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Kaiser, C. A., i D. Botstein. "Efficiency and diversity of protein localization by random signal sequences". Molecular and Cellular Biology 10, nr 6 (czerwiec 1990): 3163–73. http://dx.doi.org/10.1128/mcb.10.6.3163-3173.1990.
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Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.
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Kaiser, C. A., i D. Botstein. "Efficiency and diversity of protein localization by random signal sequences." Molecular and Cellular Biology 10, nr 6 (czerwiec 1990): 3163–73. http://dx.doi.org/10.1128/mcb.10.6.3163.
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Three randomly derived sequences that can substitute for the signal peptide of Saccharomyces cerevisiae invertase were tested for the efficiency with which they can translocate invertase or beta-galactosidase into the endoplasmic reticulum. The rate of translocation, as measured by glycosylation, was estimated in pulse-chase experiments to be less than 6 min. When fused to beta-galactosidase, these peptides, like the normal invertase signal sequence, direct the hybrid protein to a perinuclear region, consistent with localization to the endoplasmic reticulum. The diversity of function of random peptides was studied further by immunofluorescence localization of proteins fused to 28 random sequences: 4 directed the hybrid to the endoplasmic reticulum, 3 directed it to the mitochondria, and 1 directed it to the nucleus.
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Lichti, Cheryl, Anthony N. Vomund, Orion J. Peterson, Xiaoxiao Wan i Emil R. Unanue. "Analysis of the nonobese diabetic mouse islet MHC-II peptidome reveals posttranslationally modified autoantigens". Journal of Immunology 204, nr 1_Supplement (1.05.2020): 142.12. http://dx.doi.org/10.4049/jimmunol.204.supp.142.12.
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Abstract Autoimmune diabetes results from the destruction of insulin-producing beta cells, a process initiated by CD4 T cells recognizing MHC-II bound beta cell-derived peptides. We identified MHC-II bound peptides in the islets and throughout the peripheral lymphatic system in order to better understand diabetes initiation and progression. Toward this end, we performed unbiased nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis to obtain sequence information for MHC-II peptides isolated from islets, pancreatic lymph nodes and spleens of nonobese diabetic (NOD) mice. Peptides derived from beta cell proteins were further examined for their relative I-Ag7 binding strength and CD4 T cell autoreactivity. For a discussion of the major immunogenic peptides derived from the B chain of insulin and from C peptide, see the abstract of Wan et al. Herein we discuss the finding of a deamidated C peptide fragment and our search for fused peptides. We identified the deamidated insulin-1 C peptide fragment Ins1C:51–61, LQTLALEVARE. Evidence in the literature supports this C-terminal deamidation as being spontaneous rather than enzyme-mediated. Deamidation improved I-Ag7 binding compared to the wild type peptide and increased its immunogenicity, demonstrating the biological importance of this modification. The homologous peptide from insulin-2 was not identified, indicating a possible sequence bias for spontaneous deamidation. We identified only one fused peptide bound to I-Ag7, an insulin C peptide-islet amyloid polypeptide hybrid insulin peptide (HIP) that matches the previously reported HIP6.9. We also identified several free fused peptides in crinosomes and posit this as the probable site of their formation.
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Pullen, J. K., M. D. Tallquist, R. W. Melvold i L. R. Pease. "Recognition of a single amino acid change on the surface of a major transplantation antigen is in the context of self peptide." Journal of Immunology 152, nr 7 (1.04.1994): 3445–52. http://dx.doi.org/10.4049/jimmunol.152.7.3445.
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Abstract The transcripts encoding two strongly alloantigenic class I mutant molecules, Kdm4 and Kdm5, were characterized and found to encode products that differ from the parental Kd glycoprotein by single amino acid substitutions. The Kdm4 molecule has an amino acid change at position 114, an integral component of a beta-sheet associated with pockets D and E of the peptide binding site. The basis for strong alloantigenicity of the variant molecule can be attributed to differences in peptide binding that were visualized by HPLC analysis of eluted peptides. In contrast, the Kdm5 molecule differs from the parent at position 158, a component of the alpha-helix that is not associated with any of the pockets of the peptide binding site. No differences in peptide binding by Kdm5 in comparison with the parent Kd molecule were seen by HPLC, suggesting that the variant and parent molecules bind the same set of peptides. The ability of (dm4 x dm5) F1 hybrid mice to recognize and lyse BALB/c stimulator cells indicates that the alloantigenic properties determined by the 158 substitution result from the interactions of the alpha-helix regions (changed in dm5) with the pockets of the binding site (changed in dm4). We conclude that self peptides shared by the F1 hybrid and the BALB/c stimulator cells are recognized in the context of structural features of the helices of the Ag-presenting molecule as alloantigenic determinants.
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Rosloniec, E. F., D. Gay i J. H. Freed. "Epitopic analysis by anti-I-Ak monoclonal antibodies of I-Ak-restricted presentation of lysozyme peptides." Journal of Immunology 142, nr 12 (15.06.1989): 4176–83. http://dx.doi.org/10.4049/jimmunol.142.12.4176.
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Abstract Anti-I-A mAb were used as probes of functional epitopes for both the presentation of hen egg lysozyme (HEL) peptides to I-Ak-restricted T cell hybridomas and the direct binding of the HEL (46-61) peptide. When mAb directed to polymorphic regions of I-Ak were used as inhibitors of Ag presentation, several different patterns of inhibition were observed among T cells specific for the same HEL peptide as well as among T cells specific for different fragments of HEL. Although there appears to be a conserved usage of some TCR V beta gene segments among the T cell hybrids specific for the same HEL peptide, no correlation is evident between a single V gene usage and susceptibility to blocking of Ag presentation by a particular anti-I-Ak mAb. Several of the mAb demonstrated T cell "clonotypic blocking" of Ag presentation, whereas others blocked presentation to every T cell hybrid tested, regardless of the peptide specificity. When mAb directed to nonpolymorphic regions of the I-A molecule were tested for their ability to block Ag presentation, little or no inhibition was observed. In addition, Fab' fragments of inhibitory mAb functioned identically to their intact homologous counterparts in their ability to block Ag presentation indicating that "nonspecific" steric hindrance was not playing a major role in the inhibitions observed. When the polymorphic region-directed anti-I-A mAb were tested for their ability to block the direct binding of the lysozyme peptide HEL(46-61) to I-Ak, those mAb that block HEL presentation to all T cell hybrids were found to block the binding of this peptide. However, anti-I-A mAb that demonstrate selective inhibition of T cell hybrid stimulation during Ag presentation, i.e., those directed to polymorphic serologic specificities Ia.15 and Ia.19, do not block the binding of HEL(46-61) to I-Ak. These data indicate that functionally independent epitopes exist on the I-Ak molecule for the binding of antigenic peptides and for interaction with the TCR.
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Maiuolo, Jessica, Paola Costanzo, Mariorosario Masullo, Antonio D’Errico, Rosarita Nasso, Sonia Bonacci, Vincenzo Mollace, Manuela Oliverio i Rosaria Arcone. "Hydroxytyrosol–Donepezil Hybrids Play a Protective Role in an In Vitro Induced Alzheimer’s Disease Model and in Neuronal Differentiated Human SH-SY5Y Neuroblastoma Cells". International Journal of Molecular Sciences 24, nr 17 (30.08.2023): 13461. http://dx.doi.org/10.3390/ijms241713461.
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Alzheimer’s disease (AD) is the most common neurodegenerative pathology among progressive dementias, and it is characterized by the accumulation in the brain of extracellular aggregates of beta-amyloid proteins and neurofibrillary intracellular tangles consisting of τ-hyperphosphorylated proteins. Under normal conditions, beta-amyloid peptides exert important trophic and antioxidant roles, while their massive presence leads to a cascade of events culminating in the onset of AD. The fibrils of beta-amyloid proteins are formed by the process of fibrillogenesis that, starting from individual monomers of beta-amyloid, can generate polymers of this protein, constituting the hypothesis of the “amyloid cascade”. To date, due to the lack of pharmacological treatment for AD without toxic side effects, chemical research is directed towards the realization of hybrid compounds that can act as an adjuvant in the treatment of this neurodegenerative pathology. The hybrid compounds used in this work include moieties of a hydroxytyrosol, a nitrohydroxytyrosol, a tyrosol, and a homovanillyl alcohol bound to the N-benzylpiperidine moiety of donepezil, the main drug used in AD. Previous experiments have shown different properties of these hybrids, including low toxicity and antioxidant and chelating activities. The purpose of this work was to test the effects of hybrid compounds mixed with Aβ 1–40 to induce fibrillogenesis and mimic AD pathogenesis. This condition has been studied both in test tubes and by an in vitro model of neuronal differentiated human SH-SY5Y neuroblastoma cells. The results obtained from test tube experiments showed that some hybrids inhibit the activity of the enzymes AChE, BuChE, and BACE-1. Cell experiments suggested that hybrids could inhibit fibrillogenesis, negatively modulating caspase-3. They were also shown to exert antioxidant effects, and the acetylated hybrids were found to be more functional and efficient than nonacetylated forms.
