Artículos de revistas sobre el tema "Complexes de type galactose oxydase"
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1
Ramkumar, R., A. Surolia y S. K. Podder. "Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin". Biochemical Journal 308, n.º 1 (15 de mayo de 1995): 237–41. http://dx.doi.org/10.1042/bj3080237.
Resumen
The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.
2
Hatakeyama, Tomomitsu, Takuro Kamiya, Masami Kusunoki, Sachiko Nakamura-Tsuruta, Jun Hirabayashi, Shuichiro Goda y Hideaki Unno. "Galactose Recognition by a Tetrameric C-type Lectin, CEL-IV, Containing the EPN Carbohydrate Recognition Motif". Journal of Biological Chemistry 286, n.º 12 (19 de enero de 2011): 10305–15. http://dx.doi.org/10.1074/jbc.m110.200576.
Resumen
CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1–3/4(Fucα1–3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.
3
Holzer, Isabelle, Oksana Desiatkina, Nicoleta Anghel, Serena K. Johns, Ghalia Boubaker, Andrew Hemphill, Julien Furrer y Emilia Păunescu. "Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates". Molecules 28, n.º 2 (16 de enero de 2023): 902. http://dx.doi.org/10.3390/molecules28020902.
Resumen
Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC ‘click’ (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing β-galactosidase (T. gondii β-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii β-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.
4
Lyons, Christopher T. y T. Daniel P. Stack. "Recent advances in phenoxyl radical complexes of salen-type ligands as mixed-valent galactose oxidase models". Coordination Chemistry Reviews 257, n.º 2 (enero de 2013): 528–40. http://dx.doi.org/10.1016/j.ccr.2012.06.003.
5
Shimazaki, Yuichi. "Properties of the one-electron oxidized copper(II) salen-type complexes: relationship between electronic structures and reactivities". Pure and Applied Chemistry 86, n.º 2 (1 de febrero de 2014): 163–72. http://dx.doi.org/10.1515/pac-2014-5022.
Resumen
Abstract The Cu(II)-phenoxyl radical formed during the catalytic cycle of galactose oxidase (GO) attracted much attention, and the structures and properties of a number of metal-phenoxyl radical complexes have been studied. Some of the functional model systems of GO reported previously have shown that the Cu complexes oxidize primary alcohols to aldehydes and that the Cu(II)-phenoxyl radical species is formed in the catalytic cycle. Many Cu(II)-phenoxyl radical species have been produced by one-electron oxidation of the Cu(II)-phenolate complexes. On the other hand, one-electron oxidation of a Cu(II)-phenolate complex has the possibility to give different electronic structures, one of which is the Cu(III)-phenolate. From these points of view, this micro review is focused on the one-electron oxidized square-planar Cu(II) complexes of the salen-type ligands. Introduction of substituents into the phenolate moieties and conversion from a 5- to a 6-membered chelate backbone alter the electronic structure of the one-electron oxidized Cu(II) complexes and give rise to a different reactivity of benzyl alcohol oxidation. The relationship between the electronic structure and the reactivity is herein discussed.
6
Kovalchuk, Svetlana, Nina Buinovskaya, Galina Likhatskaya, Valery Rasskazov, Oksana Son, Liudmila Tekutyeva y Larissa Balabanova. "Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus". Marine Drugs 16, n.º 12 (27 de noviembre de 2018): 471. http://dx.doi.org/10.3390/md16120471.
Resumen
The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.
7
Wold, J. K., H. S. Slayter, J. F. Codington y R. W. Jeanloz. "Location of an epitopic site on epiglycanin by molecular immunoelectron microscopy". Biochemical Journal 227, n.º 1 (1 de abril de 1985): 231–37. http://dx.doi.org/10.1042/bj2270231.
