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Sivaji, N., K. V. Abhinav, and M. Vijayan. "Crystallization and biochemical characterization of an archaeal lectin fromMethanococcus voltaeA3." Acta Crystallographica Section F Structural Biology Communications 73, no. 5 (April 28, 2017): 300–304. http://dx.doi.org/10.1107/s2053230x17006173.
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A lectin fromMethanococcus voltaeA3 has been cloned, expressed, purified and characterized. The lectin appears to be specific for complex sugars. The protein crystallized in a tetragonal space group, with around 16 subunits in the asymmetric unit. Sequence comparisons indicate the lectin to have a β-prism I fold, with poor homology to lectins of known three-dimensional structure.
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Ogawa, Tomohisa, Rie Sato, Takako Naganuma, Kayeu Liu, Saho Sato, Shizuka Sakaue, Makoto Osada, Kyosuke Yoshimi, and Koji Muramoto. "Diversified Biomineralization Roles of Pteria penguin Pearl Shell Lectins as Matrix Proteins." International Journal of Molecular Sciences 22, no. 3 (January 22, 2021): 1081. http://dx.doi.org/10.3390/ijms22031081.
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Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related β-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%–50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.
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Abhinav, Koyamangalath Vadakkepat, and Mamannamana Vijayan. "Structural diversity and ligand specificity of lectins. The Bangalore effort." Pure and Applied Chemistry 86, no. 9 (September 19, 2014): 1335–55. http://dx.doi.org/10.1515/pac-2014-0607.
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AbstractStructural studies in this laboratory encompass four of the five major classes of plant lectins, including the one discovered by us. In addition to addressing issues specific to individual lectins, the work provided insights into protein folding, quaternary association and generation of ligand specificity. Legume and β-prism fold lectins constitute families of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary structure, including that involving an open structure. Strategies for generating ligand specificity include water bridges, variation in loop length, post translational modification and oligomerization. Three of the structural classes investigated have subunits with three-fold symmetry. The symmetry in the structure is reflected in the sequence to different extents in different sub-classes. The evolutionary implications of this observation have been explored. The work on lectins has now been extended to those from mycobacteria.
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Wood, Stephen D., Lisa M. Wright, Colin D. Reynolds, Pierre J. Rizkallah, Anthony K. Allen, Willy J. Peumans, and Els J. M. Van Damme. "Structure of the native (unligated) mannose-specific bulb lectin from Scilla campanulata (bluebell) at 1.7 Å resolution." Acta Crystallographica Section D Biological Crystallography 55, no. 7 (July 1, 1999): 1264–72. http://dx.doi.org/10.1107/s0907444999005326.
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The X-ray crystal structure of native Scilla campanulata agglutinin, a mannose-specific lectin from bluebell bulbs and a member of the Liliaceae family, has been determined by molecular replacement and refined to an R value of 0.186 at 1.7 Å resolution. The lectin crystallizes in space group P21212 with unit-cell parameters a = 70.42, b = 92.95, c = 46.64 Å. The unit cell contains eight protein molecules of Mr = 13143 Da (119 amino-acid residues). The asymmetric unit comprises two chemically identical molecules, A and B, related by a non-crystallographic twofold axis perpendicular to c. This dimer further associates by crystallographic twofold symmetry to form a tetramer. The fold of the polypeptide backbone closely resembles that found in the lectins from Galanthus nivalis (snowdrop) and Hippeastrum (amaryllis) and contains a threefold symmetric β-prism made up of three antiparallel four-stranded β-sheets. Each of the four-stranded β-sheets (I, II and III) possesses a potential saccharide-binding site containing conserved residues; however, site II has two mutations relative to sites I and III which may prevent ligation at this site. Our study provides the first accurate and detailed description of a native (unligated) structure from this superfamily of mannose-specific bulb lectins and will allow comparisons with a number of lectin–saccharide complexes which have already been determined or are currently under investigation.
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Sharma, Alok, Divya Chandran, Desh D. Singh, and M. Vijayan. "Multiplicity of carbohydrate-binding sites in β-prism fold lectins: occurrence and possible evolutionary implications." Journal of Biosciences 32, S2 (September 2007): 1089–110. http://dx.doi.org/10.1007/s12038-007-0111-3.
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Sharma, Alok, and Mamannamana Vijayan. "Quaternary association in β-prism I fold plant lectins: Insights from X-ray crystallography, modelling and molecular dynamics." Journal of Biosciences 36, no. 5 (November 18, 2011): 793–808. http://dx.doi.org/10.1007/s12038-011-9166-2.
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Cornil, I., R. S. Kerbel, and J. W. Dennis. "Tumor cell surface beta 1-4-linked galactose binds to lectin(s) on microvascular endothelial cells and contributes to organ colonization." Journal of Cell Biology 111, no. 2 (August 1, 1990): 773–81. http://dx.doi.org/10.1083/jcb.111.2.773.
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Cell surface carbohydrate structures acting as ligands for tissue specific mammalian lectins have been implicated in cell-cell interactions during embryogenesis, lymphocyte homing, and tumor cell metastasis. In this report, we provide evidence that beta 1-4 linked galactose (Gal) residues in N-linked oligosaccharides on the surface of blood born tumor cells serve as a ligand for binding to microvascular endothelial cells. D36W25, a class 1 glycosylation mutant of the MDAY-D2 lymphoreticular tumor cell line, lacks sialic acid and Gal in cellular glycans due to a defect in the Golgi UDP-Gal transporter. Using UDP-Gal and bovine galactosyltransferase in vitro, beta 1-4 Gal was restored to the surface of the cells and 70% of the galactosylated glycans persisted for 8 h in vitro at 37 degrees C. Compared to mock-treated D36W25 cells, galactosylated D36W25 cells showed an 80% increase in binding to microvascular endothelial cell monolayers in vitro. The enhanced binding of galactosylated D36W25 cells to endothelial cell was inhibited by the addition of lactosamine-conjugated albumin to the assay. Consistent with these observations, swainsonine and castinospermine, two inhibitors of N-linked processing that result in loss of lactosamine antennae inhibited the binding of wild-type MDAY-D2 cells to endothelial cells in vitro. Injection of radiolabeled tumor cells into the circulation of syngeneic mice, showed that galactosylation of D36W25 cells resulted in 2-3 more tumor cells retained in the lungs and livers. In addition, galactosylation of D36W25 cells increased by 30-fold the number of visible liver metastases on inspection 4 wk after tumor cell injection. These results suggest that beta 1-4Gal-binding lectins on microvascular endothelial cells can contribute to retention and secondary tumor formation of blood born tumor cells. With the increasing availability of purified glycosyltransferases, reconstruction of a variety of carbohydrate sequences on the surface of class 1 mutants provides a controlled means of studying carbohydrate-lectin interactions on viable cells.
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Naganuma, Takako, Wataru Hoshino, Yukihiro Shikanai, Rie Sato, Kaiyue Liu, Saho Sato, Koji Muramoto, Makoto Osada, Kyosuke Yoshimi, and Tomohisa Ogawa. "Novel Matrix Proteins of Pteria penguin Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins." PLoS ONE 9, no. 11 (November 6, 2014): e112326. http://dx.doi.org/10.1371/journal.pone.0112326.
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Kumazawa-Inoue, Kaori, Tomoko Mimura, Sachiko Hosokawa-Tamiya, Yukiko Nakano, Naoshi Dohmae, Akiko Kinoshita-Toyoda, Hidenao Toyoda, and Kyoko Kojima-Aikawa. "ZG16p, an animal homolog of β-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules." Glycobiology 22, no. 2 (September 23, 2011): 258–66. http://dx.doi.org/10.1093/glycob/cwr145.
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Clerch, L. B., P. L. Whitney, and D. Massaro. "Rat lung lectin synthesis, degradation and activation. Developmental regulation and modulation by dexamethasone." Biochemical Journal 245, no. 3 (August 1, 1987): 683–90. http://dx.doi.org/10.1042/bj2450683.
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Soluble lectins are widely distributed cell-agglutinating proteins. Their activity is developmentally regulated in several tissues, including the lung, but virtually nothing is known about the mechanisms of the developmental regulation or the turnover of these proteins. We studied mechanisms that might be responsible for the developmentally regulated changes in the activity of a lectin (beta-galactoside-binding protein) found in the lung, and determined if its activity or turnover could be modulated by treatment of rat pups with a glucocorticosteroid hormone (dexamethasone). Our studies on the activity and turnover of the lectin indicated that the peak of lectin activity (units/mg of protein) that occurred at age 12 days appeared to be brought about by two means: an increase in the activity of the lectin molecule itself (units/micrograms of lectin) that occurred at age 8 days, and 1.5-fold increase in the absolute rate of lectin synthesis at age 11 days. The decline in lectin activity was associated with a decrease in its rate of synthesis, return to the baseline extent of activation, and an increased rate of degradation. Treatment of rat pups with dexamethasone diminished the peak of lectin activity (units/mg of protein) by about 25%. This effect of dexamethasone was due, at least in part, to the complete prevention of activation of the lectin molecule (units/micrograms of lectin) and a premature increase in the rate of lectin degradation. Perhaps the normal fall in lectin activity after age 11 days is caused by mechanisms induced by the increase in serum corticosteroid that occurs at that age.