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Natsuume-Sakai, S., K. Sudoh, T. Kaidoh, J. Hayakawa i M. Takahashi. "Structural polymorphism of murine complement factor H controlled by a locus located between the Hc and the beta 2M locus on the second chromosome of the mouse." Journal of Immunology 134, nr 4 (1.04.1985): 2600–2606. http://dx.doi.org/10.4049/jimmunol.134.4.2600.
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Abstract Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.
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Suh, W. K., E. K. Mitchell, Y. Yang, P. A. Peterson, G. L. Waneck i D. B. Williams. "MHC class I molecules form ternary complexes with calnexin and TAP and undergo peptide-regulated interaction with TAP via their extracellular domains." Journal of Experimental Medicine 184, nr 2 (1.08.1996): 337–48. http://dx.doi.org/10.1084/jem.184.2.337.
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Newly assembled heavy chain-beta 2m heterodimers of class I histocompatibility molecules associate with the endoplasmic reticulum (ER) peptide transporter, TAP, and subsequently dissociate from TAP in parallel with their transport from the ER to the Golgi apparatus. It appears that TAP-associated class I molecules are waiting to bind appropriate peptides before they dissociate from TAP and leave the ER since binding of high affinity peptides to class I molecules in vitro leads to dissociation of TAP-class I complexes. In further support of this notion, we report that limiting peptide supply through inhibition of proteasome activities prolongs the association of mouse class I molecules with TAP and concomitantly slows their transport to the Golgi apparatus. By using a series of deletion mutants and hybrid class I molecules we demonstrate that the extracellular domains of class I molecules are sufficient for their peptide-regulated interaction with TAP. Furthermore, based on the inability of an alpha 3 domain-specific mAb to recognize TAP-class I complexes and the fact that a point mutant of the Dd molecule at residue 222 is unable to bind to TAP, it is likely that a major site of interaction with TAP resides in the membrane-proximal region of the heavy chain alpha 3 domain. Finally, we examined the relationship between the interaction of mouse heavy chain-beta 2m heterodimers with TAP and with the resident ER chaperone, calnexin. Most heterodimers that bound to TAP were found to associate simultaneously with calnexin. Upon delivery of peptide to class I molecules in permeabilized cells, dissociation from TAP was observed but the interaction with calnexin was largely maintained. Therefore, both TAP and calnexin may participate in the ER retention of peptide-deficient class I molecules. However, since release from calnexin occurs after dissociation from TAP, it appears that calnexin ultimately determines if a class I molecule is to be exported from the ER.
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Lee, J. M., D. J. McKean i T. H. Watts. "Functional mapping of MHC class II polymorphic residues. The alpha-chain controls the specificity for binding an Ad-versus an A k-restricted peptide and the beta-chain region 65-67 controls T cell recognition but not peptide binding." Journal of Immunology 146, nr 9 (1.05.1991): 2952–59. http://dx.doi.org/10.4049/jimmunol.146.9.2952.
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Abstract MHC proteins are polymorphic cell surface glycoproteins involved in the binding of peptide Ag and their presentation to T lymphocytes. The polymorphic amino acids of MHC proteins are primarily located in the N-terminal domains and are thought to influence T cell recognition both by influencing the binding of peptide Ag and by direct contact with the T cell receptor. In order to determine the relative importance of individual polymorphic amino acids in Ag presentation, a number of groups have taken the approach of interchanging polymorphic amino acids between different alleles of MHC protein in an attempt to define which of the polymorphisms influence peptide binding and which influence T cell recognition by direct contact with the TCR. The peptide OVA323-339 has been previously shown to bind to the MHC class II protein Ad and to have a much lower affinity for Ak, whereas the peptide hen egg lysozyme 46-61 binds well to Ak and poorly to Ad. In the present report, we have analyzed the ability of purified wild-type MHC class II proteins as well as the ability of three different hybrid molecules between Ad and Ak to bind and present these peptides. We find that the alpha-chain of the MHC class II protein plays a critical role in the binding of HEL46-61 and confers the specificity for binding OVA323-339, regardless of which beta-chain is present. We also find that the beta-chain region 65-67 does not control the specificity of peptide binding to the MHC protein, but is important in T cell responses to preformed MHC-peptide complexes, suggesting a role for this region in contacting the TCR.
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Lombardi, G., L. Barber, G. Aichinger, T. Heaton, S. Sidhu, J. R. Batchelor i R. I. Lechler. "Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence." Journal of Immunology 147, nr 6 (15.09.1991): 2034–40. http://dx.doi.org/10.4049/jimmunol.147.6.2034.
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Abstract A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.
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Mladenova, Kirilka, Georgi Stavrakov, Irena Philipova, Mariyana Atanasova, Svetla Petrova, Jordan Doumanov i Irini Doytchinova. "A Galantamine–Curcumin Hybrid Decreases the Cytotoxicity of Amyloid-Beta Peptide on SH-SY5Y Cells". International Journal of Molecular Sciences 22, nr 14 (15.07.2021): 7592. http://dx.doi.org/10.3390/ijms22147592.
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Misfolded amyloid beta (Aβ) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aβ cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aβ mainly through the ability to stimulate allosterically the α7 nAChRs and to regulate the calcium cytosolic concentration. Here, we examined the cytoprotective effects of two GAL derivatives, namely compounds 4b and 8, against Aβ cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aβ. GAL and curcumin (CU) were used in the study as reference compounds. It was found that 4b protects cells in a similar mode as GAL, while compound 8 and CU potentiate the toxic effects of Aβ. Allosteric stimulation of α7 nAChRs is suggested as a possible mechanism of the cytoprotectivity of 4b. These and previous findings characterize 4b as a prospective non-toxic multi-target agent against neurodegenerative disorders with inhibitory activity on acetylcholinesterase, antioxidant, and cytoprotective properties.
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DiLisio, James E., Braxton L. Jamison, Tobias Neef, Stephen D. Miller, Rocky Baker i Kathryn Haskins. "Induction of tolerance to a CD4 T cell hybrid insulin peptide epitope prolongs islet graft survival and suppresses autoreactive CD8 T cells". Journal of Immunology 208, nr 1_Supplement (1.05.2022): 174.10. http://dx.doi.org/10.4049/jimmunol.208.supp.174.10.
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Abstract Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes. Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)
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Yıldırım, Özge Çağlar, Mehmet Enes Arslan, Sena Öner, Ivana Cacciatore, Antonio Di Stefano, Adil Mardinoglu i Hasan Turkez. "Boron Nitride Nanoparticles Loaded with a Boron-Based Hybrid as a Promising Drug Carrier System for Alzheimer’s Disease Treatment". International Journal of Molecular Sciences 23, nr 15 (26.07.2022): 8249. http://dx.doi.org/10.3390/ijms23158249.
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The search for an innovative and effective drug delivery system that can carry and release targeted drugs with enhanced activity to treat Alzheimer’s disease has received much attention in the last decade. In this study, we first designed a boron-based drug delivery system for effective treatment of AD by integrating the folic acid (FA) functional group into hexagonal boron nitride (hBN) nanoparticles (NPs) through an esterification reaction. The hBN-FA drug carrier system was assembled with a new drug candidate and a novel boron-based hybrid containing an antioxidant as BLA, to constitute a self-assembled AD nano transport system. We performed molecular characterization analyses by using UV-vis spectroscopy, Fourier transform infrared spectrophotometer (FTIR), scanning electron microscope (SEM), Energy-dispersive X-ray spectroscopy (EDS) and Zeta potential investigations. Second, we tested the anti-Alzheimer properties of the carrier system on a differentiated neuroblastoma (SHSY5-Y) cell line, which was exposed to beta-amyloid (1–42) peptides to stimulate an experimental in vitro AD model. Next, we performed cytotoxicity analyses of synthesized molecules on the human dermal fibroblast cell line (HDFa) and the experimental AD model. Cytotoxicity analyses showed that even higher concentrations of the carrier system did not enhance the toxicological outcome in HDFa cells. Drug loading analyses reported that uncoated hBN nano conjugate could not load the BLA, whereas the memantine loading capacity of hBN was 84.3%. On the other hand, memantine and the BLA loading capacity of the hBN-FA construct was found to be 95% and 97.5%, respectively. Finally, we investigated the neuroprotective properties of the nano carrier systems in the experimental AD model. According to the results, 25 µg/mL concentrations of hBN-FA+memantine (94% cell viability) and hBN-FA+BLA (99% cell viability) showed ameliorative properties against beta-amyloid (1–42) peptide toxicity (50% cell viability). These results were generated through the use of flow cytometry, acetylcholinesterase (AChE) and antioxidant assays. In conclusion, the developed drug carrier system for AD treatment showed promising potential for further investigations and enlightened neuroprotective capabilities of boron molecules to treat AD and other neurodegenerative diseases. On the other hand, enzyme activity, systematic toxicity analyses, and animal studies should be performed to understand neuroprotective properties of the designed carrier system comprehensively.