Resumen
Antibodies of the IgM type present in rabbit anti-epiglycanin antiserum were purified by (NH4)2SO4 precipitation and by ion-exchange, affinity and gel-filtration chromatography. After papain treatment of this fraction, followed by gel filtration, the fraction with highest apparent Mr was incubated with epiglycanin, and the antigen-antibody complexes separated by gel filtration. These were examined by electron microscopy, using rotational shadow casting, and the photographs of the complexes were mapped for the locations of the antibody molecules on the extended epiglycanin molecules. Distribution of the frequency of attachment of immunoglobulin showed a strong tendency toward binding at one end of epiglycanin, suggesting the probable presence of only one epitope, probably a glycopeptide structure containing a beta-D-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-D-galactose chain.
8
Mimuro, J., RR Schleef y DJ Loskutoff. "Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor". Blood 70, n.º 3 (1 de septiembre de 1987): 721–28. http://dx.doi.org/10.1182/blood.v70.3.721.721.
Resumen
Abstract The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.
9
Mimuro, J., RR Schleef y DJ Loskutoff. "Extracellular matrix of cultured bovine aortic endothelial cells contains functionally active type 1 plasminogen activator inhibitor". Blood 70, n.º 3 (1 de septiembre de 1987): 721–28. http://dx.doi.org/10.1182/blood.v70.3.721.bloodjournal703721.
Resumen
The extracellular matrix (ECM) of cultured bovine aortic endothelial cells (BAEs) was analyzed by immunoblotting and reverse fibrin autography and shown to contain type 1 plasminogen activator inhibitor (PAI-1). Most PAI-1 in the ECM formed complexes with exogenously added tissue-type plasminogen activator (tPA), demonstrating that this PAI-1 was functionally active. The resulting tPA/PAI-1 complexes were recovered in the reaction solution, indicating that the PAI-1 in such complexes no longer bound to ECM. The PAI-1 could not be removed by incubating ECM in high salt (2 mol/L NaCl), sugars (1 mol/L galactose, 1 mol/L mannose), glycosaminoglycans (10 mmol/L heparin, 10 mmol/L dermatan sulfate), or epsilon-aminocaproic acid (0.1 mol/L). However, PAI-1 could be extracted from ECM by treatment with either arginine (0.5 mol/L) or potassium thiocyanate (2 mol/L), or by incubation under acidic conditions (pH 2.5). ECM depleted of PAI-1 by acid extraction was able to bind both the active and latent forms of PAI-1. In this instance, most of the bound PAI-1 did not form complexes with tPA, indicating that the latent form was not activated as a consequence of binding to ECM. Although the PAI-1 activity in conditioned medium decayed with a half-life (t 1/2) of less than 3 hours, the t 1/2 of ECM- associated PAI-1 was greater than 24 hours. These data suggest that PAI- 1 is produced by cultured BAEs in an active form and is then either released into the medium where it is rapidly inactivated or into the subendothelium where it binds to ECM. The specific binding of PAI-1 to ECM protects it from this inactivation.
10
Rossi, J. M. y S. Lindquist. "The intracellular location of yeast heat-shock protein 26 varies with metabolism." Journal of Cell Biology 108, n.º 2 (1 de febrero de 1989): 425–39. http://dx.doi.org/10.1083/jcb.108.2.425.
Resumen
An antibody highly specific for heat-shock protein (hsp)26, the unique small hsp of yeast, and mutants carrying a deletion of the HSP26 gene were used to examine the physical properties of the protein and to determine its intracellular distribution. The protein was found in complexes with a molecular mass of greater than 500 kD. Thus, it has all of the characteristics, including sequence homology and induction patterns, of small hsps from other organisms. When log-phase cells growing in glucose were heat shocked, hsp26 concentrated in nuclei and continued to concentrate in nuclei when these cells were returned to normal temperatures for recovery. However, hsp26 did not concentrate in nuclei under a variety of other conditions. For example, in early stationary-phase cells hsp26 is induced at normal growth temperatures. This protein was generally distributed throughout the cells, even after heat shock. Similarly, in cells genetically engineered to synthesize hsp26 in the presence of galactose, hsp26 did not concentrate in nuclei, with or without a heat shock. To determine if the failure of hsp26 to concentrate in the nucleus of these cells was due to the fact that the protein had been produced at 25 degrees C or to a difference in the physiological state of the cell, we investigated the distribution of the heat-induced protein in cells grown under several different conditions. In wild-type cells grown in galactose or acetate and in mitochondrial mutants grown in glucose or galactose, hsp26 also failed to concentrate in nuclei with a heat shock. We conclude that the intracellular location of hsp26 in yeast depends upon the physiological state of the cell and not simply upon the presence or absence of heat stress. Our findings may explain why previous investigations of the intracellular localization of small hsps in a variety of organisms have yielded seemingly contradictory results.