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Cook, R. G., N. F. Landolfi, V. Mehta, J. Leone, and D. Hoyland. "Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes." Journal of Immunology 139, no. 4 (August 15, 1987): 991–97. http://dx.doi.org/10.4049/jimmunol.139.4.991.
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Abstract We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.
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Sharma, Alok, and Mamannamana Vijayan. "Influence of glycosidic linkage on the nature of carbohydrate binding in β-prism I fold lectins: An X-ray and molecular dynamics investigation on banana lectin–carbohydrate complexes." Glycobiology 21, no. 1 (August 20, 2010): 23–33. http://dx.doi.org/10.1093/glycob/cwq128.
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Glavey, Siobhan V., Stephen Cunningham, Laura S. Murillo, Catherine Loughrey, Ping Wu, Michael Cairns, Satbir Kaur Gill, et al. "Glycosylation-Related Gene Expression Is Dysregulated in Multiple Myeloma and Overexpression of the Sialyltransferase ST3GAL6 Is Associated with Inferior Survival." Blood 120, no. 21 (November 16, 2012): 2931. http://dx.doi.org/10.1182/blood.v120.21.2931.2931.
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Abstract Abstract 2931 Background Alterations in glycosylation are associated with malignant transformation, with growing evidence implicating the dysregulation of glycosylation genes (glyco-genes) in multiple myeloma (MM). P selectin glycoprotein ligand-1 (PSGL1), recently reported to be important in MM cell adhesion and trafficking is over expressed by MM cells. Importantly, the generation of functional selectin ligands requires post-translational modification of scaffold proteins by glycosyltransferases and sialyltransferases generating selectin ligands such as Sialyl Lewis X (sLex). This background led us to undertake a comprehensive evaluation of the role glyco-genes in MM disease progression. Methods Analyzing publically available microarray transcriptomic datasets (Mayo Clinic GSE6477, University of Arkansas (UAMS) GSE24080, GSE2658) we sought to identify dysregulated glyco-genes in MM. The most significantly dysregulated genes were validated using real time quantitative PCR (RT_PCR) in the MM cell lines RPMI8226 and MMIR and in primary MM patient CD138 positive cells. The prognostic significance of any dysregulated genes was analyzed using Kaplan Meier survival estimates. Membrane protein extracts from MM cell lines were applied directly to lectin microarrays following fluorescent labeling to generate cell surface glycan profiles. Protein expression was assessed by immunohistochemistry (IHC) on primary MM bone marrow sections from MM patients and healthy controls. Lectin based flow cytometry was used to assess for the binding of sialic acid lectins to MM cell line RPMI8226. Results Gene expression microarray dataset analysis confirmed the over-expression of genes related to the process of glycosylation in MM. Seventy-three genes were differentially expressed (> 2 fold change in MM from combined datasets). Nineteen genes were dysregulated in smoldering MM, 12 of which were common to MM. Thirteen were differentially regulated in MGUS, 6 of which were common to both MM and MGUS. One of these genes, the sialyltransferase ST3GAL6 (ST3 beta-galactosidase, alpha-2,3-sialyltransferase 6), which plays a critical role in generation of functional selectin ligands such as sLex, was upregulated in MM (fold change = 2.67) and smoldering MM (fold change = 2.22) but was absent in MGUS. Patients from GSE24080 (n=509) were stratified into two groups based on their expression intensities for ST3GAL6. Patients with higher normalized intensity values corresponding to probeset ID 210942_s_at (ST3GAL6) were found to have a lower median overall survival compared to their lower expressing counterparts. The difference in overall survival observed was 5.13 months (p <0.001 CI 1.3, 8.9). The association with reduced survival was independently verified in the MRC Myeloma IX microarray dataset (n=260). In this dataset using the median expression of ST3GAL6 as a cutoff there was a statistically significant reduction on overall survival with higher expression of ST3GAL6 (median OS 35.7 vs. 48 months, log rank test p=0.04) RT-PCR analysis validated the over expression of glycogenes, including ST3GAL6 in MM cell lines and primary samples compared to healthy controls. We observed a trend for higher fold changes for ST3GAL6 in samples from patients with relapsed/refractory disease compared to those with responsive disease. IHC for ST3GAL6 on primary bone marrow sections from MM patients (n=57), demonstrated specific Golgi staining compared to controls. Consistent with the over expression of ST3GAL6 lectin microarray analysis of membrane protein extracts from MM cell lines RPMI8226 and MMIR showed binding to sialic acid-specific lectins Maackia amurensis agglutinin (MAA), which is specific for a2–3 linked sialic acids, and Sambucus nigra(SNA) which is specific for a2–6 linked sialic acids. This pattern was confirmed using biotinylated lectin based flow cytometry, which demonstrated a shift in allophycocyanin (APC) median fluorescence intensity for these lectins on RPMI8226 cells. Conclusions Glyco-genes, including the sialyltransferase ST3GAL6, are differentially regulated in all stages of MM with potential effects on MM biology and survival. Upregulation of ST3GAL6 may play an important role in MM cell trafficking and in this analysis is associated with inferior survival. Studies are ongoing to address the roles of ST3GAL6 over expression and altered sialylation in MM cell adhesion and trafficking. Disclosures: Morgan: Novartis: Consultancy; Celgene: Consultancy; J&J: Consultancy.
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Eluka-Okoludoh, Eboghoye, Kingsley Ekwemalor, Sarah Adjei-Fremah, Bharath Mulakala, and Mulumebet Worku. "Galectin-8 Modulates Innate and Adaptive Immune Response Genes in Bovine Neutrophils." Journal of Molecular Biology Research 9, no. 1 (January 29, 2019): 24. http://dx.doi.org/10.5539/jmbr.v9n1p24.
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Galectins (Gals) are a family of animal lectins that bind β-galactosides through a carbohydrate recognition domain. Galectin-8 is a tandem-repeat galectin, secreted intracellularly and extracellularly. It is associated with neutrophil migration and has been studied as a possible therapeutic to combat inflammation. The objective of this study was to evaluate the translational and the transcriptional effects of recombinant Galectin-8 (rGal-8) on cow neutrophils. Blood was collected aseptically from Holstein-Friesian cows (n=10) from the North Carolina A&T State University Dairy Unit. Neutrophils isolated were treated with rGal-8 (2μg), or PBS (control) and were incubated at 37°C, 5% CO2 for 1 hour. Supernatant from treated neutrophils was evaluated for total protein concentration, and galectin-8 secretion using bovine Galectin-8 Enzyme Linked-Immuno-Sorbent Assay (ELISA) kit. Total RNA was extracted, reverse transcribed, and RT-qPCR was performed using the RT² Profiler Cow Innate & Adaptive Immune Responses Array with 84 genes. The Livak method was used to calculate transcript abundance and fold change (FC>2 considered significant). Total protein concentration increased (P=0.0361) after rGal-8 treatment compared to the untreated control. Galectin-8 secretion was not significantly different in control compared to treated group (P=0.5819). Out of the 84 genes, 81 genes were differentially expressed in response to rGal-8; 14 up-regulated, 5 down-regulated, 61 genes remained unchanged. Treatment with rGal8 induced the expression of IRF7. The top five up-regulated genes include FAS, CD40, CD86, IFNGR1, STAT1; down-regulated genes were TLR9, CD14, CCR6, TICAM1, and TLR1. Selected genes were probed to validate fold change; the levels of gene expression were comparable to data from RT2 array. Exposure of bovine neutrophils to rGal-8 modified expression of immune response genes. The functional significance of the change needs further studies.
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Altieri, D. C., S. J. Stamnes, and C. G. Gahmberg. "Regulated Ca2+ signalling through leukocyte CD11b/CD18 integrin." Biochemical Journal 288, no. 2 (December 1, 1992): 465–73. http://dx.doi.org/10.1042/bj2880465.