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Hanafy, Nemany A. N., Isabel Fabregat, Stefano Leporatti i Maged El Kemary. "Encapsulating TGF-β1 Inhibitory Peptides P17 and P144 as a Promising Strategy to Facilitate Their Dissolution and to Improve Their Functionalization". Pharmaceutics 12, nr 5 (2.05.2020): 421. http://dx.doi.org/10.3390/pharmaceutics12050421.
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Transforming growth factor-beta (TGFβ1) is considered as a master regulator for many intracellular signaling pathways, including proliferation, differentiation and death, both in health and disease. It further represents an oncogenic factor in advanced tumors allowing cancer cells to be more invasive and prone to move into the metastatic process. This finding has received great attention for discovering new therapeutic molecules against the TGFβ1 pathway. Among many TGFβ1 inhibitors, peptides (P17 and P144) were designed to block the TGFβ1 pathway. However, their therapeutic applications have limited use, due to lack of selection for their targets and their possible recognition by the immune system and further due to their potential cytotoxicity on healthy cells. Besides that, P144 is a highly hydrophobic molecule with less dissolution even in organic solution. Here, we aimed to overcome the dissolution of P144, as well as design nano-delivery strategies to protect normal cells, to increase cellular penetration and to raise the targeted therapy of both P17 and P144. Peptides were encapsulated in moieties of polymer hybrid protein. Their assembly was investigated by TEM, microplate spectrum analysis and fluorescence microscopy. SMAD phosphorylation was analyzed by Western blot as a hallmark of their biological efficiency. The results showed that the encapsulation of P17 and P144 might improve their potential therapeutic applications.
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Alam, Tanweer, David A. Kenny, Torres Sweeney, Frank Buckley, Robert Prendiville, Mark McGee i Sinead M. Waters. "Expression of genes involved in energy homeostasis in the duodenum and liver of Holstein-Friesian and Jersey cows and their F1 hybrid". Physiological Genomics 44, nr 2 (styczeń 2012): 198–209. http://dx.doi.org/10.1152/physiolgenomics.00102.2011.
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Differences in feed intake and production efficiency in lactating Holstein-Friesian (HF), Jersey (JE), and JE × HF (F1) dairy cows have been reported. The liver-gut axis is important in the regulation of energy homeostasis, appetite behaviour, and production efficiency. The objectives of this study were to determine: 1) the effect of dairy cow genotype on the expression profiles of genes involved in energy homeostasis in duodenal and hepatic tissue, and 2) the association between the expression of these genes across both tissues and with economically important production efficiency traits. The expression of 27 candidate genes involved in energy homeostasis, feed intake, and energy storage was measured by qPCR. Duodenal expression of the pro-opiomelanocortin ( POMC), glucagon-like peptide 1 receptor ( GLP1R), and insulin-like growth factor 1 ( IGF1) genes was highest in HF. In contrast, hepatic expression of the leptin receptor ( LEPR), insulin-like growth factor 1 receptor ( IGF1R), protein kinase, AMP-activated, beta 1 ( AMPKB1), and POMC genes was highest in the F1 cross. In the duodenum, positive correlations were observed between mRNA expression of anorectic peptides ( POMC and GLP1R), whereas a negative correlation was detected between orexigenic (ghrelin) and anorectic (peptide YY) gene expression. A negative correlation was observed between duodenal POMC gene expression and both residual feed intake and milk production efficiency traits, while GLP1R gene expression was negatively correlated with milk production efficiency traits. A heterotic effect was observed in hepatic expression of AMKPB1, IGF1R, LEPR, POMC in the F1 genotype, possibly mediating improved feed efficiency in cross-bred cows. In conclusion, key genes involved in energy homeostasis and appetite behaviour are differentially expressed due to cow genotype in a tissue-dependent fashion. POMC and GLP1R are potential candidate genes for the identification of single nucleotide polymorphisms regulating energetic efficiency in the dairy cow, which may be incorporated into future breeding programmes.
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Braunstein, N. S., R. N. Germain, K. Loney i N. Berkowitz. "Structurally interdependent and independent regions of allelic polymorphism in class II MHC molecules. Implications for Ia function and evolution." Journal of Immunology 145, nr 6 (15.09.1990): 1635–45. http://dx.doi.org/10.4049/jimmunol.145.6.1635.
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Abstract Possible interactions between regions of allelic polymorphism in the alpha- and beta-chains of class II MHC molecules were examined by measuring the efficiency of surface expression and the reactivity with mAb of wild-type and recombinant A alpha A beta-chain pairs from the b, d, and k haplotypes. These studies revealed regions of polymorphism within the alpha- and beta-chains that interact with complementary regions in the other chain. Unexpectedly, almost all the variable segments of both the class II MHC alpha- and beta-chains either directly contributed to or were near sites of interchain interactions. The exception was the beta HV3 (hypervariable (HV] segment (residues 61-71), which appeared to neither participate in nor be affected by interchain interactions. This division of the MHC molecule into interacting vs independent regions of allelic structural variation suggests that mutagenesis experiments involving the beta HV3 segment can be analyzed in a straightforward manner, as such mutations appear unlikely to alter the conformation of other molecular segments. Furthermore, functions attributed to the beta HV3 segment either experimentally or by population analysis should have a high probability of transfer by beta HV3 exchange (either experimentally or evolutionarily), because epitopes assigned to this region of the molecule are not affected by sequences outside this segment. This is of special importance because of the apparent involvement of this region in defining a potential site of interaction with antigenic peptides and TCR. In contrast, almost all other variable segments of the MHC molecule appear to have the capacity to contribute to interactions involving at least one other variable segment. This suggests not only that the experimental analysis of the contributions of these regions to various functions requires a consideration of inter- and intrachain interaction, but also that the transfer of function by genetic exchange of these structurally dependent regions is unpredictable. Selection must therefore operate on these interacting HV segments in the context of the complete alpha beta heterodimer. These results support our earlier arguments for cis-co-evolution of alpha- and beta-chain polymorphism and the absence of selection for F1 (hybrid) class II molecules. Finally, asymmetries observed in the contributions of particular pairs of HV segments to the efficient expression of Ia alpha beta heterodimers provide a basis for understanding mechanistically how cis-co-evolution may have occurred.
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Nguyen, Hai, Perrin Guyer, Ruth A. Ettinger i Eddie A. James. "Non-Genetically Encoded Epitopes Are Relevant Targets in Autoimmune Diabetes". Biomedicines 9, nr 2 (17.02.2021): 202. http://dx.doi.org/10.3390/biomedicines9020202.
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Islet antigen reactive T cells play a key role in promoting beta cell destruction in type 1 diabetes (T1D). Self-reactive T cells are typically deleted through negative selection in the thymus or deviated to a regulatory phenotype. Nevertheless, those processes are imperfect such that even healthy individuals have a reservoir of potentially autoreactive T cells. What remains less clear is how tolerance is lost to insulin and other beta cell specific antigens. Islet autoantibodies, the best predictor of disease risk, are known to recognize classical antigens such as proinsulin, GAD65, IA-2, and ZnT8. These antibodies are thought to be supported by the expansion of autoreactive CD4+ T cells that recognize these same antigenic targets. However, recent studies have identified new classes of non-genetically encoded epitopes that may reflect crucial gaps in central and peripheral tolerance. Notably, some of these specificities, including epitopes from enzymatically post-translationally modified antigens and hybrid insulin peptides, are present at relatively high frequencies in the peripheral blood of patients with T1D. We conclude that CD4+ T cells that recognize non-genetically encoded epitopes are likely to make an important contribution to the progression of islet autoimmunity in T1D. We further propose that these classes of neo-epitopes should be considered as possible targets for strategies to induce antigen specific tolerance.
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Kuchroo, V. K., M. C. Byrne, E. Greenfield, M. J. Whitters, E. A. Nalefsky, A. Rao, M. Collins i M. E. Dorf. "Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity." Journal of Immunology 154, nr 10 (15.05.1995): 5030–38. http://dx.doi.org/10.4049/jimmunol.154.10.5030.
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Abstract Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.
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Mazzio, E., R. Badisa, S. Eyunni, S. Ablordeppey, B. George i K. F. A. Soliman. "Bioactivity-Guided Isolation of Neuritogenic Factor from the Seeds of the Gac Plant (Momordica cochinchinensis)". Evidence-Based Complementary and Alternative Medicine 2018 (31.05.2018): 1–11. http://dx.doi.org/10.1155/2018/8953958.