11
De Gaetano, Anna, Lara Gibellini, Elena Bianchini, Rebecca Borella, Sara De Biasi, Milena Nasi, Federica Boraldi, Andrea Cossarizza y Marcello Pinti. "Impaired Mitochondrial Morphology and Functionality in Lonp1wt/− Mice". Journal of Clinical Medicine 9, n.º 6 (8 de junio de 2020): 1783. http://dx.doi.org/10.3390/jcm9061783.
Resumen
LONP1 is a nuclear-encoded mitochondrial protease crucial for organelle homeostasis; mutations of LONP1 have been associated with Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome. To clarify the role of LONP1 in vivo, we generated a mouse model in which Lonp1 was ablated. The homozygous Lonp−/− mouse was not vital, while the heterozygous Lonp1wt/− showed similar growth rate, weight, length, life-span and histologic features as wild type. Conversely, ultrastructural analysis of heterozygous enterocytes evidenced profound morphological alterations of mitochondria, which appeared increased in number, swollen and larger, with a lower complexity. Embryonic fibroblasts (MEFs) from Lonp1wt/− mice showed a reduced expression of Lonp1 and Tfam, whose expression is regulated by LONP1. Mitochondrial DNA was also reduced, and mitochondria were swollen and larger, albeit at a lesser extent than enterocytes, with a perinuclear distribution. From the functional point of view, mitochondria from heterozygous MEF showed a lower oxygen consumption rate in basal conditions, either in the presence of glucose or galactose, and a reduced expression of mitochondrial complexes than wild type. In conclusion, the presence of one functional copy of the Lonp1 gene leads to impairment of mitochondrial ultrastructure and functions in vivo.
12
Kozak, Sandra, Yehudi Bloch, Steven De Munck, Aleksandra Mikula, Isabel Bento, Savvas N. Savvides y Rob Meijers. "Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis". Acta Crystallographica Section D Structural Biology 76, n.º 12 (24 de noviembre de 2020): 1244–55. http://dx.doi.org/10.1107/s2059798320013753.
Resumen
Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.
13
Chandra, Shilpi, Archana Khurana, William B. Kiosses, James Gray, Kaori Hitomi, Meng Zhao, Stewart K. Richardson et al. "A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation". Journal of Immunology 198, n.º 1_Supplement (1 de mayo de 2017): 146.20. http://dx.doi.org/10.4049/jimmunol.198.supp.146.20.
Resumen
Abstract Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid. Members of the HOPS and ESCRT complexes have been identified to play an important role in Cd1d dependent antigen presentation. Interestingly, our data show that ABCC1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. CD1d and MHC-II antigen presentation pathways both depends on antigen loading in the lysosome, therefore we also analyzed the role of these genes in MHC-II antigen presentation. Although the majority of genes identified doesn’t affect MHC-II antigen presentation, there are a few molecules that affect both antigen presentation pathways.
14
Viscardi, Valeria, Enrico Baroni, Michele Romano, Giovanna Lucchini y Maria Pia Longhese. "Sudden Telomere Lengthening Triggers a Rad53-dependent Checkpoint inSaccharomyces cerevisiae". Molecular Biology of the Cell 14, n.º 8 (agosto de 2003): 3126–43. http://dx.doi.org/10.1091/mbc.e02-11-0719.
Resumen
Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.
15
Qian, Zhong, Bo Meng, Quanhui Wang, Zhuowei Wang, Chuanqi Zhou, Qian Wang, Shuyang Tu, Liang Lin, Yanhe Ma y Siqi Liu. "Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis". Microbiology 155, n.º 5 (1 de mayo de 2009): 1717–25. http://dx.doi.org/10.1099/mic.0.025536-0.