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General mechanisms of adhesion in the immune response are coordinated by the leukocyte integrins CD11/CD18. The possible participation of these differentiation molecules in early events of transmembrane signalling was investigated. Monoclonal antibody (mAb) cross-linking of CD18, the integrin beta subunit ubiquitously expressed by all leukocytes, increased the cytosolic free Ca2+ concentration ([Ca2+]i) by 2-3-fold in monocyte THP-1 cells. Digitalized imaging in single adherent cells showed that this Ca2+ response is temporally biphasic, involves both release of Ca2+ from the intracellular stores as well as Ca2+ influx from the external compartment, and is dramatically down-modulated by terminal differentiation of THP-1 cells to a mature monocyte phenotype. Similarly, only a minor subset of 20-30% of peripheral blood monocytes heterogeneously maintain the CD18-mediated Ca(2+)-signalling properties. Cross-linking of CD18 also increased cytosolic free [Ca2+]i in a subset of approx. 15-20% of resting T lymphocytes, in a Ca2+ response that was completely abrogated during T-cell mitogenic activation with lectins or alloreactive antigen. While cross-linking of CD11a or CD11c was without effect, occupancy of CD11b increased cytosolic free [Ca2+]i in monocytic cells. This response was functionally coupled with a transient activation state of CD11b/CD18, which was reflected in its increased avidity to bind the complementary ligand fibrinogen. These results suggest that occupancy of CD18 transduces a stimulatory Ca2+ signal that is critically regulated by the state of cell activation/differentiation and by the association with the unique alpha-subunit CD11b. These intrinsic signalling properties may directly participate in regulating the oligospecific ligand recognition of leukocyte integrins.
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Vijayan, M., K. Saikrishnan, P. Kumar, Z. Dauter, K. Sekar, A. Surolia, and D. D. Singh. "Banana lectin, a β-prism I fold lectin with two carbohydrate-binding sites." Acta Crystallographica Section A Foundations of Crystallography 61, a1 (August 23, 2005): c232. http://dx.doi.org/10.1107/s0108767305090094.
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Mahanta, S. K., M. V. K. Sastry, and A. Surolia. "Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study." Biochemical Journal 265, no. 3 (February 1, 1990): 831–40. http://dx.doi.org/10.1042/bj2650831.
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Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.
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Sabotič and Kos. "CNL–Clitocybe nebularis Lectin—The Fungal GalNAcβ1-4GlcNAc-Binding Lectin." Molecules 24, no. 23 (November 20, 2019): 4204. http://dx.doi.org/10.3390/molecules24234204.
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Clitocybe nebularis lectin (CNL) is present in fruiting bodies of clouded agaric along with several similar isolectins that are all small and stable proteins. It is a beta-trefoil type lectin forming homodimers that are essential for its functionality. It binds specifically N,N’-diacetyllactosediamine (GalNAcβ1-4GlcNAc, LacDiNac) and human blood group A determinant-containing glycan epitopes. Its most probable function is to defend fruiting bodies against predators and parasites. In addition, an endogenous regulatory function is possible for CNL, as indicated by its interaction with fungal protease inhibitors sharing the beta-trefoil fold. CNL is toxic to insects, nematodes and amoebae, as well as to leukemic T-cell lines. Bivalent carbohydrate binding is essential for the toxicity of CNL, against both invertebrates and cancer-derived cell lines. In addition, CNL exhibits potent immunostimulation of human dendritic cells, resulting in a strong T helper cell type 1 response. Based on its unique characteristics, CNL is a promising candidate for applications in human and veterinary medicine as well as in agriculture, for plant protection.
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Dardevet, D., K. Komori, C. Grunfeld, S. A. Rosenzweig, and M. G. Buse. "Increased hepatic insulin proreceptor-to-receptor ratio in diabetes: a possible processing defect." American Journal of Physiology-Endocrinology and Metabolism 261, no. 5 (November 1, 1991): E562—E571. http://dx.doi.org/10.1152/ajpendo.1991.261.5.e562.
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Hepatic insulin proreceptors and receptors were studied in control and in ketotic diabetic rats 2-4 wk after streptozotocin treatment. Solubilized preparations were partially purified by wheat germ agglutinin-agarose (WGA) and lentil lectin agarose (LLA) chromatography to enrich eluates in insulin receptors and proreceptors, respectively. After phosphorylation with [gamma-32P]ATP, an approximately 190-kDa glycoprotein was identified in LLA eluates as the insulin proreceptor, based on insulin dose-dependent tyrosine autophosphorylation, immunoprecipitation with insulin receptor-specific antibodies, and high-mannose glycosylation. Mature approximately 95 kDa phosphorylated beta-subunits were present in both LLA and WGA eluates. LLA also showed phosphorylated partially processed beta-subunits (approximately 85 kDa) and proreceptors (approximately 190 kDa). Proreceptors comprised less than 1% of the total yield of hepatic insulin receptors. The incorporation of 32P into proreceptors (per gram liver or DNA) was 4.7- or 4.5-fold greater in diabetic vs. control rats, whereas receptor labeling increased only 1.8- or 1.5-fold in diabetic rats. beta-Subunit autophosphorylation per receptor was identical in control and diabetic rats. The phosphorylation data suggested a diabetes-associated 2.6-fold increase in proreceptor-to-receptor ratios. When assessed by cross-linking with 125I-labeled insulin or by immunoblotting, proreceptor-to-receptor ratios were increased 1.5- and 3.1-fold, respectively, in diabetic rats. The data suggest that uncontrolled diabetes may alter insulin receptor processing.
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Sankaranarayanan, Rajan, Kanakaraj Sekar, Rahul Banerjee, Vivek Sharma, Avadhesha Surolia, and Mamannamana Vijayan. "A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with a β-prism fold." Nature Structural Biology 3, no. 7 (July 1996): 596–603. http://dx.doi.org/10.1038/nsb0796-596.
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Vijayan, M., K. Sekar, R. Banerjee, S. K. Mahanta, A. Surolia, and R. Sankaranarayanan. "A threefold symmetric β-prism fold without internal sequence homology in the structure of the two-chain lectin jacalin." Acta Crystallographica Section A Foundations of Crystallography 52, a1 (August 8, 1996): C174. http://dx.doi.org/10.1107/s0108767396092306.
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Bourne, Yves, Véronique Roig-Zamboni, Annick Barre, Willy J. Peumans, Corinne Houlès Astoul, Els J. M. Van Damme, and Pierre Rougé. "The Crystal Structure of theCalystegia sepiumAgglutinin Reveals a Novel Quaternary Arrangement of Lectin Subunits with a β-Prism Fold." Journal of Biological Chemistry 279, no. 1 (October 15, 2003): 527–33. http://dx.doi.org/10.1074/jbc.m308218200.
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Kaplanski, G., C. Farnarier, S. Kaplanski, R. Porat, L. Shapiro, P. Bongrand, and CA Dinarello. "Interleukin-1 induces interleukin-8 secretion from endothelial cells by a juxtacrine mechanism." Blood 84, no. 12 (December 15, 1994): 4242–48. http://dx.doi.org/10.1182/blood.v84.12.4242.bloodjournal84124242.
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Inflammation is characterized by migration of neutrophils through the endothelium, and the chemokine interleukin-8 (IL-8) appears to be involved. We asked whether adherence of cells bearing a membrane-form of interleukin 1 (IL-1) induces IL-8 secretion from human umbilical vein endothelial cells (HUVEC) and fibroblasts. Human peripheral blood mononuclear cells (PBMC) were stimulated with endotoxin for 12 hours and then fixed for 4 hours with paraformaldehyde. When these cells were added to HUVEC or fibroblasts, IL-8 production was induced. This stimulation by fixed PBMC was attributed to IL-1, because pretreatment of HUVEC or fibroblasts with IL-1 receptor antagonist (IL-1Ra) reduced the induction by 95% and 80%, respectively, P < .005. Using anti-IL-1 alpha monoclonal antibodies, reduction was complete, whereas anti-IL-1 beta had no effect. IL-1 alpha was shown on the surface of monocytes by fluorescence-activated cell sorter (FACS) analysis. Blockade of IL-1 receptors on PBMC did not affect the activity of membrane-associated IL- 1 alpha, indicating that IL-1 is not anchored to the membrane through its receptors. However, PBMC treated with D-mannose before fixation resulted in a loss of activity; this loss of activity was associated with release of IL-1 alpha, not IL-1 beta, into the supernatant. Thus, anchoring of IL-1 alpha to the membrane may be via a lectin or mannose receptor-like interaction. Blockade of membrane IL-1 alpha required a 30-fold and fivefold excess of IL-1Ra compared with the amount required to block soluble IL-1 beta and IL-1 alpha, respectively. We conclude that the fixed PBMC IL-8 inducing activity is almost entirely caused by IL-1, that this represents IL-1 alpha bound to a surface lectin or mannose receptor on the monocyte, and that it functions in inflammation via juxtacrine interactions.
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Powell, L. D., S. W. Whiteheart, and G. W. Hart. "Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction." Journal of Immunology 139, no. 1 (July 1, 1987): 262–70. http://dx.doi.org/10.4049/jimmunol.139.1.262.
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Abstract The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.
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Ford, Bradley, Denis Verger, Karen Dodson, Ender Volkan, Maria Kostakioti, Jennifer Elam, Jerome Pinkner, Gabriel Waksman, and Scott Hultgren. "The Structure of the PapD-PapGII Pilin Complex Reveals an Open and Flexible P5 Pocket." Journal of Bacteriology 194, no. 23 (September 21, 2012): 6390–97. http://dx.doi.org/10.1128/jb.06651-11.
