Extracellular histones: a unifying mechanism driving platelet-dependent extracellular vesicle release and thrombus formation in COVID-19.

BACKGROUND: COVID-19 can cause profound inflammation and coagulopathy, and while many mechanisms have been proposed, there is no known common pathway leading to a prothrombotic state. OBJECTIVES: From the beginning of the COVID-19 pandemic, elevated levels of extracellular histones have been found in plasma of patients infected with SARS-CoV-2. We hypothesized that platelet activation triggered by extracellular histones might represent a unifying mechanism leading to increased thrombin generation and thrombosis. METHODS: We utilized blood samples collected from an early clinical trial of hospitalized COVID-19 patients (NCT04360824) and recruited healthy subjects as controls. Using plasma samples, we measured the procoagulant and prothrombotic potential of circulating extracellular histones and extracellular vesicles (EVs). Platelet prothrombotic activity was assessed via thrombin generation potential and platelet thrombus growth. Circulating EVs were assessed for thrombin generation potential in vitro in plasma and enhancement of thrombotic susceptibility in vivo in mice. RESULTS: Compared with controls, COVID-19 patients had elevated plasma levels of citrullinated histone H3, cell-free DNA, nucleosomes, and EVs. Plasma from COVID-19 patients promoted platelet activation, platelet-dependent thrombin generation, thrombus growth under venous shear stress, and release of platelet-derived EVs. These prothrombotic effects of COVID-19 plasma were inhibited by an RNA aptamer that neutralizes both free and DNA-bound histones. EVs isolated from COVID-19 plasma enhanced thrombin generation in vitro and potentiated venous thrombosis in mice in vivo. CONCLUSION: We conclude that extracellular histones and procoagulant EVs drive the prothrombotic state in COVID-19 and that histone-targeted therapy may prove beneficial.

Endogenous SOD2 (Superoxide Dismutase) Regulates Platelet-Dependent Thrombin Generation and Thrombosis During Aging.

BACKGROUND: Reactive oxygen species (ROS) contribute to platelet hyperactivation during aging. Several oxidative pathways and antioxidant enzymes have been implicated; however, their mechanistic contributions during aging remain elusive. We hypothesized that mitochondria are an important source of platelet ROS and that mitochondrial SOD2 (superoxide dismutase) protects against mitochondrial ROS-driven platelet activation and thrombosis during aging. METHODS: We studied littermates of platelet-specific SOD2-knockout (SOD2fl/flPf4Cre, pSOD2-KO) and control (SOD2fl/fl) mice at young (4-5 months) or old (18-20 months) ages. We examined agonist-induced platelet activation, platelet-dependent thrombin generation potential, and susceptibility to in vivo thrombosis. RESULTS: Platelet αIIbß3 activation, aggregation, and adhesion were increased to similar extents in aged mice of both genotypes compared with young mice. In contrast, the age-dependent increases in mitochondrial and total cellular ROS, calcium elevation, and phosphatidylserine exposure were augmented in platelets from pSOD2-KO mice compared with control mice. Aged pSOD2-KO mice showed increased platelet-dependent thrombin generation compared with aged control mice. In vivo, aged pSOD2-KO mice exhibited enhanced susceptibility to carotid artery and pulmonary thrombosis compared to aged control mice. Adoptive transfer of platelets from aged pSOD2-KO but not aged control mice increased thrombotic susceptibility in aged host mice, suggesting a prothrombotic effect of platelet pSOD2 deficiency. Treatment with avasopasem manganese (GC4419), a SOD mimetic, decreased platelet mitochondrial pro-oxidants, cellular ROS levels, and inhibited procoagulant platelet formation and arterial thrombosis in aged mice. CONCLUSIONS: Platelet mitochondrial ROS contributes to age-related thrombosis and endogenous SOD2 protects from platelet-dependent thrombin generation and thrombosis during aging.

Review of the endocrine organ-like tumor hypothesis of cancer cachexia in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of solid tumors, associated with a high prevalence of cachexia (~80%). PDAC-derived cachexia (PDAC-CC) is a systemic disease involving the complex interplay between the tumor and multiple organs. The endocrine organ-like tumor (EOLT) hypothesis may explain the systemic crosstalk underlying the deleterious homeostatic shifts that occur in PDAC-CC. Several studies have reported a markedly heterogeneous collection of cachectic mediators, signaling mechanisms, and metabolic pathways, including exocrine pancreatic insufficiency, hormonal disturbance, pro-inflammatory cytokine storm, digestive and tumor-derived factors, and PDAC progression. The complexities of PDAC-CC necessitate a careful review of recent literature summarizing cachectic mediators, corresponding metabolic functions, and the collateral impacts on wasting organs. The EOLT hypothesis suggests that metabolites, genetic instability, and epigenetic changes (microRNAs) are involved in cachexia development. Both tumors and host tissues can secrete multiple cachectic factors (beyond only inflammatory mediators). Some regulatory molecules, metabolites, and microRNAs are tissue-specific, resulting in insufficient energy production to support tumor/cachexia development. Due to these complexities, changes in a single factor can trigger bi-directional feedback circuits that exacerbate PDAC and result in the development of irreversible cachexia. We provide an integrated review based on 267 papers and 20 clinical trials from PubMed and ClinicalTrials.gov database proposed under the EOLT hypothesis that may provide a fundamental understanding of cachexia development and response to current treatments.