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Nerve growth factor (NGF) is an endogenously produced protein with the capacity to induce central nervous system (CNS) neuronal differentiation and repair. NGF signaling involves its binding to tropomyosin-related kinase (Trk) receptors, internalization, and initiation of phosphorylation cascades which cause microtubule reorganization and neuronal outgrowth. Because NGF cannot cross the blood-brain barrier, its therapeutic use is limited. Synthetic peptides that can act as NGF receptor agonists (NGF mimetics) are known to attenuate neurodegenerative pathologies in experimental models of Alzheimer’s disease and Parkinson’s disease; however, the existence of plant-based NGF mimetics is uncertain. For this reason, we recently completed a high throughput screening of over 1100 nutraceuticals (vitamins, herbal plant parts, polyphenolics, teas, fruits, and vegetables) to identify neuritogenic factor using a PC-12 cell model. Remarkably we found only one, commonly known as the seed of Gac plant (Momordica cochinchinensis) (MCS). In the current study, we further investigated this seed for its neuritogenic effect using bioactivity-guided chemical separations. The data show no biological neuritogenic activity in any chemical solvent fraction, where activity was exclusive to the crude protein. MSC crude proteins were then separated by 1D electrophoresis, where the active neuritogenic activity was confirmed to have a molecular mass of approximately 17 kDa. Subsequently, the 17kDa band was excised, digested, and run on a UPLC-MS/MS with a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with data evaluated diverse tools such as X! Tandem, OMS, and K-score algorithms. Proteomic evaluation of the 17kDa band confirmed evidence for 11S globulin subunit beta, napin, oleosin, Momordica trypsin inhibitors (TI) MCoTI-I /II, and many isoforms of Two Inhibitor Peptide Topologies (TIPTOPs). While all peptides identified correspond to the genus/species, Momordica cochinchinensis and Cucumis Sativus, a significant limitation of the analysis is the nonexistence of full annotation for the Momordica cochinchinensis proteome. In conclusion, these findings demonstrate that there is a stable protein within MCS having a mass of 17kDa with the capacity to induce neurite outgrowth. Future work will be required to establish the therapeutic value of the MCS for the treatment of neurodegenerative diseases.
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Soos, M. A., C. E. Field i K. Siddle. "Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity". Biochemical Journal 290, nr 2 (1.03.1993): 419–26. http://dx.doi.org/10.1042/bj2900419.
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Hybrid insulin/insulin-like growth factor-I (IGF-I) receptors have previously been described in human placenta, but it has not been possible to study their properties in the presence of classical insulin receptors and type I IGF receptors. To facilitate the purification of hybrids, we produced an anti-peptide monoclonal antibody IGFR 1-2, directed against the C-terminal peptide of the type I IGF receptor beta-subunit. The antibody bound native human and rat type I IGF receptors, and reacted specifically with the beta-subunit on immunoblots. Solubilized placental microsomal membranes were depleted of classical type I IGF receptors by incubation with an immobilized monoclonal antibody IGFR 24-55, which reacts well with type I receptors but very poorly with hybrid receptors. Residual hybrid receptors were then isolated by incubation with immobilized antibody IGFR 1-2, and recovered by elution with excess of synthetic peptide antigen. Binding properties of hybrids were compared with those of immuno-affinity-purified insulin receptors and type I IGF receptors, by using the radioligands 125I-IGF-I and 125I-insulin. Hybrids bound approx. 20 times as much 125I-IGF-I as 125I-insulin at tracer concentrations (approx. 0.1 nM). The binding of 125I-insulin, but not 125I-IGF-I, to hybrids increased after treatment with dithiothreitol to reduce disulphide bonds between the alpha-subunits. Hybrids behaved very similarly to type I receptors with respect to the inhibition of 125I-IGF-I binding by unlabelled IGF-I and insulin. By contrast, the affinity of hybrids for insulin was approx. 10-fold lower than that of classical insulin receptors, as assessed by inhibition of 125I-insulin binding by unlabelled hormone. It is concluded that the properties of insulin receptors, but not IGF receptors, are markedly affected by assembly as hybrid compared with classical structures, and that hybrids are more likely to be responsive to IGF-I than insulin under physiological conditions.
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Nanda, N. K., K. K. Arzoo i E. E. Sercarz. "In a small multideterminant peptide, each determinant is recognized by a different V beta gene segment." Journal of Experimental Medicine 176, nr 1 (1.07.1992): 297–302. http://dx.doi.org/10.1084/jem.176.1.297.
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Given the vast potential for diversification of the T cell receptor (TCR) repertoire and the fact that V(a) beta mice exist in the wild, it would have been predicted that in spite of the absence of 10 TCR V beta gene segments, V(a) beta mice would still have been able to produce an antigen-specific T cell response to all determinants. We have recently shown that Vb beta mice, with a wild-type TCR V beta repertoire, respond to peptide 110-121 of sperm whale myoglobin, with a majority of T cells expressing TCR V beta 8.2 and restricted to a hybrid I-A(d)/I-E(d) major histocompatibility complex molecule, and a smaller number of T cells expressing TCR V beta 8.1 and restricted to the I-A(d) molecule. However, V(a) beta mice, lacking members of the TCR V beta 8 gene family, responded only with I-A(d)-restricted T cells. Thus, it appeared that the I-A(d)-restricted response was less constrained, or more plastic. We now show that the two separate panels of I-A(d)-restricted T cell hybrids derived from V(a) beta or Vb beta mice in fact recognize distinct determinants within the same peptide 110-121. The determinant recognized by V(a) beta T cells is NH2 terminal (core: 110-118) with an absolute requirement for the residue Ala-110 for a successful interaction with TCRs. On the other hand, Vb beta T cells recognize the COOH-terminal region (core: 112-118) on the same peptide with an absolute requirement for COOH-terminal residue 118. In the dominance hierarchy displayed by the three distinct determinants of peptide 110-121, V(a) beta mice cannot recognize the two most dominant: the hybrid I-A(d)/I-E(d)-restricted determinant and the COOH-terminal, I-A(d)-restricted determinant. They instead respond with T cells specific for a third, distinctly NH2-terminal determinant. Our results show a strict association between recognition of a particular specificity and TCR V beta usage. This evidence suggests that even when a small peptide induces a heterogenous group of TCR V beta S, this need not be considered evidence for plasticity. Rather, at the level of individual determinants within the peptide, the results can point in the opposite direction, towards serious constraints in recognition at the level of V beta expression.
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Arcasoy, M. O., M. Romana, M. E. Fabry, E. Skarpidi, R. L. Nagel i B. G. Forget. "High levels of human gamma-globin gene expression in adult mice carrying a transgene of deletion-type hereditary persistence of fetal hemoglobin." Molecular and Cellular Biology 17, nr 4 (kwiecień 1997): 2076–89. http://dx.doi.org/10.1128/mcb.17.4.2076.
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Persistent expression of the gamma-globin genes in adults with deletion types of hereditary persistence of fetal hemoglobin (HPFH) is thought to be mediated by enhancer-like effects of DNA sequences at the 3' breakpoints of the deletions. A transgenic mouse model of deletion-type HPFH was generated by using a DNA fragment containing both human gamma-globin genes and HPFH-2 breakpoint DNA sequences linked to the core sequences of the locus control region (LCR) of the human beta-globin gene cluster. Analysis of gamma-globin expression in six HPFH transgenic lines demonstrated persistence of gamma-globin mRNA and peptides in erythrocytes of adult HPFH transgenic mice. Analysis of the hemoglobin phenotype of adult HPFH transgenic animals by isoelectric focusing showed the presence of hybrid mouse alpha2-human gamma2 tetramers as well as human gamma4 homotetramers (hemoglobin Bart's). In contrast, correct developmental regulation of the gamma-globin genes with essentially absent gamma-globin gene expression in adult erythroid cells was observed in two control non-HPFH transgenic lines, consistent with autonomous silencing of normal human gamma-globin expression in adult transgenic mice. Interestingly, marked preferential overexpression of the LCR-distal (A)gamma-globin gene but not of the LCR-proximal (G)gamma-globin gene was observed at all developmental stages in erythroid cells of HPFH-2 transgenic mice. These findings were also associated with the formation of a DNase I-hypersensitive site in the HPFH-2 breakpoint DNA of transgenic murine erythroid cells, as occurs in normal human erythroid cells in vivo. These results indicate that breakpoint DNA sequences in deletion-type HPFH-2 can modify the developmentally regulated expression of the gamma-globin genes.
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Allen, P. M., D. J. McKean, B. N. Beck, J. Sheffield i L. H. Glimcher. "Direct evidence that a class II molecule and a simple globular protein generate multiple determinants." Journal of Experimental Medicine 162, nr 4 (1.10.1985): 1264–74. http://dx.doi.org/10.1084/jem.162.4.1264.