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ABSTRACTP pili are hairlike polymeric structures that mediate binding of uropathogenicEscherichia colito the surface of the kidney via the PapG adhesin at their tips. PapG is composed of two domains: a lectin domain at the tip of the pilus followed by a pilin domain that comprises the initial polymerizing subunit of the 1,000-plus-subunit heteropolymeric pilus fiber. Prior to assembly, periplasmic pilin domains bind to a chaperone, PapD. PapD mediates donor strand complementation, in which a beta strand of PapD temporarily completes the pilin domain's fold, preventing premature, nonproductive interactions with other pilin subunits and facilitating subunit folding. Chaperone-subunit complexes are delivered to the outer membrane usher where donor strand exchange (DSE) replaces PapD's donated beta strand with an amino-terminal extension on the next incoming pilin subunit. This occurs via a zip-in–zip-out mechanism that initiates at a relatively accessible hydrophobic space termed the P5 pocket on the terminally incorporated pilus subunit. Here, we solve the structure of PapD in complex with the pilin domain of isoform II of PapG (PapGIIp). Our data revealed that PapGIIp adopts an immunoglobulin fold with a missing seventh strand, complemented in parallel by the G1 PapD strand, typical of pilin subunits. Comparisons with other chaperone-pilin complexes indicated that the interactive surfaces are highly conserved. Interestingly, the PapGIIp P5 pocket was in an open conformation, which, as molecular dynamics simulations revealed, switches between an open and a closed conformation due to the flexibility of the surrounding loops. Our study reveals the structural details of the DSE mechanism.
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Rosa, José César, Lewis Joel Greene, Paulo SÉRgio Lopes De Oliveira, Richard Garratt, Leila Beltramini, Katheryn Resing, and Maria-Cristina Roque-Barreira. "KM+, a mannose-binding lectin from artocarpus integrifolia: Amino acid sequence, predicted tertiary structure, carbohydrate recognition, and analysis of the β-prism fold." Protein Science 8, no. 1 (December 31, 2008): 13–24. http://dx.doi.org/10.1110/ps.8.1.13.
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Ledoux, S., N. Rebai, A. Dagenais, I. T. Shaw, J. Nalbantoglu, R. P. Sekaly, and N. R. Cashman. "Amyloid precursor protein in peripheral mononuclear cells is up-regulated with cell activation." Journal of Immunology 150, no. 12 (June 15, 1993): 5566–75. http://dx.doi.org/10.4049/jimmunol.150.12.5566.
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Abstract The deposition of beta/A4 protein in extraneural organs of patients with Alzheimer disease suggests that this peptide may in part be derived from a peripheral precursor. We studied expression of amyloid precursor protein (APP) in PBMC. APP expression was detectable in resting PBMC by northern blot analysis, immunoblotting studies, and immunohistochemistry. By reverse transcription-polymerase chain reaction, the 751 and 770 APP transcripts containing the Kunitz protease inhibitor (KPI) domain were approximately 10-fold more abundant than the 695 transcript lacking the KPI domain. Activation of PBMC with the lectin PHA-P was associated with an increase in apparent intracellular APP content by cytofluorometry, and an increase in the proportion of the 695 APP transcript lacking the KPI domain. We conclude that resting and activated PBMC express APP and could contribute to a circulating pool of this protein. In addition, PBMC APP is up-regulated with mitogenic stimulation and may participate in the regulation of activation of these cells.
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Dutta, Somnath, Budhaditya Mazumdar, Kalyan K. Banerjee, and Amar N. Ghosh. "Three-Dimensional Structure of Different Functional Forms of the Vibrio cholerae Hemolysin Oligomer: a Cryo-Electron Microscopic Study." Journal of Bacteriology 192, no. 1 (October 23, 2009): 169–78. http://dx.doi.org/10.1128/jb.00930-09.
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ABSTRACT Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.
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Hirai, K., A. L. De Weck, and B. M. Stadler. "Characterization of a human basophil-like cell promoting activity." Journal of Immunology 140, no. 1 (January 1, 1988): 221–27. http://dx.doi.org/10.4049/jimmunol.140.1.221.
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Abstract Biologic and biochemical properties of a human basophil-like cell promoting activity (BaPA), which induces growth of metachromatically staining cells from normal bone marrow cells in a liquid culture system have been examined. In order to study this T cell factor, an assay was developed based on the intracellular histamine content of the cultured human bone marrow cells. Many lymphokines, including granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin 1 alpha and 1 beta, interleukin 2, and interferon-alpha and gamma, did not exhibit any significant activity in the assay. By employing this assay, BaPA was purified approximately 500-fold from lectin-stimulated spleen cell-conditioned medium. BaPA has a molecular weight of 23,000 on high performance liquid chromatography gel filtration and displays isoelectric points between 5.8 and 7.3. It is heat stable up to 80 degrees C for 30 min and resistant to 6 M guanidine hydrochloride, whereas it is rather sensitive to sulfhydryl reagents. BaPA has no stimulating activity on mouse bone marrow cells.
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Issad, T., J. M. Tavaré, and R. M. Denton. "Analysis of insulin receptor phosphorylation sites in intact rat liver cells by two-dimensional phosphopeptide mapping. Predominance of the tris-phosphorylated form of the kinase domain after stimulation by insulin." Biochemical Journal 275, no. 1 (April 1, 1991): 15–21. http://dx.doi.org/10.1042/bj2750015.
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1. Insulin receptors were partially purified from rat liver by chromatography on wheat-germ-lectin-Sepharose. Incubation with [gamma-32P]ATP in the presence of insulin resulted in increased phosphorylation of the beta-subunit on both tyrosine and serine residues. Two-dimensional mapping of tryptic peptides showed that, in agreement with previous studies using preparations of receptors from other sources, the tyrosine residues involved were the three tyrosines in the kinase domain (corresponding to tyrosines 1158, 1162 and 1163 of the human receptor) plus two tyrosines close to the C-terminus (corresponding to tyrosines 1328 and 1334). 2. The effects of insulin on the phosphorylation of receptors within intact rat liver cells were determined by incubating cells in the presence of [32P]Pi for 50 min and then with or without insulin for a further 10 min. The labelled receptors were then rapidly isolated by sequential use of wheat-germ-lectin-Sepharose chromatography and immuno-isolation using a monoclonal antibody to the C-terminal end of the beta-subunit. 3. Insulin was found to increase overall phosphorylation of the receptor nearly 3-fold. Two-dimensional mapping was then carried out in combination with phosphoamino acid analysis. This revealed that the pattern of phosphorylation of the receptors in cells incubated in the absence and presence of insulin exhibited a number of marked differences from that observed in previous studies on intact cells, which had been restricted to cells expressing very high levels of insulin receptors such as certain hepatoma-derived cells or cells transfected with insulin receptor cDNA. The differences in the effects of insulin included a larger increase in the proportion of receptors being phosphorylated on the three tyrosine residues of the kinase domain, no apparent phosphorylation of the two tyrosine residues close to the C-terminus and no increase in either threonine or overall serine phosphorylation. 4. The receptors appeared to be phosphorylated on a number of different serine residues in cells incubated in the absence of insulin. Evidence for both increases and decreases in the phosphorylation of specific serine residues on addition of insulin was obtained. 5. It is concluded that care should be taken when extrapolating findings on the phosphorylation of the insulin receptor within cultured cells to more physiological situations.
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Gong, Z. Y., and B. Brandhorst. "Autogenous regulation of tubulin synthesis via RNA stability during sea urchin embryogenesis." Development 102, no. 1 (January 1, 1988): 31–43. http://dx.doi.org/10.1242/dev.102.1.31.
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When pluteus embryos of Lytechinus pictus were treated with colcemid, the incorporation of [35S]methionine into tubulin declined by 5- to 15-fold within 4 h. This was mostly accounted for by a rapid decline in the concentration of alpha- and beta-tubulin mRNA in the cytoplasm. Treatment with other microtubule depolymerizing agents (colchicine, nocodazole, low concentrations of vinblastine) had similar effects. Treatment of embryos with the microtubule-stabilizing agent, taxol, or high concentrations of vinblastine resulted in increased synthesis of tubulin. The concentration of tubulin mRNA increases during development and becomes increasingly sensitive to colcemid and decreasingly sensitive to taxol. The transcriptional activity of tubulin genes, estimated by an RNA run-on assay in isolated nuclei, was not altered after colcemid treatment. On the other hand, the rate of decay of tubulin mRNA in prism embryos treated with actinomycin D was increased several fold by colcemid treatment, while taxol treatment led to an increased half-life of tubulin mRNA. These observations suggest that tubulin synthesis is autogenously regulated at the level of mRNA stability by the level of unpolymerized tubulin. The increasing polymerization of microtubules and declining level of unpolymerized tubulin during embryogenesis would result in a stabilization of tubulin mRNA accounting for the increasing concentration of tubulin mRNA and rate of tubulin synthesis, as well as the increasing sensitivity of tubulin synthesis to microtubule-depolymerizing agents. Evidence is also presented for a rapid influence of the level of unpolymerized tubulin on the efficiency of translation of tubulin mRNA.
