). Fmax was not related to HU dosage, but ▵F (Fmax − Fi) was strongly correlated to ▵MCV (MCVmax − MCVi). HbF increase resulted from the increase of both F cells and F/F cell. In this rather short series, Fi and Fmax were not significantly associated with age, gender, or β-globin haplotype. Neither Fmax nor ▵F was related to bone marrow reserve, as measured by baseline reticulocyte or neutrophil counts. However, Fmax was highly dependent on Fi. When patients are individualized into three groups according to Fmax (group 1, Fmax >20% [12 patients]; group 2, 10% < Fmax < 20% [11 patients]; group 3, Fmax <10% [5 patients]), Fi is significantly different between groups, being the highest in group 1. In addition, the best responders (group 1) were significantly different from patients in the two other groups with higher levels of total hemoglobin, decreased bilirubin, and decreased polymer tendency.
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Wen, Xueyan, Songrong Li, Mengchan Guo, Hongzhan Liao, Yongmin Chen, Xi Kuang, Xiaoping Liao, Lin Ma, and Qifu Li. "miR‑181a‑5p inhibits the proliferation and invasion of drug‑resistant glioblastoma cells by targeting F‑box protein 11 expression." Oncology Letters 20, no. 5 (September 14, 2020): 1. http://dx.doi.org/10.3892/ol.2020.12098.
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Zeng, Guichao, Luoyi Gao, Keiji Suetake, Ratan Mani Joshi, and Robert K. Yu. "Variations in gene expression patterns correlated with phenotype of F-11 tumor cells whose expression of GD3-synthase is suppressed." Cancer Letters 178, no. 1 (April 2002): 91–98. http://dx.doi.org/10.1016/s0304-3835(01)00817-5.
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Zeng, Guichao, Luoyi Gao, and Robert K. Yu. "Reduced cell migration, tumor growth and experimental metastasis of rat F-11 cells whose expression of GD3-synthase is suppressed." International Journal of Cancer 88, no. 1 (2000): 53–57. http://dx.doi.org/10.1002/1097-0215(20001001)88:1<53::aid-ijc8>3.0.co;2-7.
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Pittenger, M. F., A. Kistler, and D. M. Helfman. "Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon." Journal of Cell Science 108, no. 10 (October 1, 1995): 3253–65. http://dx.doi.org/10.1242/jcs.108.10.3253.
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The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.
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Giona, Fiorina, Maria Caterina Putti, Maria Luisa Moleti, Mauro Nanni, Anna Maria Testi, Stefania Varotto, Enrico Marco Gottardi, et al. "Imatinib Mesylate Induces High Complete Cytogenetic and Molecular Response Rates in Children and Adolescents with Philadelphia Chromosome-Positive (Ph+) Chronic Myelogenous Leukemia (CML) in Chronic Phase (CP)." Blood 112, no. 11 (November 16, 2008): 4273. http://dx.doi.org/10.1182/blood.v112.11.4273.4273.