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We have examined the individual contributions of the I-A kappa alpha chain, the I-A kappa beta chain, and the foreign antigen hen egg-white lysozyme (HEL) in the formation of the determinant being recognized by the T cell receptor. As functional probes we have used (a) a panel of 10 HEL-specific T cell hybridomas, (b) a panel of antigen-presenting cells (APC) possessing mutations in either the I-A kappa alpha or I-A kappa beta chains, and (c) proteolytic fragment of HEL and related synthetic peptides. The ability of the I-A kappa beta and I-A kappa alpha mutant cell lines to present antigen to the 10 T cell hybridomas divided these T cells into six distinct groups. These HEL-specific T cells therefore appear to recognize several distinct domains on the I-A kappa molecule. The 10 T cell hybrids were then shown to recognize at least three distinct determinants on the HEL molecule, with 8 of the 10 hybrids recognizing one of two major determinants HEL(46-61) or HEL(34-45). Combining the response patterns to the panel of I-A kappa mutant APC lines with the antigen specificity revealed that the 10 T cell hybrids recognized at least eight unique determinants formed by the I-A kappa alpha chains, I-A kappa beta chains, and HEL peptides. This analysis provides direct evidence that a large number of different determinants or T cell receptor ligands can be generated from a single Ia molecule and a simple globular protein.
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Wither, J., J. Pawling, L. Phillips, T. Delovitch i N. Hozumi. "Amino acid residues in the T cell receptor CDR3 determine the antigenic reactivity patterns of insulin-reactive hybridomas." Journal of Immunology 146, nr 10 (15.05.1991): 3513–22. http://dx.doi.org/10.4049/jimmunol.146.10.3513.
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Abstract We examined TCR gene usage in a panel of beef insulin/I-Ad-restricted T cell hybrids obtained from BALB/c mice. These hybrids demonstrated several distinct patterns of reactivity defined by their ability to respond to species variants of insulin. Correlation of TCR-alpha and -beta-gene usage with these patterns of reactivity demonstrated that TCR gene usage was restricted within Ag reactivity groups. In particular, V-J junctional regions (CDR3 equivalent) were restricted with conserved junctional amino acid motifs present in both TCR-alpha- and -beta-chains. Comparison of TCR gene usage in hybrids expressing identical V alpha and V beta gene segments but demonstrating different patterns of reactivity revealed that changes in either J alpha and/or J beta gene segment usage could alter antigenic reactivity. Indeed, single or limited amino acid differences within the CDR3 region were sufficient to markedly alter fine specificity. These data demonstrate the critical role for CDR3 in determining antigenic reactivity in beef insulin-reactive hybrids and are compatible with the current model of TCR/peptide/MHC interaction.
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Kijimoto-Ochiai, S., Y. U. Katagiri, T. Hatae i H. Okuyama. "Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method". Biochemical Journal 257, nr 1 (1.01.1989): 43–49. http://dx.doi.org/10.1042/bj2570043.
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The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a ‘bisecting’ acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.
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Kim, G. D., I. C. Carr i G. Milligan. "Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and β2-adrenoceptor-expressing NG108-15 cells". Biochemical Journal 308, nr 1 (15.05.1995): 275–81. http://dx.doi.org/10.1042/bj3080275.
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Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.
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Lai, M. Z., Y. J. Jang, L. K. Chen i M. L. Gefter. "Restricted V-(D)-J junctional regions in the T cell response to lambda-repressor. Identification of residues critical for antigen recognition." Journal of Immunology 144, nr 12 (15.06.1990): 4851–56. http://dx.doi.org/10.4049/jimmunol.144.12.4851.
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Abstract The T cell response to lambda-repressor is directed to a 15 amino acid peptide (P12-26) of the protein in A/J mice. Previous studies have demonstrated a preferential use of V alpha 2 and V beta 1 amongst the T cell hybridomas specific for P12-26 in the context of I-Ek. By using the polymerase chain reaction, the sequences of a panel of the T cells using V alpha 2 and V beta 1 were determined. A highly conserved alpha-chain V-J junctional sequence was found in six of the eight T cell hybrids. This consensus alpha-chain VJ sequence may be combined with different members of V alpha 2, indicating a more restricted selection on the junctional region than on the V element in these T cells. In contrast, greater diversities were found on the V-D-J region of beta-chains despite the same V beta 1 and J beta 2.1 were used. However, a highly conserved glutamic acid residue was found at the same position of beta-chains where a similar conservation was identified in cytochrome c-specific T cells. The correlation of the TCR sequence with the fine specificities of these T cells suggests that a single amino acid deletion in the V alpha-J alpha region may reduce the P12-26 response and abolish the recognition of an altered peptide [Phe22] P12-26. In addition, three amino acid difference in the V-D-J region of the beta-chain also determine the P12-26 reactivity. Thus the V(D)J junctional regions of both alpha- and beta-chains may be critical for the recognition of the peptide Ag presented by the specific MHC molecule.
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Schlauder, G. G., M. P. Bell, B. N. Beck, A. Nilson i D. J. McKean. "The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms." Journal of Immunology 135, nr 3 (1.09.1985): 1945–54. http://dx.doi.org/10.4049/jimmunol.135.3.1945.
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Abstract Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.
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Fruth, U., N. Solioz i J. A. Louis. "Leishmania major interferes with antigen presentation by infected macrophages." Journal of Immunology 150, nr 5 (1.03.1993): 1857–64. http://dx.doi.org/10.4049/jimmunol.150.5.1857.
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Abstract Impairment of the Ag-presenting capacity of macrophages harboring intracellular Leishmania might represent one of the several mechanisms by which these parasites can evade host-protective T cell responses. Thus, the present study was designed to investigate the ability of macrophages, parasitized with Leishmania major, to present Ag to relevant T cell hybridomas. Results show that bone marrow-derived macrophages from BALB/c mice, after infection with L. major, have a greatly reduced capacity to present OVA, beta-galactosidase, and L. major-derived Ag to specific T cell hybrids derived from mice immunized with those Ag. In contrast, after pulsing with relevant peptide, macrophages containing L. major have a normal Ag-presenting capacity. The inhibition of presentation of native Ag did not appear to result from decreased endocytosis or catabolism. Inasmuch as the inhibition of presentation could not be attributed to an impaired processing of the Ag or an unavailability of MHC class II molecules on the surface of infected cells, these results could indicate that the presence of L. major interferes with the intracellular loading of MHC class II molecules with antigenic peptides.
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Zhou, P., G. D. Anderson, S. Savarirayan, H. Inoko i 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, nr 3 (1.02.1991): 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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Zamvil, S. S., P. A. Nelson, D. J. Mitchell, R. L. Knobler, R. B. Fritz i L. Steinman. "Encephalitogenic T cell clones specific for myelin basic protein. An unusual bias in antigen recognition." Journal of Experimental Medicine 162, nr 6 (1.12.1985): 2107–24. http://dx.doi.org/10.1084/jem.162.6.2107.
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Class II-restricted T cell clones specific for myelin basic protein (MBP) have been generated from PL/J and (PL/J X SJL/J)F1 [((PLSJ)F1] mice following sensitization to rat MBP. Of 17 T cell clones generated from (PLSJ)F1 mice, 5 are I-Au(A alpha uA beta u) restricted, one is restricted to I-As(A alpha sA beta s), 10 are restricted to hybrid I-A(u X s)F1 (A alpha sA beta u) determinants, and one clone is restricted to hybrid I-E(u X s) (E alpha uE beta s) molecules. Thus, of 16 I-A-restricted T cell clones generated from (PLSJ)F1 mice, only one is I-As-restricted, reflecting a lack of priming to MBP in association with I-As. T cell clones restricted to I-Au and to I-E (E alpha u E beta s) molecules recognize mouse (self) MBP. Furthermore, only the five T cell clones restricted to I-Au molecules recognize a determinant in common with mouse (self) MBP within the encephalitogenic N-terminal peptide. Three such I-Au restricted T cell clones, derived independently, cause paralysis in 100% of (PL/J X SJL/J)F1 mice tested. Acute, chronic unremitting, and chronic relapsing paralysis are all induced following injection of these clones. Administration of greater numbers of cloned T cells causes acute and fatal experimental allergic encephalomyelitis, while administration of lower numbers of cloned T cells is associated with chronic unremitting and relapsing paralysis. Paralysis induced with T cell clones shares many clinical, immunologic, and histologic aspects with human demyelinating diseases such as multiple sclerosis. Histopathology reveals perivascular lymphocytic infiltration, demyelination, and remyelination. These studies demonstrate the utility of T cell clones for analyzing the association between class II major histocompatibility complex molecules and disease susceptibility.
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Beckers, W., L. Villa, S. Gonfloni, L. Castagnoli, S. M. Newton, G. Cesareni i P. Ghiara. "Increasing the immunogenicity of protein antigens through the genetic insertion of VQGEESNDK sequence of human IL-1 beta into their sequence." Journal of Immunology 151, nr 4 (15.08.1993): 1757–64. http://dx.doi.org/10.4049/jimmunol.151.4.1757.