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Ruffet, E., N. Paquet, S. Frutiger, G. J. Hughes, and J. C. Jaton. "Structural and electron-microscopic studies of jacalin from jackfruit (Artocarpus integrifolia) show that this lectin is a 65 kDa tetramer." Biochemical Journal 286, no. 1 (August 15, 1992): 131–34. http://dx.doi.org/10.1042/bj2860131.
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The 133-amino-acid sequences of the alpha-subunit of jacalin (a lectin from Artocarpus integrifolia) and of the slightly larger alpha'-subunit were determined. The alpha'- and alpha-subunits, in the approximate ratio of 1:3, were found to be virtually identical in their primary structures, except for one valine for isoleucine substitution at position 113. Although both alpha'- and alpha-chains were glycosylated, the extent of glycosylation in the alpha'-chain was much greater than that in the alpha-subunit. In the alpha'-polypeptide, all molecules contained an N-linked oligosaccharide at position 74 and some contained sugar at position 43. The alpha- and alpha'-subunits were found to be strongly non-covalently associated with three distinct beta-subunits containing 20 amino acids each. Electron-microscopic visualization of native jacalin disclosed a structure composed of four alpha-type subunits with a clear-cut 4-fold symmetry. Analytical-ultracentrifugation studies of jacalin revealed an average molecular mass of 65 kDa, a value compatible with a tetrameric structure of the alpha(alpha')-subunits. The recalculated number of sugar-binding sites per jacalin molecule, given a molecular mass of 65 kDa, would yield 0.8 sites per alpha(alpha')-promoter, i.e. about twice the value previously determined [Appukutan & Basu (1985) FEBS Lett. 180, 331-334; Ahmed & Chatterjee (1989) J. Biol. Chem. 264, 9365-9372].
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Uriarte, M., W. Stalmans, S. Hickman, and M. Bollen. "Phosphorylation and nucleotide-dependent dephosphorylation of hepatic polypeptides related to the plasma cell differentiation antigen PC-1." Biochemical Journal 293, no. 1 (July 1, 1993): 93–100. http://dx.doi.org/10.1042/bj2930093.
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A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the phosphorylation of the membrane proteins is that the action of a protein kinase is initially offset by nucleotide-stimulated dephosphorylation.
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Nyholm Kyvsgaard, Julie, Christina Ellervik, Emilie Bundgaard Lindkvist, Christian Bressen Pipper, Flemming Pociot, Jannet Svensson, and Steffen Ullitz Thorsen. "Perinatal Whole Blood Zinc Status and Cytokines, Adipokines, and Other Immune Response Proteins." Nutrients 11, no. 9 (August 22, 2019): 1980. http://dx.doi.org/10.3390/nu11091980.
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(1) Background: Zinc is an essential micronutrient and zinc deficiency is associated with immune dysfunction. The neonatal immune system is immature, and therefore an optimal neonatal zinc status may be important. The aim of this study was to investigate the possible association between neonatal whole blood (WB)-Zinc content and several immune markers. (2) Methods: In total, 398 healthy newborns (199 who later developed type 1 diabetes and 199 controls) from the Danish Newborn Screening Biobank had neonatal dried blood spots (NDBS) analyzed for WB-Zinc content and (i) cytokines: Interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, IL-12 (p70), interferon gamma, tumor necrosis factor alpha, and transforming growth factor beta; (ii) adipokines: leptin and adiponectin; (iii) other immune response proteins: C-reactive protein (CRP), and mannose-binding lectin (MBL), and soluble triggering receptors expressed on myeloid cells1 (sTREM-1). WB-Zinc content was determined using laser ablation inductively coupled plasma mass spectrometry. For each analyte, the relative change in mean level was modelled by a robust log-normal model regression. (3) Results: No association was found between WB-Zinc content and all the immune response markers in either the unadjusted or adjusted models overall or when stratifying by case status. (4) Conclusions: In healthy Danish neonates, WB-Zinc content was not associated with cytokines, adipokines, CRP, MBL or sTREM, which does not indicate a strong immunological function of neonatal zinc status.
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Ashur-Fabian, Osnat, Adi Har-Zahav, Aviv Shaish, Ninette Amariglio, Dror Harats, Gideon Rechavi, and Martin Ellis. "Imatinib Mesylate Affects the Expression of Lipid Metabolism Genes in K562, a Chronic Myeloid Leukemia Cell Line." Blood 112, no. 11 (November 16, 2008): 4238. http://dx.doi.org/10.1182/blood.v112.11.4238.4238.
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Abstract Background: Imatinib mesylate (IM) is a tyrosine kinase inhibitor designed to inhibit the BCR-ABL fusion protein, the hallmark of chronic myeloid leukemia (CML). Interestingly, IM treatment possesses a rapid lipid-lowering effect on patients with hyperlipidemia, particularly those receiving 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR) inhibitors. While it is known that IM may competitively inhibit statin metabolism, the lipid-lowering effect of IM is not fully understood. Therefore, we have studied the effects of IM on a family of genes critically involved in lipid metabolism, including low density lipoprotein receptor (LDL-R), HMGCoAR, cholesteryl ester transfer protein (CETP), apolipoprotein B (apoB) and apolipoprotein B editing complex (apobec1), in K562, a CML cell line model. Methods: Cell cultures and treatments: K562 cells were grown in DMEM supplemented with 10% FCS. Cells were treated with IM or DMSO (1mM) and were harvested after 24–96 hours. Untreated cells served as a control as did the DMSO-treated cells. RNA extraction and cDNA synthesis: RNA was extracted using Trizol reagent method and reverse transcribed using random primers and M-MLV. Real-time PCR assays: Real-time PCR assays were conducted using assays on demand for the different genes (Applied Biosystems, Foster City, CA, USA) and performed on Applied Biosystems 7900HT Prism® real-time PCR instrument with a 100ng equivalent of cDNA, in a total volume of 20 ml 1X Taqman master mix (Applied Biosystems). Results were quantified using the delta-delta cycle threshold method, normalized to actin beta and expressed as “fold-change from control”. Statistics: Results were analyzed using unpaired students’ t-test. Results: LDL-R, HMGcoAR, CETP, apobec1 and apoB genes expression were evaluated in untreated K562 cells (control) and following treatment with DMSO (negative control) or IM for 24 to 96 hours. IM treatment of K562 cells significantly affected the expression of lipid-related genes. Namely, a time-dependent increase in LDL-R (3–6 fold), HMGcoAR (2–5 fold) and CETP (4–50 fold) expressions were documented following IM treatment. No increase in expression of these genes was observed in untreated and DMSO treated controls. Basal apobec1 and apoB expression detected in untreated K562 cells was abolished following IM but not DMSO treatment. Discussion: Putative mechanisms for the decrease in serum cholesterol and triglycerides observed in some patients treated with IM include reduced intestinal absorption and inhibition of platelet derived growth factor receptor (PDGFR), resulting in decreased lipoprotein lipase (LPL) synthesis. Our novel observation proposes another explanation, namely a direct effect of IM on central lipid-related genes. Conclusions: The direct effect of IM on lipid-related genes, together with the fact that in cell culture IM inhibits proliferation and induces apoptosis, suggest a possible role of lipids in apoptosis/cell cycle pathways. This possibility is currently the subject of further investigation.
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Wallgren, A., R. Festin, C. Gidlof, M. Dohlsten, T. Kalland, and TH Totterman. "Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells." Blood 82, no. 4 (August 15, 1993): 1230–38. http://dx.doi.org/10.1182/blood.v82.4.1230.1230.
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Abstract In vitro studies have indicated that chronic lymphocytic leukemia of B- cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA- induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)- alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)
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Wallgren, A., R. Festin, C. Gidlof, M. Dohlsten, T. Kalland, and TH Totterman. "Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells." Blood 82, no. 4 (August 15, 1993): 1230–38. http://dx.doi.org/10.1182/blood.v82.4.1230.bloodjournal8241230.
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In vitro studies have indicated that chronic lymphocytic leukemia of B- cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA- induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)- alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)
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Altieri, D. C., and T. S. Edgington. "Identification of effector cell protease receptor-1. A leukocyte-distributed receptor for the serine protease factor Xa." Journal of Immunology 145, no. 1 (July 1, 1990): 246–53. http://dx.doi.org/10.4049/jimmunol.145.1.246.