DNase 1 Protects From Increased Thrombin Generation and Venous Thrombosis During Aging: Cross-Sectional Study in Mice and Humans.

Background Human aging is associated with increased risk of thrombosis, but the mechanisms are poorly defined. We hypothesized that aging induces peroxide-dependent release of neutrophil extracellular traps that contribute to thrombin generation and thrombosis. Methods and Results We studied C57BL6J mice and littermates of glutathione peroxidase-1 transgenic and wild-type mice at young (4 month) and old (20 month) ages and a healthy cohort of young (18-39 years) or middle-aged/older (50-72 years) humans. In plasma, we measured thrombin generation potential and components of neutrophil extracellular traps (cell-free DNA and citrullinated histone). Aged wild-type mice displayed a significant increase in thrombin generation that was decreased in aged glutathione peroxidase-1 transgenic mice. Both aged wild-type and aged glutathione peroxidase-1 transgenic mice demonstrated similar elevation of plasma cell-free DNA compared with young mice. In contrast, plasma levels of citrullinated histone were not altered with age or genotype. Release of neutrophil extracellular traps from neutrophils in vitro was also similar between young and aged wild-type or glutathione peroxidase-1 transgenic mice. Treatment of plasma or mice with DNase 1 decreased age-associated increases in thrombin generation, and DNase 1 treatment blocked the development of experimental venous thrombi in aged C57BL6J mice. Similarly, thrombin generation potential and plasma cell-free DNA, but not citrullinated histone, were higher in middle-aged/older humans, and treatment of plasma with DNase 1 reversed the increase in thrombin generation. Conclusions We conclude that DNase 1 limits thrombin generation and protects from venous thrombosis during aging, likely by hydrolyzing cell-free DNA.

Natural anthraquinone compound emodin as a novel inhibitor of aurora A kinase: A pilot study.

Aurora kinase A (AURKA) carries out an essential role in proliferation and involves in cisplatin resistance in various cancer cells. Overexpression of AURKA is associated with the poor prognosis of cancer patients. Thus, AURKA has been considered as a target for cancer therapy. Developing AURKA inhibitors became an important issue in cancer therapy. A natural compound emodin mainly extracted from rhubarbs possesses anti-cancer properties. However, the effect of emodin on AURKA has never been investigated. In the present study, molecular docking analysis indicated that emodin interacts with AURKA protein active site. We also found nine emodin analogues from Key Organic database by using ChemBioFinder software. Among that, one analogue 8L-902 showed a similar anti-cancer effect as emodin. The bindings of emodin and 8L-902 on AURKA protein were confirmed by cellular thermal shift assay. Furthermore, emodin inhibited the AURKA kinase activity in vitro and enhanced the cisplatin-DNA adduct level in a resistant ovarian cancer cell line. It seems that emodin may have the potential to inhibit cancer cell growth and enhance cisplatin therapy in cancer with resistance. Collectively, our finding reveals a novel AURKA inhibitor, emodin, which may be vulnerable to ovarian cancer therapy in the future.

Potential natural products that target the SARS-CoV-2 spike protein identified by structure-based virtual screening, isothermal titration calorimetry and lentivirus particles pseudotyped (Vpp) infection assay.

BACKGROUND AND AIM: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells through the binding of the viral spike protein with human angiotensin-converting enzyme 2 (ACE2), resulting in the development of coronavirus disease 2019 (COVID-19). To date, few antiviral drugs are available that can effectively block viral infection. This study aimed to identify potential natural products from Taiwan Database of Extracts and Compounds (TDEC) that may prevent the binding of viral spike proteins with human ACE2 proteins. METHODS: The structure-based virtual screening was performed using the AutoDock Vina program within PyRX software, the binding affinities of compounds were verified using isothermal titration calorimetry (ITC), the inhibitions of SARS-CoV-2 viral infection efficacy were examined by lentivirus particles pseudotyped (Vpp) infection assay, and the cell viability was tested by 293T cell in MTT assay. RESULTS AND CONCLUSION: We identified 39 natural products targeting the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in silico. In ITC binding assay, dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone showed dissociation constant (K d) = 0.468 µM, 1.712 µM, 6.650 µM, 2.86 µM, 3.761 µM and 4.27 µM, respectively. In Vpp infection assay, the compounds have significantly and consistently inhibition with the 50-90% inhibition of viral infection efficacy. In cell viability, torvoside K, epimedin, amentoflavone, and saikosaponin C showed IC50 > 100 µM; dioscin and celastrol showed IC50 = 1.5625 µM and 0.9866 µM, respectively. These natural products may bind to the viral spike protein, preventing SARS-CoV-2 from entering cells. SECTION 1: Natural Products. TAXONOMY CLASSIFICATION BY EVISE: SARS-CoV-2, Structure-Based Virtual Screening, Isothermal Titration Calorimetry and Lentivirus Particles Pseudotyped (Vpp) Infection Assay, in silico and in vitro study.