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Abstract The immunogenicity of two recombinant protein Ag containing the immunostimulatory sequence of human IL-1 beta 163-171 (VQGEESNDK) genetically engineered into their structure has been evaluated. The IL-1 beta sequence was inserted into the loop between alpha helices D and E of recombinant human ferritin H chain and into the hypervariable region of recombinant flagellin from Salmonella muenchen. The chimeric proteins were injected into mice and the level of humoral immune response developed against the native proteins was assessed by measuring the number of Ag-specific plaque forming cells/spleen or as the level of serum IgG response. The response was compared to that of mice receiving injections with wild-type protein Ag not containing the VQGEESNDK sequence or with hybrid constructs containing unrelated foreign peptide sequences of the same length. A significantly higher immune response was observed in mice immunized with chimeric constructs containing the human IL-1 beta 163-171 sequence. These data suggest that the insertion of the VQGEESNDK sequence may prove useful to increase the immune response against poorly immunogenic recombinant proteins.
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Hughes, S. R., S. Goyal, J. E. Sun, P. Gonzalez-DeWhitt, M. A. Fortes, N. G. Riedel i S. R. Sahasrabudhe. "Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers." Proceedings of the National Academy of Sciences 93, nr 5 (5.03.1996): 2065–70. http://dx.doi.org/10.1073/pnas.93.5.2065.
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Lunn, C. A., i M. Inouye. "Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli." Journal of Biological Chemistry 262, nr 17 (czerwiec 1987): 8318–24. http://dx.doi.org/10.1016/s0021-9258(18)47566-7.
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NIELSEN, Daniel, Hanna GYLLBERG, Pernilla ÖSTLUND, Tomas BERGMAN i Katarina BEDECS. "Increased levels of insulin and insulin-like growth factor-1 hybrid receptors and decreased glycosylation of the insulin receptor alpha- and beta-subunits in scrapie-infected neuroblastoma N2a cells". Biochemical Journal 380, nr 2 (1.06.2004): 571–79. http://dx.doi.org/10.1042/bj20040010.
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We have previously shown that ScN2a cells (scrapie-infected neuroblastoma N2a cells) express 2-fold- and 4-fold-increased levels of IR (insulin receptor) and IGF-1R (insulin-like growth factor-1 receptor) respectively. In addition, the IR α- and β-subunits are aberrantly processed, with apparent molecular masses of 128 and 85 kDa respectively, as compared with 136 and 95 kDa in uninfected N2a cells. Despite the 2-fold increase in IR protein, the number of 125I-insulin-binding sites was slightly decreased in ScN2a cells [Östlund, Lindegren, Pettersson and Bedecs (2001) Brain Res. 97, 161–170]. In order to determine the cellular localization of IR in ScN2a cells, surface biotinylation was performed, showing a correct IR trafficking and localization to the cell surface. The present study shows for the first time that neuroblastoma N2a cells express significant levels of IR–IGF-1R hybrid receptors, and in ScN2a cells the number of hybrid receptors was 2-fold higher than that found in N2a cells, potentially explaining the apparent loss of insulin-binding sites due to a lower affinity for insulin compared with the homotypic IR. Furthermore, the decreased molecular mass of IR subunits in ScN2a cells is not caused by altered phosphorylation or proteolytic processing, but rather by altered glycosylation. Enzymic deglycosylation of immunoprecipitated IR from N2a and ScN2a cells with endoglycosidase H, peptide N-glycosidase F and neuraminidase all resulted in subunits with increased electrophoretic mobility; however, the 8–10 kDa shift remained. Combined enzymic or chemical deglycosylation using anhydrous trifluoromethane sulphonic acid treatment ultimately showed that the IR α- and β-subunits from ScN2a cells are aberrantly glycosylated. The increased formation of IR–IGF-1R hybrids in ScN2a cells may be part of a neuroprotective response to prion infection. The degree and functional significance of aberrantly glycosylated proteins in ScN2a cells remain to be determined.
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Faulstich, D., S. Auerbach, L. Orci, M. Ravazzola, S. Wegchingel, F. Lottspeich, G. Stenbeck, C. Harter, F. T. Wieland i H. Tschochner. "Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex." Journal of Cell Biology 135, nr 1 (1.10.1996): 53–61. http://dx.doi.org/10.1083/jcb.135.1.53.
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Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.
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Donovan, K. A., S. K. Singh, C. S. David i L. R. Pease. "Molecular analysis and repair of a defective Ef alpha gene." Journal of Immunology 142, nr 11 (1.06.1989): 4034–40. http://dx.doi.org/10.4049/jimmunol.142.11.4034.
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Abstract The molecular basis for the defective expression of Ef alpha was determined by analysis of the 5' region of a full length Ef alpha gene. The gene was isolated from a genomic library prepared from the A.CA/SnDv mouse strain. DNA sequence analysis of the 5' portion of the Ef alpha gene, which encodes the 5' regulatory sequences and the signal peptide, revealed the presence of a stop codon in the exon encoding the signal peptide. The remainder of the sequences were highly related to sequences found in previously characterized, functional E alpha alleles. Previous studies indicate that the f allele is transcribed at rates comparable to the rates of functional alleles and that mRNA accumulates in the cytoplasm. Primer extension analysis demonstrated that Ef alpha transcripts initiate identically to the functional Ek alpha allele, mapping the defect in the Ef alpha gene 3' of the transcriptional initiation site. To determine whether the stop codon in the signal peptide was the only major defect in this gene, reciprocal chimeric genes were constructed in which the 5' regions, including the first exons, of the defective f allele and the functional k allele were exchanged. The hybrid genes were inserted into an SV40 promotor driven expression vector for cotransfection with an Ek beta gene. Surface I-E expression was demonstrated using I-E specific mAb in Cos-7 and L cell lines transfected with the hybrid gene consisting of the 5' region of the k allele and the 3' portion of the f allele. Therefore, the single stop codon present in the exon encoding the leader peptide of the Ef alpha gene appears to be the only defect preventing this gene from expressing a functional E alpha-chain.
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Ruberti, G., V. Paragas, D. Kim i C. G. Fathman. "Selection for amino acid sequence and J beta element usage in the beta chain of DBA/2V beta b- and DBA/2V beta a-derived myoglobin-specific T cell clones." Journal of Immunology 151, nr 11 (1.12.1993): 6185–94. http://dx.doi.org/10.4049/jimmunol.151.11.6185.
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Abstract Previous experiments in our laboratory have demonstrated that there is a marked restriction on the TCR beta-chain usage in DBA/2 mice in response to the sperm whale myoglobin (SpWMb) determinant 110-121, predominated by the use of the V beta 8.2 gene element. We analyzed the response of mice that had been genetically depleted of V beta 8+ T cells by generating a DBA/2 line that carries the V beta a TCR haplotype. Despite the very limited TCR repertoire expressed by DBA/2V beta a mice, they made an excellent response after immunization with the SpWMb 110-121 peptide. Data presented in this manuscript demonstrate that there is an equally restricted TCR V beta-chain utilization in the T-cell response to the determinant SpWMb 110-121 in DBA/2V beta a mice. Unexpectedly, there was a shift of MHC restriction of this determinant to T cells in the V beta a strain when compared with the V beta b strain of DBA/2 mice. We had previously demonstrated that DBA/2 mice utilized both the hybrid E alpha dA beta d MHC molecule as well as the conventional A alpha dA beta d molecule as presenting elements in response to SpWMb 110-121. Data presented in this manuscript demonstrate that the T-cell response in DBA/2V beta a mice is entirely restricted by the A alpha dA beta d MHC class II molecule. By analyzing a panel of SpWMb 110-121-specific T-cell clones from DBA/2V beta a mice, we were able to study the TCR repertoire expressed on T cells from mice that lack the V beta 8.2 gene. The V beta usage by the panel of clones analyzed was remarkably homogeneous. Thirteen of the 17 clones analyzed used the V beta 1 gene segment. Perhaps more striking was the junctional region nucleotide and amino acid sequences that were shared among these clones and that were similar to the V beta 8.2 clones analyzed previously. All clones assayed used the J beta 2.6 element, as did the great majority of the V beta 8.2 clones analyzed from DBA/2 (and B10.D2) V beta b mice. Importantly, in each strain of mice, irrespective of the V beta utilized, each TCR appeared to have selected an acidic amino acid in the beta-chain at position 100.(ABSTRACT TRUNCATED AT 400 WORDS)
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Tsiagbe, V. K., J. Asakawa, A. Miranda, R. M. Sutherland, Y. Paterson i G. J. Thorbecke. "Syngeneic response to SJL follicular center B cell lymphoma (reticular cell sarcoma) cells is primarily in V beta 16+ CD4+ T cells." Journal of Immunology 150, nr 12 (15.06.1993): 5519–28. http://dx.doi.org/10.4049/jimmunol.150.12.5519.