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Abstract Mitogenesis, cell differentiation and immune-inflammatory responses are regulated by the coordinated assembly of proteases with specific cellular receptors. We have investigated the possibility that immune effector cells may express a high-affinity protease receptor. To address this hypothesis, we have generated mAb to factor V and its activated form Va, a circulating plasma protein that binds the serine protease of the coagulation cascade, factor Xa. Further, by flow microfluorimetry screening, we have isolated a panel of these mAb that recognize a surface molecule expressed on transformed monocytic cells. We now show that these mAb bind to blood monocytes, to CD3- CD16+ CD56+ NK cells, and with considerable heterogeneity, to neutrophils. A small subset of CD3+ cells (5 to 10%) was also identified by these probes and further phenotypically characterized by two-color flow microfluorimetry as predominantly coexpressing CD2, CD4 or CD8, CD57, CD11b, and alpha/beta TCR. This subset of CD3+ cells was expanded in vitro by both lectin- or Ag-specific stimulation. In addition, long term alloreactive stimulation resulted in approximately 8- to 10-fold increased expression of the molecule recognized by these mAb. Functional analyses were performed on a selected T cell clonal derivative of the transformed cell line HuT 78. These cells bound 125I-factor Xa in a specific reaction saturated at 194,000 +/- 26,000 molecules/cell with a Kd approximately 10 to 20 nM and inhibited by the mAb panel described above. These data suggest that immune effector cells express a dynamically regulated protease receptor that is immunologically related to the plasma coagulation protein factor V and its activated form Va. We propose the term effector cell protease receptor-1 to tentatively identify this molecule, and we speculate on its possible involvement in specialized protease-mediated effector functions.
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Desai, Ankit A., Tong Zhou, Mehdi Nouraie, Victor R. Gordeuk, Joe GN Garcia, and Roberto F. Machado. "Genomic Assessment of Mortality in Sickle Cell Disease- A Novel Association with Cytokine Imbalance and T Cell Dysregulation." Blood 118, no. 21 (November 18, 2011): 1081. http://dx.doi.org/10.1182/blood.v118.21.1081.1081.
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Abstract Abstract 1081 Rationale: Sickle cell disease (SCD) is characterized by systemic complications and significant clinical variability. While some patients never experience severe clinical events, others suffer lifelong morbidity and accelerated mortality. To address this variability, we prospectively followed patients with SCD at steady-state to assess outcomes and leveraged microarray analysis of RNA isolated from peripheral blood mononuclear cells (PBMCs) to identify novel gene expression patterns and signaling pathways that could be associated with survival in SCD. Methods: Forty-two adult patients with SCD at steady-state (no VOC or ACS episodes for >3 weeks) were followed for 5 years (median follow-up 1154 ± 317 days). At baseline enrollment, patients underwent phlebotomy for plasma and serum collection. The plasma was utilized to extract mRNA from PBMCs. The serum was used to assess targeted cytokine levels using high-sensitive cytokine kits. Microarray expression analysis of PBMC mRNA utilized an FDR<10% and a fold-change >1.2. Student t-tests were performed for statistical significance. Results: Five out of forty-two patients (∼12%) died over the course of the study (median follow-up 1093 ± 320 days for surviving patients vs 1246 ± 325 days for patients who died, p=0.42). Although there were trends towards older age, higher tricuspid regurgitation jet velocity, NT-pro-BNP levels, AST levels, and lifetime number of transfusions in the patients that died, there were no statistically significant differences between groups (Table 1). Patients who died (n=5) exhibited 278 differentially regulated genes in comparison to those who survived (n=37). Gene ontology analysis revealed significantly represented T-cell receptor and immune mediated pathways, comprising seven out of the top ten pathways (p<0.05). Using a fold-change of 1.5 threshold on the transcripts, we identified a 14 gene signature which discriminated the survival of patients with 100% accuracy. Multiple genes within this molecular signature for survival also included immune-mediate pathways including CD160, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), interleukin 1, beta (Il1B), killer cell lectin-like receptor subfamily G, member 1 (KLRG1), CD3d molecule, delta (CD3D), and killer cell lectin-like receptor subfamily F, member 1 (KLRF1). Given this robust representation of immune-mediate pathways in the signature and ontology analysis, we measured targeted plasma cytokine levels (based on candidates known to be involved in SCD) in all patients. IL-6 (5.7 ± 4.8 vs 11.7 ± 4.8 pg/mL, p=0.006) and IL-8 levels (3.5 ± 2.3 vs 6.2 ± 2.3 pg/mL, p=0.009) were significantly higher during steady-state in patients who later died than in those who survived. Interestingly, the anti-inflammatory cytokine IL-10 (35.2 ± 109.6 vs 16.1 ± 15.6 pg/mL) trended higher in the survival group while tumor necrosis factor levels trended lower (38.2 ± 28.0 vs 48.8 ± 38.5 pg/mL) but this did not reach statistical significance. Conclusion: These preliminary data suggest that a PBMC genomic signature is a potential prognostic biomarker in SCD. Patients who subsequently died demonstrated a unique PBMC-gene expression profile with significant representation of specific T-cell and immune-mediated pathways. Concurrently, these at-risk patients also demonstrated higher serum IL-6 and IL-8 levels. Given that high circulating levels of these type 2 cytokines suppress both humoral and cell-mediated immune functions, we speculate that T-cells may play novel role in the variable morbidity and mortality observed in SCD. Disclosures: No relevant conflicts of interest to declare.
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Mulakala, Bharath Kumar, Eluka-Okoludoh Eboghoye, Kingsley Ekwemalor, and Mulumebet Worku. "PSIX-20 Galectins expression and secretion in cow milk." Journal of Animal Science 99, Supplement_3 (October 8, 2021): 266–67. http://dx.doi.org/10.1093/jas/skab235.488.
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Abstract The objective of this study was to assess the expression and secretion of Galectins (Gal) in cow milk. Cow milk contains a range of proteins of moderate or low abundance that contribute to host defense. However, host defense proteins in milk were not fully discovered. Galectins belong to the lectin family that recognizes specific carbohydrates on cells and involved in innate immune responses. Holstein Friesian cows (n = 16) from North Carolina A&T diary unit were used for this study. Based on the Dairy Heard farm index, eight cows each were assigned to the high or low SCC group. Total RNA was isolated from somatic cells converted to cDNA, for real-time PCR. Cow-specific Gal primers were designed using the NCBI Primer 3 tool. Housekeeping genes RPLP0, UCHL5, and beta-actin were served as internal controls. Total whey protein concentration was determined using a BCA kit. Secretion of Gal was assessed using a specific ELISA kit. Data were analyzed using the Proc ANOVA procedure in SAS 9.4. Galectin was transcribed and secreted in milk. Transcription of Galectin was different in both HSCC and LSCC groups. Total protein concentration remained the same in both groups. Secretion of galectins was different between the HSCC and LSCC group but not significantly. The observed difference in HSCC and LSCC cows warrants further study using more animals; this will aid in a better definition of the role of Gal in the milk host defense. These may also aid as the diagnostic biomarkers for certain infections.
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Jung, Jinwon, Juhee Kim, Bora Lee, Jung A. Kwon, Suyoun Lee, Byeongmin Yoo, Minji Ko, et al. "Abstract 6296: Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload." Cancer Research 83, no. 7_Supplement (April 4, 2023): 6296. http://dx.doi.org/10.1158/1538-7445.am2023-6296.
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Abstract C-type lectin domain family 12 member A (CLEC12A, CLL-1) is a single-pass transmembrane protein of 265 amino acids that is found on monocytes and AML blasts. We have created and characterized antibody-drug conjugates (ADCs) based on a humanized CLEC12A specific antibody to determine whether CLEC12A may be exploited as a therapeutic target for AML. The payload is a proprietary PBD prodrug with a beta-glucuronide trigger, and the linker-payload was conjugated through farnesyltransferase-mediated functionalization. The ADC demonstrated sub-nM cytotoxicity in vitro against several CLEC12A-positive cell lines including HL-60 and PL21. Leu234Ala/Leu235Ala (LALA) mutations were employed to reduce Fc gamma receptor binding and to avoid on-target toxicity. The LALA mutation-bearing ADC displayed a nearly 50-fold decrease in cytotoxicity towards CD34+ hematopoietic stem cells. Its potency was reduced but still sufficient with IC50 values of 61 pM and 15 pM against HL60 and PL21, respectively. Regardless of LALA mutation, the ADCs demonstrated potent antitumor activities with a complete regression at a dose of 0.5 mpk against a subcutaneous HL-60 SCID mouse xenograft model. When the LALA-mutated ADC was tested against a disseminated NSGA mouse model of HL-60-luc, all treated animals survived without clinical symptoms for three weeks after treatment, whereas vehicle-treated animals exhibited morbidity 19 days after treatment or 35 days after tumor implantation. The bone marrow of ADC-treated animals appeared to have nearly fully recovered, whereas that of vehicle-treated animals showed necrosis or tumor growth. In cynomolgus monkeys, the LALA-mutated ADC had a half-life of 82 hours at a dose of 0.2 mpk, and target-mediated drug disposition appeared weak or negligible. Our preclinical studies have shown that CLEC12A targeting ADC can be used as a therapeutics for treating AML. Citation Format: Jinwon Jung, Juhee Kim, Bora Lee, Jung A Kwon, Suyoun Lee, Byeongmin Yoo, Minji Ko, Ilhwan Ryu, Donghoon Yeom, Kyoungjae Lee, Jaehyun Eom, Hanbyul Lee, Jinhyung Ahn, Eunsil Sung, Weonkyoo You, Sang Hoon Lee, Myeong Joo Kim, Keon Woo Kwon, Hyun Joo Bae, Yun-Hee Park, Ho Young Song, Chul-Woong Chung. Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6296.
