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Background: Diabetes mellitus is an endocrine disorder characterized by a state of hyperglycemia. It is caused by defect in the insulin secretion, insulin action or even both. Long-term complications of diabetes are many as nephropathy, retinopathy and neuropathy. Diabetes mellitus is considered the most common cause of renal failure worldwide. Diabetes results in many histological changes in the kidney. Most cell types of the kidney as podocytes, tubular cells and mesangial cells affect in diabetes.The most characteristic change in diabetic nephropathy (DN) is glomerular alteration as glomerular expansion and thickened glomerular basement membrane.Many alterations of the respiratory functions aredetected in diabetic patients. Several characteristic histological changes had been described in the lung of diabetic animals. Diabetic lung is characterized with increased connective tissue at the expense of alveolar air spaces.This review focuses on histological changes on the kidney and lung of diabetic animal model. Conclusion: Kidney and lung are target organs for diabetes. DN is one of common chronic complications of diabetes. DN is the most common indication for renal replacement therapy worldwide. Several histopathological changes were reported in lung of diabetic animals. It is essential to provide novel therapeutic strategies that could prevent complication of diabetes on kidney and lung.

Introduction: Bisphenol A (BPA), a well-known industrial chemical, has adverse effects on the brain even at relatively low exposure levels in rodents, primates and humans. Omega-3-fatty acids participate in a number of neuronal processes including neurogenesis, neuron differentiation, and neuro-protection. Aim of the work: To investigate the possible protective action of omega-3-fatty acids on the injurious histological effect induced by Bisphenol A on rat hippocampus. Materials and Methods: Eighteen adult male Wistar rats were divided into 3 equal groups; control, BPA (1.2mg/kg daily for 3 weeks, intraperitoneally) and the third group were given omega-3 (300mg/kg orally) in addition to BPA in the aforementioned dose and duration. Hippocampus sections were processed for hematoxylin and eosin staining, GFAB staining and electron microscopic examination. Results: BBPA administration resulted into several histological alterations. Glial fibrillary acidic protein-positive cells were more abundant in the hippocampus of BPA-treated animals compared with the control animals. Ultra-structurally, the hippocampus of BPA-treated group showed nerve cells having nuclei with irregular outline and dilated perinuclear envelop, dilated RER and Golgi, swollen mitochondria with destroyed cristae. Some of the myelinated and unmyelinated nerve fibers showed degenerative changes. Concomitant administration of omega-3-fatty acids ameliorated these effects. Conclusions: OOmega-3-fatty acids partially minimized the severity of BPA- induced hippocampus injurious histological effects in Wistar rat. [ABSTRACT FROM AUTHOR]

Introduction: Titanium dioxide nanoparticles (Tio2 NPs) are manufactured worldwide in large quantities for use in a wide range of applications. TiO2 NPs proved to damage liver function and induce an oxidative stress attack leading to liver toxicity. Milk thistle is an herbal supplement used to treat liver and biliary disorders. Silymarin, an active ingredient of milk thistle, is a strong antioxidant that promotes liver cell regeneration and stabilizes cell membranes. Aim: To investigate the biochemical, the histological and the histochemical changes in the liver after administration of different doses of Tio2 NPs and to evaluate the possible protective role of Milk thistle against these effects. Materials and Methods: Fifty adult male rats were divided into five groups; group I control, groups IIa and IIIa injected IP by 100mg/kg and 150mg/kg TiO2 for 2 weeks, respectively and groups IIb and 111b treated by oral milk thistle 4 weeks; one week prior, 2 weeks concomitant with IP 100mg/kg and 150mg/kg TiO2, respectively and the forth week after injection. Thereafter, rats were sacrificed and liver as well as blood samples were collected for estimation of serum alanine aminotransferase (ALT) and alkaline phosphatase (ALPs). Liver samples were processed for examination by TEM and immunohistochemical staining with P53 and PCNA antibodies counted statistically positive cells. Results: There was an increase in ALT and ALPs activities. Groups 11a and 111a treated by TiO2 showed signs of apoptosis and degeneration in the hepatocytes with nuclear changes and a significant increase in P53 and PCNA antibodies positive cells. These changes were ameliorated by concomitant injection with milk thistle with TiO2 in groups IIb and IIIb. Conclusion: The alterations observed might be an indication of hepatocyte injury due to TiO2 NPs toxicity that interacts with proteins and enzymes in hepatic tissue leading to generation of reactive oxygen species induce hepatocytes apoptosis. Milk thistle, has a hepatoprotective effect probably by its antioxidant effect. [ABSTRACT FROM AUTHOR]

Liver sinusoids are lined by fenestrated endothelial cells surrounded by perisinusoidal cells, Kupffer cells, and pit cells, as well as large granular lymphocytes. The functional ability of the liver cells can be substantially modified by exposure to toxins. In the current work, we assessed the histopathological and ultrastructural effects of a time-course exposure to aflatoxin B1 (AFB1) on the hepatic structures of rats. A total of 30 adult female Wistar rats were randomly divided into three groups: a control group, a group orally administered 250 µg/kg body weight/day of AFB1 for 5 days/week over 4 weeks, and a group that received the same AFB1 treatment but over 8 weeks. Histopathological and ultrastructural examinations of hepatocytes revealed massive vacuolar degeneration and signs of necrosis. Furthermore, the rat liver of the treated group exhibited damage to the sinusoidal endothelium, invasion of the space of Disse with hyperactive Kupffer cells, and some immune cells, as well as Ito cells overloaded with lipids. In addition, damaged telocytes were observed. Taken together, our results indicate that AFB1 induces irreversible adverse effects on the livers of rats.