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Abstract The growth of SJL B cell lymphomas (RCS, reticulum cell sarcoma) in vivo and in vitro is known to depend on cytokine production by RCS-responsive host CD4+ T cells. The high frequency of RCS responsive cells in normal SJL lymph nodes prompted us to prepare a set of 21 RCS-specific T cell hybridomas. Like normal SJL T cells, these hybridoma cells respond to RCS, but not to normal syngeneic B cells; produce IL-2, IL-4, and IL-5; and promote growth of RCS in gamma-irradiated syngeneic hosts. A superantigen-like stimulation by RCS cells was borne out by the fact that all the RCS-specific hybridomas used V beta 16 in their TCR. RCS cells did not stimulate I-As-restricted, V beta 17a+ KLH-specific, or V beta 1+ heme-specific hybridomas, but were excellent Ag presents for these cells. Preincubation of RCS cells with high concentrations of the KM core peptide (high affinity for I-As) did not interfere with the ability of RCS to stimulate RCS-specific hybridomas. The relative representation of mRNA for V beta 1, 4, 10, 15, 16, and 17a was evaluated in RNA extracted from normal SJL lymph node cells responding to Con A or to RCS cells. Only V beta 16 was specifically enriched in the response to RCS. Moreover, the degree of responsiveness to RCS cells in lymph node cells from F1 hybrids of SJL and I-E transgenic SJL mice, corresponds to the relative abundance of V beta 16 in mRNA, but not of V beta 17a mRNA.
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Sun, Ting, Mengyao Li, Feng Zhao i Lin Liu. "Surface Plasmon Resonance Biosensors with Magnetic Sandwich Hybrids for Signal Amplification". Biosensors 12, nr 8 (22.07.2022): 554. http://dx.doi.org/10.3390/bios12080554.
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The conventional signal amplification strategies for surface plasmon resonance (SPR) biosensors involve the immobilization of receptors, the capture of target analytes and their recognition by signal reporters. Such strategies work at the expense of simplicity, rapidity and real-time measurement of SPR biosensors. Herein, we proposed a one-step, real-time method for the design of SPR biosensors by integrating magnetic preconcentration and separation. The target analytes were captured by the receptor-modified magnetic nanoparticles (MNPs), and then the biotinylated recognition elements were attached to the analyte-bound MNPs to form a sandwich structure. The sandwich hybrids were directly delivered to the neutravidin-modified SPR fluidic channel. The MNPs hybrids were captured by the chip through the neutravidin–biotin interaction, resulting in an enhanced SPR signal. Two SPR biosensors have been constructed for the detection of target DNA and beta-amyloid peptides with high sensitivity and selectivity. This work, integrating the advantages of one-step, real-time detection, multiple signal amplification and magnetic preconcentration, should be valuable for the detection of small molecules and ultra-low concentrations of analytes.
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Winther, M. D., G. Allen, R. H. Bomford i F. Brown. "Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals." Journal of Immunology 136, nr 5 (1.03.1986): 1835–40. http://dx.doi.org/10.4049/jimmunol.136.5.1835.
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Abstract A fusion protein consisting of beta-galactosidase (GZ) to which was attached at its N-terminus the amino acid sequence corresponding to residues 142-160 of the immunogenic protein VP1 of foot-and-mouth disease virus (FMDV) has been expressed in E. coli. A chemically synthesized section of DNA corresponding to the amino acid sequence 142-160 was inserted into a vector (pXY410) designed to express fusion proteins with the carboxy terminal 1015 amino acids of GZ. The hybrid protein immunopurified by a GZ-specific monoclonal antibody was soluble, retained full GZ activity, and induced virus-neutralizing antibody in guinea pigs and mice. There were significant differences between the responses of individual mice to the FMDV peptide sequence, although the titers against GZ were uniformly high. This variable pattern did not change after hyperimmunization and was demonstrable in a range of mouse strains of different haplotype. The same results were obtained whether the response was measured by virus neutralization or by RIA against the FMDV peptide sequence. The possible reasons for the variable recognition of the FMDV epitopes by individual mice are discussed.
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Nagler-Anderson, C., L. A. McNair i A. Cradock. "Self-reactive, T cell receptor-gamma delta+, lymphocytes from the intestinal epithelium of weanling mice." Journal of Immunology 149, nr 7 (1.10.1992): 2315–22. http://dx.doi.org/10.4049/jimmunol.149.7.2315.
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Abstract Intraepithelial T lymphocytes (IEL) are dispersed throughout the intestinal epithelial lining but their role in cellular immune defense is unknown. Their location suggests that their highly activated state may be due to constant exposure to bacterial Ag. To study IEL specificity and function we have prepared a panel of IEL-T cell hybridomas from both adult and weanling C57B1/6 mice. Many of these expressed TCR-gamma delta, a cell type rare in peripheral lymph nodes and spleen but predominant at epithelial surfaces. We have identified a subset of gamma delta T cells from weanling mice which is self reactive, i.e., these hybrids secrete IL-2 spontaneously, without antigenic stimulation or a requirement for APC. Self-reactive TCR-gamma delta+ hybrids and lines, all of which bear a particular TCR (V gamma 1.1C gamma 4V delta 6), have previously been derived from neonatal thymus and the skin. Northern blot and immunoprecipitation analyses suggest that the self-reactive IEL hybrids also bear a C gamma 4/V delta 6 TCR. Antibody inhibition experiments showed that the self-reactivity of the IEL hybrids is TCR mediated. Spontaneous IL-2 production was blocked by soluble anti-CD3 and anti-TCR-gamma delta antibodies but not by antibodies to the TCR-alpha beta. The self-reactive IEL hybrids lack class II MHC and the class I-like proteins CD1 and TLA but express class I MHC. IEL hybrids may also require the vitronectin receptor as an accessory molecule for their activation because spontaneous IL-2 production is blocked by antibody to the vitronectin receptor as well as by the extracellular matrix protein active site peptide RGDS, but not the control peptide RGES. V gamma 1.1C gamma 4V delta 6 T cells in the thymus, skin, and intestine may represent a small and unique subpopulation of lymphocytes with a potential for autoimmune reactivity at peripheral sites.
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Yu, Wen Qing, Gui Ping Zheng, De Wen Qiu, Feng Chao Yan, Wen Zhi Liu i Wan Xue Liu. "Draft genome sequence, disease-resistance genes, and phenotype of a Paenibacillus terrae strain (NK3-4) with the potential to control plant diseases". Genome 61, nr 10 (październik 2018): 725–34. http://dx.doi.org/10.1139/gen-2018-0113.
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Paenibacillus terrae NK3-4 is a plant growth-promoting rhizobacterium that may be useful for controlling plant diseases. We conducted a genomic analysis and identified the genes mediating antimicrobial functions. Additionally, an extracellular antifungal protein component was isolated and identified. The draft genome sequence was assembled into 54 contigs, with 5 458 568 bp and a G+C content of 47%. Moreover, 4 690 015 bp encoded 5090 proteins, 7 rRNAs, and 54 tRNAs. Forty-four genes involved in antimicrobial functions were detected. They mainly encode 19 non-ribosomal peptide synthetases (NRPSs); one polyketide synthase/NRPSs hybrid enzyme; four Zn-dependent metalloproteases; three antilisterial bacteriocin subtilosin biosynthesis proteins (AlbA); four serine proteases; five pectate lyases; three beta-glucanases; and four 1,4-beta-xylanases. These include four novel NRPSs that have not been found in any species of Paenibacillus. Furthermore, five proteins exhibiting antifungal activity were identified from the antifungal extracellular protein component based on MS/MS and the strain NK3-4 predicted protein library. On the basis of these features, we propose that strain NK3-4 represents a promising biocontrol agent for protecting plant from diseases. The draft genome sequence described herein may provide the genetic basis for the characterization of the molecular mechanisms underlying the biocontrol functions. It may also facilitate the development of rational strategies for improving the strain.
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Riberdy, J. M., i P. Cresswell. "The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation." Journal of Immunology 148, nr 8 (15.04.1992): 2586–90. http://dx.doi.org/10.4049/jimmunol.148.8.2586.