Resumen
On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 °C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.
16
Prandini, M. H., A. Reboul y M. G. Colomb. "Biosynthesis of complement C1 inhibitor by Hep G2 cells. Reactivity of different glycosylated forms of the inhibitor with C1s". Biochemical Journal 237, n.º 1 (1 de julio de 1986): 93–98. http://dx.doi.org/10.1042/bj2370093.
Resumen
The biosynthesis of C1 Inh (C1 inhibitor) was studied in a human hepatoma cell line (Hep G2) by metabolic labelling, immunoprecipitation with anti-(C1 Inh) serum, analysis on SDS/polyacrylamide gel slabs and fluorography. Two forms of C1 Inh are secreted by Hep G2: a minor form of Mr 90,000 and a major form of Mr approximately 100,000. The latter form is also found in small amounts intracellularly in co-existence with an 80,000-Mr form. Accumulation of the 80,000-Mr C1 Inh is favoured when the cells are labelled at 23 degrees C instead of 37 degrees C or when they are treated with monensin. In the presence of tunicamycin, a compound that blocks the formation of N-asparagine-linked oligosaccharide chains, a decrease in Mr of both secreted and intracellular major forms is observed, indicating that secreted and intracellular C1 Inh contain N-linked oligosaccharide units. The 100,000 Mr secreted C1 Inh is sensitive to endoglycosidase F but resistant to endoglycosidase H, and it incorporates [3H]galactose, [3H]glucosamine and [3H]galactosamine, indicating the presence of both N-linked oligosaccharides of the complex type and O-linked oligosaccharides. The intracellular C1 Inh contains N-linked oligosaccharide units of the high-mannose type as demonstrated by endoglycosidase H-sensitivity. The functional activity of C1 Inh during its biosynthesis was tested by studying its reactivity towards C1s. Both secreted and intracellular C1 Inh form covalent-like complexes with purified plasma C1s. The underglycosylated C1 Inh secreted in presence of tunicamycin is still reactive with purified C1s. These results clearly show that sugars are not essential for this inhibitory activity of C1 Inh.
17
Hayes, Patti, Claire Fergus, Magda Ghanim, Cansu Cirzi, Lyubomyr Burtnyak, Callum J. McGrenaghan, Francesca Tuorto, Derek P. Nolan y Vincent P. Kelly. "Queuine Micronutrient Deficiency Promotes Warburg Metabolism and Reversal of the Mitochondrial ATP Synthase in Hela Cells". Nutrients 12, n.º 3 (24 de marzo de 2020): 871. http://dx.doi.org/10.3390/nu12030871.
Resumen
Queuine is a eukaryotic micronutrient, derived exclusively from eubacteria. It is incorporated into both cytosolic and mitochondrial transfer RNA to generate a queuosine nucleotide at position 34 of the anticodon loop. The transfer RNA of primary tumors has been shown to be hypomodified with respect to queuosine, with decreased levels correlating with disease progression and poor patient survival. Here, we assess the impact of queuine deficiency on mitochondrial bioenergetics and substrate metabolism in HeLa cells. Queuine depletion is shown to promote a Warburg type metabolism, characterized by increased aerobic glycolysis and glutaminolysis, concomitant with increased ammonia and lactate production and elevated levels of lactate dehydrogenase activity but in the absence of significant changes to proliferation. In intact cells, queuine deficiency caused an increased rate of mitochondrial proton leak and a decreased rate of ATP synthesis, correlating with an observed reduction in cellular ATP levels. Data from permeabilized cells demonstrated that the activity of individual complexes of the mitochondrial electron transport chain were not affected by the micronutrient. Notably, in queuine free cells that had been adapted to grow in galactose medium, the re-introduction of glucose permitted the mitochondrial F1FO-ATP synthase to operate in the reverse direction, acting to hyperpolarize the mitochondrial membrane potential; a commonly observed but poorly understood cancer trait. Together, our data suggest that queuosine hypomodification is a deliberate and advantageous adaptation of cancer cells to facilitate the metabolic switch between oxidative phosphorylation and aerobic glycolysis.