Dichlorodiphenyltrichloroethane (DDT), a persistent organochlorine pesticide, has been linked to adverse biological effects in organisms. However, there is limited knowledge about its toxic effects on marine organisms and the underlying molecular mechanisms. This study investigated the toxic effects of DDT in the hooded oyster Saccostrea cucullata. The oysters were exposed to DDT at concentrations of 0, 10, 50, 100, 500, 1000 and 2000 µg/L for 96 h and the LC 50 (96 h) was 891.25 µg/L. Two sublethal concentrations (10 and 100 µg/L) were used to investigate the histopathological effects and the proteome response. Histopathological results showed that DDT caused the alteration of mantle tissue. This included the induction of mucocytes in the epithelium and the inflammatory effect in the connective tissue indicated by the enlargement of blood sinus and hemocyte aggregation within the sinus. Proteomic results showed that, amongst approximately 500 protein spots that were detected across 2DE gels, 51 protein spots were differentially expressed (P < 0.01; fold change > 1.2). Of these, 29 protein spots were identified by LC-MS/MS. These included 23 up-regulated, 5 down-regulated and 1 fluctuating spots. Thus, we observed that stress response and cytoskeletal proteins are the central targets of DDT action. Furthermore, DDT alters the expression of proteins involved in energy metabolism, calcium homeostasis and other proteins of unknown function. Additionally, proteomic results clearly elucidated the molecular response of the histopathological changes which were driven by the alteration of cytoskeletal proteins. Our results improve the current knowledge of toxicity of the DDT to histology and molecular response of oyster proteome to DDT exposure. In addition, histopathological changes will be beneficial for the development of an appropriate guideline for health assessment of this species in ecotoxicological context., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Vitamin B 3 (nicotinic acid, nicotinamide) is an essential water-soluble vitamin and cellular energy metabolism depends on the vitamin B 3 -derived cofactors. Inaccessible covalently-linked nicotinic acid in food such as maize can cause vitamin B 3 deficiency in animals since maize is also deficient in tryptophan, the precursor of nicotinic acid. A sensitive and reproducible GC-FID-based method for the quantification of the sum of the three forms of vitamin B 3 from animal liver was developed. Free nicotinic acid, free nicotinamide and nicotinamide moiety of NAD + /NADP + (and their riboside precursors) were simultaneously derivatized as methyl nicotinate. Reaction time and temperature and the extraction procedure for methyl nicotinate were optimized. Starting from wild boar liver, removal of proteins, solvent exchange, derivatization, and chloroform extraction resulted in sufficient enrichment and baseline separation of methyl nicotinate. The within-laboratory reproducibility of the full procedure was determined with RSD <10%. On-column limit of detection and lower limit of quantification for methyl nicotinate were both sub-picomole. The accuracy of the method was determined from the recoveries of the pre-extraction spiked-in vitamin B 3 standards. The overall recovery for the full procedure was 16% but very consistent (RSD = 7%), enabling determination of apparent vitamin B 3 concentrations for relative quantitative comparison., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

In recent years, thanks to advances in Mass Spectrometry (MS)-based quantitative proteomics, studies on signaling pathways have moved from a detailed description of individual components to system-wide analysis of entire signaling cascades, also providing spatio-temporal views of intracellular pathways. Quantitative proteomics that combines stable isotope labeling by amino acid in cell culture (SILAC) with enrichment strategies for post-translational modification-bearing peptides and high-performance tandem mass spectrometry represents a powerful and unbiased approach to monitor dynamic signaling events. Here we provide an optimized SILAC-based proteomic workflow to analyze temporal changes in phosphoproteomes, which involve a generic three step enrichment protocol for phosphopeptides. SILAC-labeled peptides from digested whole cell lysates are as a first step enriched for phosphorylated tyrosines by immunoaffinity and then further enriched for phosphorylated serine/threonine peptides by strong cation exchange in combination with titanium dioxide-beads chromatography. Analysis of enriched peptides on Orbitrap-based MS results in comprehensive and accurate reconstruction of temporal changes of signaling networks.

Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A, and 2B, which may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated with drug resistance or sensitivity, and drug transportation. The NetworKIN algorithm predicted the protein kinases CK2a, CDK1, PLK1, and ATM as likely candidates involved in the hyperphosphorylation of the topoisomerases. Up-regulation of protein and/or phosphorylation levels of topoisomerases in TIMP-1 high expressing cells may be part of the mechanisms by which TIMP-1 confers resistance to treatment with the widely used topoisomerase inhibitors in breast and colorectal cancer.

Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resolution tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissociation (CID) and used these spectra to devise a quantitative filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissociation (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements observed above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.