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Vennegoor, C. J., E. van de Wiel-van Kemenade, R. J. Huijbens, F. Sanchez-Madrid, C. J. Melief, and C. G. Figdor. "Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway." Journal of Immunology 148, no. 4 (February 15, 1992): 1093–101. http://dx.doi.org/10.4049/jimmunol.148.4.1093.
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Abstract Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.
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Chorich, Lynn P., Michael P. Diamond, Janet E. Hall, Kenneth S. Korach, Lawrence C. Layman, Yin Li, Haitao Liu, and Robert A. Roman. "OR06-3 ESR1 Pathogenic Variant With Incomplete Estrogen Insensitivity." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A677—A678. http://dx.doi.org/10.1210/jendso/bvac150.1401.
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Abstract Introduction Estrogen is vital to human reproduction and acts primarily through two receptors: estrogen receptor alpha (ERα, encoded by ESR1) and estrogen receptor beta (ERβ, encoded by ESR2). Surprisingly, very few human ESR1 pathogenic variants have been recognized, despite the existence of >1000 different androgen receptor pathogenic variants causing androgen insensitivity syndrome. Functional analysis of an ESR1 homozygous missense variant (p.Glu375His) in the ERα ligand-binding domain (LBD) showed a 240-fold decrease in estrogen response in a patient with complete estrogen insensitivity and abnormal pubertal development. We predict that less severe ESR1 variants will present with unexplained infertility. Previously, we performed whole exome sequencing on 200 females with unexplained infertility and identified 4 likely pathogenic ESR1 heterozygous missense variants confirmed by Sanger sequencing, including one variant (p.Thr313Met) in the LBD. In this study, we performed in vitro functional analysis to evaluate if the p.Thr313Met likely pathogenic variant found in a patient with unexplained infertility causes incomplete estrogen insensitivity. Hypothesis We hypothesize that the p.Thr313Met likely pathogenic variant in the ERa LBD will result in incomplete estrogen insensitivity. Methods The p.Thr313Met variant plasmid was constructed using site-directed mutagenesis (SDM), and confirmed by Sanger sequencing. An in vitro cell model was used to evaluate estrogenic activity. HepG2 cells were transiently transfected with WT ESR1, p.Thr313Met, or p.Glu375His plasmid, along with the Firefly estrogen response element (ERE) luciferase reporter (3X ERE TATA luc) and Renilla luciferase (pRL-TK luc) plasmids using the Effectene transfection protocol. Cells were treated with 17β-estradiol (0.01, 0.1, 1, 10, and 100 nM). A dual luciferase reporter assay was used to compare relative Firefly and Renilla luciferase activity between WT ESR1, p.Thr313Met, and p.Glu375His plasmids using a luminometer. Estrogen dose response curves were generated, and a two-way ANOVA with Tukey's test was performed to compare the relative luciferase activity across WT and variant plasmids using GraphPad Prism v.9. Differences were considered significant at p<0.05. Results Estrogen response curves were generated for WT, p.Thr313Met, and p.Glu375His plasmids. The p.Thr313Met variant showed lower overall activity with a right shifted dose response curve with a statistically significant decrease in relative luciferase activity in comparison to WT (p<0.05). The p.Thr313Met variant was more active at higher doses (1 and 10 nM) with a left shifted dose curve when compared to the p.Glu375His variant (p<0.05). Conclusion We have identified a human ESR1 variant that impairs estrogen signaling in vitro, suggesting incomplete estrogen insensitivity as a putative mechanism for unexplained infertility. The identification of ESR1 pathogenic variants and the evaluation of their functionality could provide improved diagnosis and genetic counseling for patients with unexplained infertility. Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.
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Sivaji, Nukathoti, Kaza Suguna, Avadhesha Surolia, and Mamannamana Vijayan. "Structural and related studies on Mevo lectin from Methanococcus voltae A3: the first thorough characterization of an archeal lectin and its interactions." Glycobiology, July 11, 2020. http://dx.doi.org/10.1093/glycob/cwaa063.
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Abstract Crystallographic and solution studies of Mevo lectin and its complexes, the first effort of its kind on an archeal lectin, reveal a structure similar to β-prism I fold lectins from plant and animal sources, but with a quaternary association involving a ring structure with seven-fold symmetry. Each subunit in the heptamer carries one sugar binding site on the first Greek key motif. The oligomeric interface is primarily made up of a parallel β-sheet involving a strand of Greek key I of one subunit and Greek key ΙΙΙ from a neighboring subunit. The crystal structures of the complexes of the lectin with mannose, αMan(1,2)αMan, αMan(1,3)αMan, a mannotriose and a mannopentose revealed a primary binding site similar to that found in other mannose specific β-prism I fold lectins. The complex with αMan(1,3)αMan provides an interesting case in which a few subunits have the reducing end at the primary binding site, while the majority have the nonreducing end at the primary binding site. The structures of complexes involving the trisaccharide and the pentasaccharide exhibit cross-linking among heptameric molecules. The observed arrangements may be relevant to the multivalency of the lectin. Phylogenetic analysis of amino acid sequences indicates that Mevo lectin is closer to β-prism I fold animal lectins than with those of plant origin. The results presented here reinforce the conclusion regarding the existence of lectins in all three domains of life. It would also appear that lectins evolved to the present form before the three domains diverged.
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Moore, Jordan T., Mohamed Ewees, Jay L. Zweier, Daniel Gallego-perez, and Yousef Hannawi. "Abstract P54: Endothelial Cells and Pericytes Collection Using Laser Capture Microdissection in Spontaneously Hypertensive Stroke Prone Rats." Stroke 52, Suppl_1 (March 2021). http://dx.doi.org/10.1161/str.52.suppl_1.p54.
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Introduction: Neurovascular unit (NVU) dysfunction plays a key role in cerebral small vessel disease (cSVD) pathogenesis. Spontaneously Hypertensive Rats - Stroke Prone (SHRSP) is a relevant model for cSVD where prominent impairment of the NVU has been identified. Previous studies of SHRSP and Wistar-Kyoto control rats (WKY) were performed at tissue level failing to capture the NVU cellular components where the early pathological events occur. Therefore, we developed a novel method for selective endothelial cell (EC) and pericytes capture using Laser Capture Microdissection (LCM). Methods: 10 SHRSP and 10 WKY male rats were studied at 16 weeks of age. Prior to brain collection, lectin was administered using intracardiac injection for vessel identification. Rapid frozen immunohistochemistry (IHC) staining protocol was optimized by selecting a panel of antibodies that showed first specific staining pattern for EC and pericytes in a routine overnight IHC protocol. LCM collection was subsequently optimized to achieve collection of 1.5-2 million μm 2 of tissue in 30 minutes to ensure RNA stability. RNA was isolated from the LCM samples using PicoPure RNA isolation kit (ThermoFisher Scientific, Inc.) and RNA quantity was measured using NanoDrop technology. qRT-PCR we performed to measure the expression of smooth muscle actin (ACTA2) (pericytes), von Willebrand Factor (vWF) (EC). Results: Following optimization of overnight and rapid staining protocols, platelet-derived growth factor (PDGRF) beta (ABCAM, Inc.) (1:10 concentration) showed best results for perdicytes identification while RCA lectin (Vector labs, Inc.) (1:20 concentration) showed best results for EC. Co-staining with CD31 antibody was performed to confirm the specificity of RCA lectin binding to EC. RNA isolation protocol achieved good RNA collection quantity (4-8) ng/μl. qRT-PCR showed higher expression of ACTA2 (9.5-fold increase) in PDGRF+ elements, 2.8-fold increase of vWF (lectin+ elements) compared to (PDGRF-, lectin -) elements confirming the enrichment of these elements by EC and pericytes. Conclusion: LCM is feasible in achieving good quality collection of EC and pericyte in SHRSP and WKY allowing for investigation of the proteomic and transcriptomic changes that result in cSVD.