18
Bakunina, Irina, Lubov Slepchenko, Stanislav Anastyuk, Vladimir Isakov, Galina Likhatskaya, Natalya Kim, Liudmila Tekutyeva, Oksana Son y Larissa Balabanova. "Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants". Marine Drugs 16, n.º 10 (24 de septiembre de 2018): 349. http://dx.doi.org/10.3390/md16100349.
Resumen
A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.
19
Dubel, N. I., L. M. Grytsyk y A. R. Grytsyk. "The study of the polysaccharide composition of the herb of Alchemilla L. genus species growing in the territory of the Precarpathian region". News of Pharmacy 104, n.º 2 (8 de octubre de 2022). http://dx.doi.org/10.24959/nphj.22.93.
Resumen
Medicines of plant origin containing polysaccharides are used in pharmaceutical practice since they exhibit a wide spectrum of thepharmacological activity. Species of the Alchemilla L. genus of the Rosaceae family are of important scientific and practical importance; they contain different groups of biologically active substances (BAS), including phenolic compounds and polysaccharides. The lack of information in the literature on the quantitative content of polysaccharides in this raw material indicates the topicality of research in this direction. Aim. To isolate and study the polysaccharide composition of the herb of the Alchemilla L. genus species growing in the territory of the Precarpathian region. Materials and methods. To isolate polysaccharide fractions and study their monomer composition, we used herb of 6 species of the Alchemilla L. genus (Alchemilla (A.) flabellata Buser., A. subcrenata Buser., A. phegophila Juz., A. microdonta Juz., A. hebescens Juz., A. turkulensis Pawі.) harvested during the mass flowering phase in various areas of the Ivano-Frankivsk region within 2020-2021. The quantitative content of polysaccharide fractions in the raw material studied was determined by the gravimetric method after successive extraction of the raw material with purified water R, hydrochloric acid solution and sodium hydroxide solution, followed by precipitation with 96 % ethanol R. The qualitative monomer composition of polysaccharides was determined by the ascending paper chromatography(PC) and thin-layer chromatography (TLC) in different solvent systems compared to authentic samples of neutral and acidic monosaccharides. Results and discussion. It was found that the total content of polysaccharide fractions in the herb of the Alchemilla species studied ranged from 7.73 % to 15.35 %, depending on the type of Alchemilla species. The yield of water-soluble polysaccharides (WSP) ranged from 2.62 % to 5.49 %, pectin substances (PS) – from 1.41 % to 2.13 %, hemicellulose (HC) A – from 0.45 % to 2.96 % and HC B – from 2.51 % to 6.44 %. The maximum amount of WSP and HC A was observed in the herb of Alchemilla turkulensis Pawł. (5.49 % and 2.96 %, respectively), the highest amount of PS and HC B was detected in the herb of Alchemilla phegophila Juz. (2.13 % and 6.44 %, respectively). The composition of monosaccharides was determined by the methods of PC and TLC compared to authentic samples. Glucose and arabinose were identified in the hydrolysates of the WSP of the herb of the Alchemilla L. genus species. The monomer composition of PS of the raw material studied was represented by glucose, arabinose and galactose. Glucose, galactose and xylose were found in the hydrolyzates of HC A fraction; glucose, galactose, arabinose, xylene, rhamnose, glucuronic and galacturonic acids were identified in HC B fraction. Conclusions. For the first time, polysaccharide complexes have been isolated from 6 species of the Alchemilla L. genus harvested in different areas of the Ivano-Frankivsk region. The monomer composition of sugars has been determinedby the methods of PC and TLC compared to authentic samples of monosaccharides in the hydrolyzates of WSP, PS, HC A and B fractions of species of the Alchemilla L. genus growing in the territory of Precarpathian region. The research results obtained are of practical importance for the further study of the pharmacological activity of the raw material studied and can be used in the development of quality control methods for the medicinal plant raw material and substances obtained from it.