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Abstract .174xCEM.T2 (T2) is a human cell hybrid that has a large homozygous deletion within the MHC, including all of the functional class II genes. We have generated stable HLA-DR3 and H-2 I-Ak transfectants of T2 that express parental levels of class II molecules at the cell surface. T2.Ak transfectants fail to stimulate a hen egg lysozyme (HEL)-specific, I-Ak-restricted T cell when incubated with intact HEL. However, stimulation occurs if the appropriate HEL peptide is provided. The T2 cell line therefore has a defect in class II-restricted Ag processing. Biosynthetic studies demonstrate that the kinetics of I-Ak transport in T2.Ak are similar to the parental rates of transport, although the percentage of I-Ak molecules transported appears somewhat lower. I-Ak glycoproteins in T2.Ak associate normally with the I-chain, which appears to be proteolytically cleaved after transport through the Golgi apparatus in a similar fashion to that in the parent cell line, .174xCEM.T1 (T1). The DR alpha beta heterodimers in T2 differ from the parental phenotype in two ways. First, HLA-DR3 expressed in T2 does not have the epitope recognized by the DR3-specific mAb 16.23, although DR3 expressed in the parent does have the epitope. Second, the alpha beta subunits in the parent remain associated when exposed to SDS at room temperature, although those in T2 dissociate.
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Copier, J., M. J. Kleijmeer, S. Ponnambalam, V. Oorschot, P. Potter, J. Trowsdale i A. Kelly. "Targeting signal and subcellular compartments involved in the intracellular trafficking of HLA-DMB." Journal of Immunology 157, nr 3 (1.08.1996): 1017–27. http://dx.doi.org/10.4049/jimmunol.157.3.1017.
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Abstract Evidence suggests that peptide loading onto MHC class II molecules occurs in a specialized late endocytic compartment (MIIC) where HLA-DM predominantly resides and in which MHC class II transiently accumulates before transport to the cell surface. We examined the targeting signals and compartments involved in the intracellular trafficking of human HLA-DM by expressing hybrid molecules comprising the cytoplasmic domain of DMB and luminal and transmembrane domains of CD8 in HeLa cells. A tyrosine-based tetrapeptide motif present in the cytoplasmic domain of DMB targeted hybrid molecules to intracellular vesicles. Mutation of the tyrosine residue to alanine resulted in redistribution of hybrid molecules to the cell surface. Correct intracellular targeting of HLA-DM was crucial for normal function in B cells. Immunoelectron microscopy on ultrathin cryosections showed that CD8-DMB molecules accumulated in late endocytic compartments sharing characteristics with lysosomes, like MHC class II compartments in APCs. Thus far, the exit of DMB from the Golgi complex has not been elucidated. Interestingly, we found that although the mannose 6-phosphate receptor and CD8-DMB contain similar tyrosine signals, no co-localization was observed in the trans-Golgi network, suggesting that these proteins are differentially sorted at this site. Co-transfection of CD8-DMB, HLA-DR alpha, HLA-DR beta, and an invariant chain revealed that HLA-DR molecules accumulated together with CD8-DMB in these lysosomal compartments. The similarity of these lysosomal-like compartments in wild-type and transfected cells suggests that they are part of the normal endocytic pathway in non-APCs.
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Mezeiova, Eva, Katarina Chalupova, Eugenie Nepovimova, Lukas Gorecki, Lukas Prchal, David Malinak, Kamil Kuca, Ondrej Soukup i Jan Korabecny. "Donepezil Derivatives Targeting Amyloid-β Cascade in Alzheimer's Disease". Current Alzheimer Research 16, nr 9 (29.10.2019): 772–800. http://dx.doi.org/10.2174/1567205016666190228122956.
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: Alzheimer's Disease (AD) is a neurodegenerative disorder with an increasing impact on society. Because currently available therapy has only a short-term effect, a huge number of novel compounds are developed every year exploiting knowledge of the various aspects of AD pathophysiology. To better address the pathological complexity of AD, one of the most extensively pursued strategies by medicinal chemists is based on Multi-target-directed Ligands (MTDLs). Donepezil is one of the currently approved drugs for AD therapy acting as an acetylcholinesterase inhibitor. In this review, we have made an extensive literature survey focusing on donepezil-derived MTDL hybrids primarily targeting on different levels cholinesterases and amyloid beta (Aβ) peptide. The targeting includes direct interaction of the compounds with Aβ, AChE-induced Aβ aggregation, inhibition of BACE-1 enzyme, and modulation of biometal balance thus impeding Aβ assembly.
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Munson, K., N. Lambrecht, J. M. Shin i G. Sachs. "Analysis of the membrane domain of the gastric H(+)/K(+)-ATPase". Journal of Experimental Biology 203, nr 1 (1.01.2000): 161–70. http://dx.doi.org/10.1242/jeb.203.1.161.
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A structure of the catalytic or alpha subunit of the H(+)/K(+)-ATPase, with ten transmembrane segments, and of the beta subunit, with a single such segment, was established using a combination of tryptic cleavage and peptide sequencing and in vitro translation. Sites at which covalent ligands bind to external surfaces were also defined by cleavage, separation and sequencing. Cys813 was found to be the common covalent binding site for all the substituted pyridyl methylsulfinyl benzimidazoles. The binding region of a K(+)-competitive reagent, the 1,2 α -imidazo-pyridine SCH 28080, was defined by the kinetic effects of site-specific mutations. Amino acids substitutions in membrane-spanning segments M1, M3, M4 and M6 were found to influence the apparent inhibitor constant, K(i), to varying degrees, some having a large effect, some a moderate effect and some a slight effect, whereas some mutations had no effect. We interpret changes in K(i) without effects on the apparent Michaelis constant, K(m), as affecting SCH 28080 binding only. Mutation of Cys813 significantly affected the K(i) for SCH 28080, explaining the prevention of benzimidazole inhibition by the imidazo-pyridine.A model of the α subunit was constructed with a vestibule on the luminal surface of the pump bounded by M1-M6 and containing the SCH 28080 binding region. The cation binding site is suggested to be more towards the cytoplasmic face of the enzyme's membrane domain. This model predicts the membrane peptide associations for the catalytic subunit. Biochemical and yeast two-hybrid methods place the beta subunit in association with M8, whereas similar methods place M5/6 in proximity to M9/10. These results, when combined with analysis of the two-dimensional crystals of the sarcoplasmic reticular Ca(2+) and Neurospora crassa H(+)-ATPases, provide the basis for a tentative model of the arrangement of the six core segments of the gastric H(+)/K(+)-ATPase.
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Kjer-Nielsen, L., J. D. Perera, L. F. Boyd, D. H. Margulies i J. McCluskey. "The extracellular domains of MHC class II molecules determine their processing requirements for antigen presentation." Journal of Immunology 144, nr 8 (15.04.1990): 2915–24. http://dx.doi.org/10.4049/jimmunol.144.8.2915.
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Abstract We have evaluated the relative contributions of the extracellular and cytoplasmic domains of MHC class II molecules in determining the Ag-processing requirements for class II-restricted Ag presentation to T cells. Hybrid genes were constructed to encode a heterodimeric I-Ak molecule in which the extracellular portion of the molecule resembled wild type I-Ak but where the connecting stalk, transmembrane and cytoplasmic domains of both the alpha- and beta-chain were derived from the class I molecule H-2Dd. Mutant I-Ak molecules were expressed as heterodimeric membrane glycoproteins reactive with mAb specific for wild type I-Ak. Fibroblast and B lymphoma cells expressing either wild type or mutant I-Ak molecules were able to process and present hen egg lysozyme (HEL) and conalbumin to Ag-specific, I-Ak-restricted, T cell hybridomas or clones. The mutant-expressing cells presented native and peptide Ag less efficiently than the wild type-expressing cells, suggesting that the disparity in presentation efficiency was not due to a difference in Ag processing. CD4 interaction was intact on the mutant I-Ak molecules. Presentation of native Ag by mutant and wild type-I-Ak-expressing cells was abolished by preincubation with chloroquine, or after paraformaldehyde fixation. After transfection of a cDNA encoding the gene for HEL, neither mutant nor wild type-I-Ak-expressing cells presented endogenously synthesized HEL to a specific T hybrid. Newly synthesized mutant I-Ak molecules were associated with invariant chain. These data demonstrate the ability of hybrid class II molecules to associate intracellularly with invariant chain and degraded foreign Ag in a conventional class II-restricted processing pathway indicating that the extracellular domains of class II molecules play a dominant role in controlling these Ag-processing requirements.
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DiLisio, James E., Kaitlin Reyes, K. Scott Beard, Tobias Neef, Stephen D. Miller, Rocky L. Baker i Kathryn Haskins. "Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope". Journal of Immunology 210, nr 1_Supplement (1.05.2023): 238.04. http://dx.doi.org/10.4049/jimmunol.210.supp.238.04.
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Abstract An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B
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Hasib, Annie, Ming T. Ng, Victor A. Gault, Dawood Khan, Vadivel Parthsarathy, Peter R. Flatt i Nigel Irwin. "An enzymatically stable GIP/xenin hybrid peptide restores GIP sensitivity, enhances beta cell function and improves glucose homeostasis in high-fat-fed mice". Diabetologia 60, nr 3 (21.12.2016): 541–52. http://dx.doi.org/10.1007/s00125-016-4186-y.
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