To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosaccharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

An activity-based isotope-coded affinity tagging (AB-ICAT) strategy for proteome-wide quantitation of active retaining endoglycosidases has been developed. Two pairs of biotinylated, cleavable, AB-ICAT reagents (light H(8) and heavy D(8)) have been synthesized, one incorporating a recognition element for cellulases and the other incorporating a recognition element for xylanases. The accuracy of the AB-ICAT methodology in quantifying relative glycosidase expression/activity levels in any two samples of interest has been verified using several pairs of model enzyme mixtures where one or more enzyme amounts and/or activities were varied. The methodology has been applied to the biomass-degrading secretomes of the soil bacterium, Cellulomonas fimi, under induction by different polyglycan growth substrates to obtain a quantitative profile of the relative expression/activity levels of individual active retaining endoglycanases per C. fimi cell. Such biological profiles are valuable in understanding the strategies employed by biomass-degrading organisms in exploiting environments containing different biomass polysaccharides. This is the first report on the application of an activity-based ICAT method to a biological system.

Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.

The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 A crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-His-Asp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc)(2) for activity and proceeds through a tetrahedral oxyanion intermediate.

Glycosynthases are synthetic enzymes derived from retaining glycosidases in which the catalytic nucleophile has been replaced. The mutation allows irreversible glycosylation of sugar acceptors using glycosyl fluoride donors to afford oligosaccharides without any enzymatic hydrolysis. Glycosynthase technology has proven fruitful for the facile synthesis of useful oligosaccharides, therefore the expansion of the glycosynthase repertoire is of the utmost importance. Herein, we describe for the first time a glycosynthase, derived from a retaining xylanase, that synthesizes a range of xylo-oligosaccharides. The catalytic domain of the retaining endo-1,4-beta-xylanase from Cellulomonas fimi (CFXcd) was successfully converted to the corresponding glycosynthase by mutation of the catalytic nucleophile to a glycine residue. The mutant enzyme (CFXcd-E235G) was found to catalyze the transfer of a xylobiosyl moiety from alpha-xylobiosyl fluoride to either p-nitrophenyl beta-xylobioside or benzylthio beta-xylobioside to afford oligosaccharides ranging in length from tetra- to dodecasaccharides. These products were purified by high performance liquid chromatography in greater than 60% combined yield. 1H and 13C NMR spectroscopic analyses of the isolated p-nitrophenyl xylotetraoside and p-nitrophenyl xylohexaoside revealed that CFXcd-E235G catalyzes both the regio- and stereo-selective synthesis of xylo-oligosaccharides containing, exclusively, beta-(1 --> 4) linkages.

New functional proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Glycosidases play critical roles in biology, yet a robust method for functional analysis of their activities and identities in biological proteomes is still lacking. An aryl 2-deoxy-2-fluoro xylobioside inactivator was conjugated through cleavable and noncleavable linker arms to a biotin tag, thereby yielding two new active-site-directed reagents for activity-based profiling of retaining beta-glycanases in complex proteomes. Crucially, these tagged reagents possess high specificity for their target enzymes with kinetic parameters similar to those of the untagged reagent. Western blotting showed that these reagents bind and covalently label active retaining beta-glycanases both in pure enzyme samples and in the secreted proteome of the soil bacterium Cellulomonas fimi. Such reagents therefore show great promise for future activity-based targeting of glycanases.

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.

The endo-type chitin deacetylase (EC 3.5.1.41) from a deuteromycete, Colletotrichum lindemuthianum (ATCC 56676), catalyses the hydrolysis of the acetamido group of GlcNAc (2-acetamido-2-deoxy-D-glucose) residues in chitin or chito-oligosaccharides with a degree of polymerization (n) equal to or greater than 2. The steady-state kinetic parameters for the initial deacetylation reactions of (GlcNAc)(2-6) were determined using a direct, continuous spectrophotometric assay in combination with ESI-MS (electrospray ionization MS) analysis of the products. The dependence of the observed K(m) and k(cat)/K(m) on n suggests the presence of four enzyme subsites (-2, -1, 0 and +1) that interact with GlcNAc residues from the non-reducing end to the reducing end of the substrate. The turnover number (k (cat), 7 s(-1)) is independent of n and represents the intrinsic rate constant (k(int)) for the hydrolysis of the acetamido group in subsite 0. The subsite affinities for the GlcNAc residues were calculated from the observed k(cat)/K(m) values (A (-2), -11.0; A (-1), -1.5; A (0), -7.7; A (+1), -12.5 kJ x mol(-1)). The increments in the subsite affinities due to the recognition of the acetamido groups were calculated [DeltaDelta G ((N-acetyl))=3.3, 0, 4.0 and 0 kJ x mol(-1) for subsites -2, -1, 0 and +1 respectively]. The steady-state kinetic parameters for the second deacetylation reaction of (GlcNAc)(4) were also determined using (GlcNAcGlcNAcGlcNGlcNAc) as the substrate. The comparison of the experimental and theoretical values (calculated using the subsite affinities) suggests that the mono-deacetylated substrate binds strongly in a non-productive mode occupying all four subsites, thereby inhibiting the second deacetylation reaction.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.