Aurora B kinase: a potential drug target for cancer therapy.

BACKGROUND: Ensuring genetic integrity is essential during the cell cycle to avoid aneuploidy, one of the underlying causes of malignancies. Aurora kinases are serine/threonine kinase that play a vital role in maintaining the genomic integrity of the cells. There are three forms of aurora kinases in the mammalian cells, which are highly conserved and act together with several other proteins to control chromosome alignment and its equal distribution to daughter cells in mitosis and meiosis. METHODS: We provide here a detailed analysis of Aurora B kinase (ABK) in terms of its expression, structure, function, disease association and potential therapeutic implications. RESULTS: ABK plays an instrumental in mitotic entry, chromosome condensation, spindle assembly, cytokinesis, and abscission. Small-molecule inhibitors of ABK are designed and synthesized to control cancer progression. A detailed understanding of ABK pathophysiology in different cancers is of great significance in designing and developing effective therapeutic strategies. CONCLUSION: In this review, we have discussed the physiological significance of ABK followed by its role in cancer progression. We further highlighted available small-molecule inhibitors to control the tumor proliferation and their mechanistic insights.

Rosmarinic acid restrains protein glycation and aggregation in human serum albumin: Multi spectroscopic and microscopic insight - Possible Therapeutics Targeting Diseases.

Protein aggregation and glycation are directly associated with many pathological conditions including several neurodegenerative disorders. This study investigates the potential of naturally occurring plant product, Rosmarinic acid (RA), to inhibit the glycation and aggregation process. In this study, we report that varying concentrations of methylglyoxal (MG) induce advanced glycation end products (AGEs) and aggregates formation in HSA in vitro on day 6 and day 8, respectively. AGEs specific fluorescence confirmed the formation of AGEs in HSA in the presence of MG and further characterized the inhibitory potential of RA. It was found that the presence of RA prevented AGEs formation in vitro. Further, aggregates of HSA were characterized employing multi spectroscopic and microscopic techniques and RA was found to inhibit this process. This study proposes that RA could be a potential natural molecule to treat disorders where AGEs and aggregates of proteins play a pivotal role.

Deciphering the Nature of Caffeic Acid to Inhibit the HSA Aggregation Induced by Glyoxal.

BACKGROUND: Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions. OBJECTIVE: In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA). METHODS: Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy. RESULTS: Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property. CONCLUSION: The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

An Update on the Association of Protein Kinases with Cardiovascular Diseases.

BACKGROUND: Protein kinases are the enzymes involved in phosphorylation of different proteins which leads to functional changes in those proteins. They belong to serine-threonine kinases family and are classified into the AGC (Protein kinase A/ Protein kinase G/ Protein kinase C) families of protein and Rho-associated kinase protein (ROCK). The AGC family of kinases are involved in G-protein stimuli, muscle contraction, platelet biology and lipid signaling. On the other hand, ROCK regulates actin cytoskeleton which is involved in the development of stress fibres. Inflammation is the main signal in all ROCK-mediated disease. It triggers the cascade of a reaction involving various proinflammatory cytokine molecules. METHODS: Two ROCK isoforms are found in mammals and invertebrates. The first isoforms are present mainly in the kidney, lung, spleen, liver, and testis. The second one is mainly distributed in the brain and heart. RESULTS: ROCK proteins are ubiquitously present in all tissues and are involved in many ailments that include hypertension, stroke, atherosclerosis, pulmonary hypertension, vasospasm, ischemia-reperfusion injury and heart failure. Several ROCK inhibitors have shown positive results in the treatment of various disease including cardiovascular diseases. CONCLUSION: ROCK inhibitors, fasudil and Y27632, have been reported for significant efficiency in dropping vascular smooth muscle cell hyper-contraction, vascular inflammatory cell recruitment, cardiac remodelling and endothelial dysfunction which highlight ROCK role in cardiovascular diseases.

Role of phytocystatin in combating metal ion induced conformational alterations in glutathione reductase.