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AHAMAD, ADIL, and S. H. ANSARI. "A REVIEW ON MULTIPURPOSE MEDICINAL PROPERTIES OF TRADITIONALLY USED PSIDIUM GUAJAVA LEAVES." Asian Journal of Pharmaceutical and Clinical Research, August 7, 2022, 9–22. http://dx.doi.org/10.22159/ajpcr.2022.v15i8.43179.
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Psidium guajava is an important food crop and medicinal plant available in tropical and subtropical countries. P. guajava (Guava), belonging to the family of Myrtaceae. P. guajava Linn. (Guava) is used not only as food but also as folk medicine in subtropical areas around the world because of its pharmacologic activities. It contains important phytoconstituents such as tannins, triterpenes, flavonoid, quercetin, pentacyclic triterpenoid, guajanoic acid, saponins, carotenoids, lectins, leucocyanidin, ellagic acid, amritoside, beta-sitosterol, uvaol, oleanolic acid, and ursolic acid. Conventionally, guava is used for the treatment of various ailments such as antioxidant, hepatoprotective, anti-allergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardioactive, anti-cough, antidiabetic, anti-inflammatory, and antinociceptive activities, supporting its traditional uses.
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Azimudin, Ridha, S. Preetha, J. Selvaraj, Sridevi G, and Jeevitha M. "Effect of Carica Seed Extract on Inhibitory Kappa B Kinase Beta and mTOR mRNA Expression in Lung Cancer Cells (A549 Cells)." Journal of Pharmaceutical Research International, January 27, 2022, 10–17. http://dx.doi.org/10.9734/jpri/2022/v34i5b35410.
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Background: Lung cancer is considered as one of the most common causes for cancer-related death globally. Carica papaya is one of the most well-known traditional medicines to treat diseases, and is also known to treat cancer and help in cancer prevention. The study investigates the effect of Carica seed extract on inhibitory kappa B kinase beta and mTOR mRNA expression in lung cancer cells (A549 cells).
Methods: Cell viability test was assessed using MTT assay. mRNA expression of inhibitory kappa B kinase beta and mTOR mRNA was analyzed by real-time PCR. The results was analysed statistically by ANOVA and Duncan multiple range test with graphpad prism version 5 software. p<0.05 was considered significant.
Results: It was observed that there wasmaximum inhibition (50%) at 400-500µg/ml of Carica seed extract. It was also found that the fold change over control of mTOR mRNA expression was significant at 500µg/ml of Carica seed extract and the fold change over control of IKKB mRNA expression was significant (p<0.05) at 400µg/ml in cancer cells treated with Carica seed extract.
Conclusion: Thus concluding that Carica seed extract has been found to have significant anticancer property on A549 lung cancer cell lines, and can be used as a natural product in combating lung cancer.
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"$beta;-galactoside-binding lectins in hearts and cardiac myocytes of adult rats and rabbits C. K�nig and G. Gercken. Institute of Biochemistry and Food Chemistry, University of Hamburg, 2000 Hamburg 13, F.R.G." Journal of Molecular and Cellular Cardiology 21 (July 1989): S28. http://dx.doi.org/10.1016/0022-2828(89)91297-2.
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Iribarren, Carlos, Malini Chandra, Mark A. Hlatky, Stephen Fortmann, Thomas Quertermous, and Alan S. Go. "Abstract P016: Galectin-3 and Incident Heart Failure among Patients with Pre-existing Coronary Artery Disease: the ADVANCE Study." Circulation 129, suppl_1 (March 25, 2014). http://dx.doi.org/10.1161/circ.129.suppl_1.p016.
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Galecin-3 (Gal-3) is a novel beta-galactoside-binding lectin implicated in pro-fibrotic pathways that are upregulated during cardiac remodeling. Gal-3 has been associated with poor prognosis among patients with heart failure and with development of heart failure after acute coronary syndrome. In this study we sought to ascertain: 1) the cross-sectional demographic, behavioral and clinical correlates of serum levels of Gal-3, and 2) its independent predictive value for incident heart failure among 1,312 participants in The Atherosclerotic Disease, VAscular FunctioN and GenetiC Epidemiology (ADVANCE) Study whose initial presentation of coronary artery disease (CAD) between 10/2001 and 12/2003 was acute myocardial infarction (n=871; mean ± SD age; 62 ± 8 years; 23% female) or stable angina (n=441; mean ± SD age; 61 ± 8 years ; 34% female). Gal-3 was measured in frozen serum collected at the baseline examination (2002 to 2004) with the ARCHITECT assay (Abbott Diagnostics, Abbott Park, IL). Follow-up for incident heart failure using ICD-9 hospital principal discharge diagnostic codes 428, 402, 404 and 398 through 12/31/2012 resulted in 74 events (63 among AMI patients, 11 among stable angina patients). The mean ± SD Gal-3 concentration was 16.9 ± 5.3 ng/mL among AMI patients and 16.1 ± 4.4 ng/mL among stable angina patients. The significant independent correlates of Gal-3 were CAD presentation, age, gender, physical activity and estimated glomerular filtration rate (eGFR). In Cox regression with adjustment for age, gender, race and CAD presentation, each 1 SD increment of Gal-3 was associated with 1.64-fold increased hazard of heart failure (95% CI, 1.40 to 1.93; p<0.0001). After further adjustment for smoking status, activity level, alcohol intake, body mass index, LDL-C, HDL-C, triglycerides, diabetes, hypertension, hs-CRP and e-GFR, Gal-3 remained associated with incident heart failure (HR=1.44; 95% CI, 1.18 to 1.75; p=0.0003). Also in fully-adjusted analysis, and relative to quartile 1, the HRs were 1.10 (95% CI, 0.45 to 2.86) for quartile 2, 1.29 (95% CI, 0.53 to 3.11) for quartile 3 and 2.39 (95% CI, 1.06 to 5.37) for quartile 4 of Gal-3. Our results support the value of Gal-3 as a biomarker of heart failure development among patients with pre-existing CAD.
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Ali Sheikh, M. S., A. Alduraywish, A. Almaeen, U. Salma, L. Fei, X. Ke, and T. Yang. "The clinical impact of serum sLOX-1 level in coronary artery disease patients as inferred from its implication in the in vitro protective effects of metoprolol against hypoxic injury of HUVECs." European Heart Journal 41, Supplement_2 (November 1, 2020). http://dx.doi.org/10.1093/ehjci/ehaa946.1388.
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Abstract Background Ischemic coronary artery disease (CAD) is a major public health problem across the world. Early detection and appropriate management significantly reduced CAD-induced morbidities and death. Endothelial cells are pathogenically implicated. Purpose Our study was designed to investigate the role of the soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) in the in vitro protective effect of Metoprolol against hypoxia-induced injury of Human umbilical vein endothelial cells (HUVECs). Secondly, the clinical significance of variations in serum levels of sLOX-1 in patients with CAD was assessed. Methods In vitro, hypoxic injury model of HUVECs was established in an atmosphere of 1% O2, 95% N2, and 5% CO2 for 24 hours. The protective effect and mechanism of action of the cardio-selective beta-blocker Metoprolol at 10–6 μM concentration was investigated. Consented stable atherosclerotic CAD (n=150) and unstable angina pectoris patients (n=75) along with 150 healthy volunteer subjects were voluntarily enrolled in this ethically approved study. Invasive coronary angiogram with ≥50% stenosis at least in one major coronary artery was used for diagnosis. ESC/ACC/AHC/practical protocols were used for categorizing patients into stable or unstable CAD. Serum sLOX-1 level was measured using specific ELISA kit. The diagnostic significance of serum sLOX-1 levels was assessed by analyzing its area under the curve (AUC). Results In vitro hypoxic conditions induced high rate of cellular apoptosis, high levels of LOX-1 expression, reactive oxygen species (ROS) generation and LDH release from HUVECs after 24 hours incubation, compared to normoxic control cells. Metoprolol significantly decreased LOX-1 levels, and prevented the release of LDH and generation of ROS. This culminated into marked improvement in cellular viability of hypoxia-exposed HUVECs (p<0.001). Compared to healthy subjects, serum levels of sLOX-1s were significantly elevated in atherosclerotic stable and unstable CAD patients (p<0.001). Serum sLOX-1 levels were increased by 4.21 folds in stable CAD patients and by 6.373 folds in atherosclerotic unstable angina patients vs. healthy participants. Moreover, the levels in the two patients' groups were significantly different (p<0.001). In stable angina CAD patients, sLOX-1 AUC = 0.929; and in unstable CAD patients, AUC = 0.944, indicating that serum sLOX-1 levels clearly differentiated patients from healthy participants with high specificity and sensitivity. Conclusions Extrapolated from HUVECs hypoxia-induced injury model and the protective effect of Metoprolol, elevation of the circulating levels of sLOX-1 correlated with increased risks for atherosclerotic CAD and is a highly sensitive and specific biomarker for early detection of the disease. Funding Acknowledgement Type of funding source: None