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Roberts, Eleanor. "New Horizons in IgA Nephropathy: A Focus on Current Treatment and Emerging Solutions". European Medical Journal, 14 de diciembre de 2023, 14–23. http://dx.doi.org/10.33590/emj/10303661.
Resumen
IgA nephropathy (IgAN) is a common form of glomerular disease, with wide heterogeneity of symptom occurrence and progression. Diagnosis is based on kidney biopsy findings. IgAN initiates in the mucosa with development of galactose-deficient IgA1 (Gd-IgA1) and anti-Gd-IgA1 autoantibodies, leading to deposition of these complexes in glomerular mesangium with resulting fibrosis, inflammation, tubulointerstitial scarring, and glomerular injury. This can lead to chronic kidney disease (CKD), kidney failure, and death. IgAN treatment involves optimised supportive care, including individualised strategies to address symptoms, such as high blood pressure control and cardiovascular risks. Drug treatment includes renin-angiotensin-aldosterone system (RAAS) inhibitors and immunosuppressant therapies. While the latter can successfully lower proteinuria, and have a positive effect on estimated glomerular filtration rate (eGFR), adverse effects can limit treatment duration, and increasing proteinuria and decreasing eGFR can return following treatment discontinuation. New formulations of immunosuppressant therapies include delayed-release budesonide with targeted release in the lower part of the small intestine where Gd-IgA1 production occurs. Although treatment with this drug can reduce proteinuria and sustain eGFR levels, similar to other immunosuppressant therapies, effects seem to be predominantly limited to the active treatment period. Targeting a different mechanism, sparsentan is a dual endothelin A receptor (ETA) and angiotensin II receptor type 1 (AT1) blocker that targets endothelin-1 (ET-1) and angiotensin II, both involved in IgAN progression. Initial Phase III trial results show significant differences, favouring sparsentan, compared with the AT1 blocker irbesartan, on proteinuria, with similar adverse event profiles. These agents, and several other drugs in development, will widen the armamentarium of therapies for people with IgAN, which, when used in combination, can target different aspects of IgAN pathogenesis for a more individualised treatment approach.
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Cervantes, Vanessa y Editte Gharakhanian. "Investigating Env9's Mechanism of Membrane Insertion in Saccharomyces cerevisiae". FASEB Journal 30, S1 (abril de 2016). http://dx.doi.org/10.1096/fasebj.30.1_supplement.lb50.
Resumen
Tail‐anchored proteins have vital roles within the cell such as vesicular transport and apoptosis, and often times their specific membrane localization is crucial to their function. These proteins have hydrophobic regions near their C‐terminus, making them identifiable to protein insertion machinery. One pathway which targets C‐tail anchored proteins is the well‐characterized Guided Entry of Tail‐Anchored Protein (GET) complex, consisting of Get1‐5 and Sgt2. Interestingly, a second mechanism has been proposed named the SRP‐independent (SND) pathway which similarly targets tail‐anchored proteins. Our protein of interest, Env9, is one of four novel endosome to vacuole interface (ENV) gene products and our data suggests it possesses a hydrophobic region near its C‐terminus. Env9 localizes to lipid droplet membranes however, its insertion mechanism is unknown. We hypothesize Env9 is targeted to membranes by either GET or SND pathways because of both its c‐terminal hydrophobic domain and its interactions with components of both complexes. Negative genetic interactions with both ENV8 (GET4) and with ENV10 (SND2) have been reported in systematic studies and have been observed in our laboratory. In order to investigate these interactions, we used both microscopic and biochemical approaches. Env9 was tagged with either GFP (green fluorescent protein) under an inducible galactose promoter or HA (hemagglutinin) under a constitutive PGK promoter to analyze localization in GET and SND mutants via confocal microscopy and western blot analysis, respectively. Preliminary microscopy results show slight disruption in GET mutants whereas SND mutants appear to have wild‐type Env9 localization patterns. In preliminary western blot analysis, tagged double mutants Δget4/env9 and Δsnd2/env9 continue to localize Env9‐HA to the P13 pellet that includes lipid droplets, but also vacuoles and aggregated protein complexes. These results suggest Env9 may be targeted to membranes via the GET complex and specifically implicate the GET complex in the process of insertion of Env9 into the ER membrane. These moderate phenotypes may also suggest compensation by one complex in the absence of the other in order to accurately target vital C‐tail anchored proteins. Additionally, the compensatory effect may be further stimulated by the highly overexpressed constructs used in these studies. We are currently exploring the insertion of Env9 expressed at native levels. Additionally, we are examining Env9 localization in GET double and triple mutants, as well as determining protein‐protein interactions via other biochemical approaches such as co‐immunoprecipitation.Support or Funding InformationAcknowledgements: This project was supported by NIH AREA #R‐15 GM85794‐02, NSF‐MRI grant #DBI0722757 for confocal microscopy, and NIH 2R25GM071638‐09A1 for the Research Initiative for Scientific Advancement Fellowship funding. We'd like to thank Dr. Maya Schuldiner at the Weizmann Institute of Science for her donations of the GET and SND mutants.