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Abstract Imatinib mesylate (IM), a BCR-ABL tyrosine kinase inhibitor, is an effective therapy for CML in adults and has shown efficacy in children with Ph+ leukemias. The aim of this study was to evaluate the efficacy of IM in Ph+ CML patients (pts) in CP aged <18 years at diagnosis, previously untreated or resistant to Interferon (IFN). In all pts, IM therapy, started at a dose of 340 mg/m2/day, was modulated according to the hematologic parameters. Cytogenetic studies were performed on bone marrow (BM) cells at baseline and, during IM therapy, every 3 months (mo). Complete cytogenetic response (CCyR) was also confirmed by FISH. BCR-ABL transcripts were measured in the peripheral blood (PB) cells every mo and in the BM cells every 3 mo by real-time quantitative PCR (RQ-PCR). Molecular response (MolR) was defined as major in the presence of a BCRABL: ABL ratio <0.05% and as complete with a ratio <0.001. Between February 2001 and October 2007, 13 Ph+ CML pts (9 M and 4 F; median age 128/12 years) in CP were recruited from 2 pediatric centers (Rome and Padua). Eight of the 13 pts (7 M and 1 F; median age 11 years) received IM at diagnosis and 5 (3 F and 1 M; median age 146/12 years) after IFN therapy given at a mean dose of 6.000.000 UI/day for a median of 18 mo. All but 1 pt tolerated well IM treatment. The mean dose of IM administered was 326 mg/m2/day for untreated pts and 227 mg/m2/day for those resistant to IFN. The characteristics and followup of the pts are summarized in the Table: Sex/Age at diagn/Age at treat (yrs) IFN duration/%Ph+ IM mg/m2/day CCyR/time (mo) Max Bcr-Abl:Abl (%)/time (mo) (BM) Max Bcr-Abl:Abl (%)/time (mo) (PB) CCyR duration (mo) Follow-up .F/11/146/12 40 mo/100 193.5 4 0/60 0/4 +80 Alive CCyR, Bcr-Abl:Abl (%)BM 0 PB 0.0023 F/179/12/1810/12 9 mo/100 182 6 1.27/9 0.89/9 +7 Lost to follow-up in CCyR at + 13 mo M/91/12/117/12 26 mo/50 208 3 0/36 0/12 +65 Alive CCyR, Bcr-Abl:Abl (%)BM 0 PB 0 F/89/12/910/12 13 mo/50 350 3 0/44 0/66 +66 Alive CCyR, Bcr-Abl:Abl (%)BM 0.009 PB 0 M/172/12/189/12 18 mo/80 205 9 0.029/68 0.114/72 +82 Alive CCyR, Bcr-Abl:Abl (%)BM 0.05 PB 0.15 M/126/12 −/100 310 4 0/42 0/30 +66 Alive CCyR, Bcr-Abl:Abl (%): BM 0 PB 0 M/161/12 −/100 327 n.e. n.e. n.e. n.e. IM tox; alive CCyR after SCT (sibl) (+40 mo) M/144/12 −/100 291 4 0/42 0/24 +61 Alive CCyR, Bcr-Abl:Abl (%) BM 0 PB 0 M/811/12 −/100 357.5 6 0.044/9 0.057/9 CyRel/33 Alive CCyR after SCT (+ 8 mo) M/95/12 −/100 326 3 0.013/12 0.028/9 +12 Alive CCyR,Bcr-Abl:Abl (%) BM 0.013 PB 0.15 M/410/12 −/100 328.5 3 0.02/9 0/12 BMT/+13 SCT (sibl) in CCyR->Alive in CCyR +43 mo M/137/12 −/100 349 6 0.012/30 0.025/30 +32 Alive CCyR, Bcr-Abl:Abl (%) BM 0.012 PB 0.025 F/94/12 −/100 320 3 0.009/9 0.003/9 +7 Alive CCyR, Bcr-Abl:Abl (%) BM 0.02 PB 0.003 Twelve of the 13 pts (92%) achieved a CCyR after a median of 4 mo (range 3–9). Eleven of the latter 12 pts were evaluated for MolR: 11/11 (100%) pts achieved a MolR, 6 major (54.5%) and 5 complete (45.5%), on BM cells after a median of 36 mo (range 9–68) and 9/11 pts (82%) on PB cells, 4 major (44.4%) and 5 complete (55.6%), after a median of 12 mo (range 4–66). To date, 12 evaluable pts are alive in CCyR: 3 after a stem cell transplantation (SCT) and 9 still receiving IM for a median time of 68 mo (range 10–89). MolR persists on BM cells in 9/9 pts (100%), 4 complete (44%), and on PB cells in 7/9 pts (78%), 4 complete. Our experience indicates that IM is highly effective in children and adolescents with Ph+ CML in CP, capable also of inducing high and persistent CCyR and MolR rates also in pts resistant to IFN.