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Post, Tomas, Yutaka Negishi, Laura Wingens, Elmar Pieterse, Ilse Rood, Luuk Hilbrands, Musa Mhlanga, Klaas Mulder, Nils Rother y Raphael Duivenvoorden. "#4458 SINGLE CELL RNA SEQUENCING OF CIRCULATING LEUKOCYTES FROM IGA NEPHROPATHY PATIENTS IDENTIFIES AN IMPORTANT ROLE FOR INNATE IMMUNE CELLS". Nephrology Dialysis Transplantation 38, Supplement_1 (junio de 2023). http://dx.doi.org/10.1093/ndt/gfad063c_4458.
Resumen
Abstract Background and Aims IgA-nephropathy (IgAN) is a slowly progressive disease characterized by mesangial deposition of IgA1 that causes kidney injury and can eventually lead to end stage renal disease. A key factor underlying the development of IgAN is the presence of galactose-deficient IgA1 (Gd-IgA1) in the circulation. The formation of IgA and IgG antibodies against Gd-IgA1 results in the formation of immune complexes which deposit in the kidney mesangium. Production of Gd-IgA1 hints to defects in B cell regulation. However, the exact immunological dysregulation leading to the formation of Gd-IgA1, and the subsequent antibody formation against it, remains elusive. Here we use single cell RNA sequencing (scRNA-seq) of circulating leukocytes for in depth analysis of immune cell populations in patients with active IgAN to gain a better understanding of the pathogenesis of this disease. Method Peripheral blood mononuclear cells were isolated for scRNA-seq from the blood of four patients with active IgAN, and four controls, matched for age and estimated glomerular filtration rate (eGFR). Cell hashing (Biolegend) of individual samples was performed before pooling samples for scRNA-seq. scRNA expression libraries were performed using the chromium X machine (10xgenomics) according to 10XGenomics single cell 5’ V2 user guide. Libraries were sequenced on an Illumina Nextseq500 and reads were mapped to GrCh38 using CellRanger. Differential gene expression analysis was performed for each immune cell type (FDR < 0.05). Reactome pathway enrichment analysis was performed on the differentially expressed genes. Results Identification of immune cell subtypes in IgAN patients and matched controls did not show a significant difference in cell numbers or ratios. B cells showed only minor changes in gene expression between IgAN patients and controls, and T cell populations showed changes in a limited set of genes, whereas interestingly, most differentially expressed genes were found in monocytes and NK cells (Figure 1A). Analysis of the differentially expressed genes showed enrichment for genes in interferon signaling pathways, mostly in monocytes and NK cells (Figure 1B). Gene enrichment analysis showed differential expression of type I IFN genes and antigen presentation genes, also mainly in monocytes and NK cells. Conclusion Our data shows the specific endotype of circulating immune cells from patients with active IgAN. While gene expression changes in T and B cells are discrete, changes in monocytes and NK cells appear especially prominent. In these cell types, we observed an important role for interferon signaling. Further analysis of scRNA-seq data in a larger patient cohort will follow, as well as analysis of T and B cell receptor clones in IgAN patients, with which we aim to gain new insights into immune dysregulation in IgAN.