Glutathione reductase (GR) is a flavoprotein that catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione (2GSH) in the presence of coenzyme NADPH. The importance of glutathione stems from the fact that it serves an important role in various metabolic processes. Plants growing in highly polluted areas are exposed to higher concentration of metal ions; thereby feeling abiotic stress and affecting various regulatory enzyme activities. In this study, effect of metal ions has been studied on GR. Phytocystatins show an increased expression in abiotic stress conditions. Here in, the effect of cystatin isolated from yellow mustard seeds (YMP) on heavy metals induced conformational changes in GR was investigated making use of GR activity assay, UV-absorption spectroscopy, fluorescence spectroscopy, FTIR, CD, ITC and SEM analysis. The results obtained clearly reveals that metal ions like Cu2+ and Zn+2 induces concentration dependent conformational changes in GR; YMP restores these alterations in way decreasing the effective concentration of metal ions.

Deciphering the toxic effects of iprodione, a fungicide and malathion, an insecticide on thiol protease inhibitor isolated from yellow Indian mustard seeds.

Pesticides are being used globally to improve agricultural production. They are applied specifically to combat with pathogens that are a major threat for reduced optimum yield of crops. This study was carried out to see the effect of commercially used pesticides on a specific plant protein viz. phytocystatin isolated from yellow mustard seeds (YMP). Phytocystatin is a thiol proteinase inhibitor, which regulates endogenous and exogenous cysteine proteinases and plays a vital physiological role in plants. Different classes of pesticides like fungicide (iprodione) of dicarboximide class and an insecticide (malathion) of class organophosphate are retorted for our study. In the presence of these pesticides, biophysical and biochemical changes were observed in phytocystatin. These changes were evaluated making use of caseinolytic activity assay, UV-vis spectroscopy, fluorescence spectroscopy, FTIR, and circular dichroism. Isothermal titration calorimetry was employed to see interaction pattern of these pesticides with phytocystatin. The results obtained clearly depict that the pesticides bind with the phytocystatin thereby changing its native conformation and reducing its intrinsic property of inhibition on cysteine proteinase as evident by reduced anti-papain inhibition in the presence of pesticides. Furthermore, CD and FTIR spectroscopy results clearly show a decrease in α-helical content upon interaction with malathion and iprodione. Among the two pesticides, iprodione has far more pronounced effect on YMP evident from striking changes in UV, Fluorescence, CD and FTIR spectroscopy. 2,4-dinitrophenylhydrazine spectrophotometric assay was also carried out to check production of ROS, generation of ROS was observed in the presence of these pesticides thus implying that ROS might be responsible for changes in native structure of phytocystatin induced by pesticides.

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach.

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 104  M-1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes.

Methylglyoxal induced glycation and aggregation of human serum albumin: Biochemical and biophysical approach.

Serum protein glycation and formation of advanced glycation end products (AGEs) correlates with many diseases viz. diabetes signifying the importance of studying the glycation pattern of serum proteins. In our present study, methylglyoxal was investigated for its effect on the structure of human serum albumin (HSA); exploring the formation of AGEs and aggregates of HSA. The analytical tools employed includes intrinsic and extrinsic fluorescence, UV spectroscopy, far UV circular dichroism, Thioflavin T fluorescence, congo red binding, polyacrylamide gel electrophoresis (PAGE). UV and fluorescence spectroscopy revealed the structural transition of native HSA evident by new peaks and increased absorbance in UV spectra and quenched fluorescence in the presence of MG. Far UV CD spectroscopy revealed MG induced secondary structural alteration evident by reduced α-helical content. AGEs formation was confirmed by AGEs specific fluorescence. Increased ThT fluorescence and CR absorbance of 10mM MG incubated HSA suggests that glycated HSA results in the formation of aggregates of HSA. SEM and TEM were reported to have an insight of these aggregates. Molecular docking was also utilized to see site specific interaction of MG-HSA. This study is clinically significant as HSA is a clinically relevant protein which plays a crucial role in many diseases.

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight.

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 104 M-1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.

Global transition of human serum albumin to prefibrillar aggregates induced by temsirolimus: Insight into implications of anti-renal cancer drug.

In our study, we have characterized the prefibrillar aggregates of human serum albumin (HSA) induced by temsirolimus, anti-renal cancer drug. Molecular docking was retorted to confirm binding of HSA and temsirolimus. Temsirolimus caused the structural transition of native HSA to non-native species after prolonged incubation of 20 days. These non-native species were characterized as prefibrillar aggregates as evident by decreased intrinsic fluorescence and enhanced 8-anilino-1-naphthalene-sulphonic acid (ANS) fluorescence. Further, enhanced thioflavin T fluorescence and shift in congo red (CR) spectra of temsirolimus-incubated HSA as compared to native HSA are suggestive of global transition of HSA in presence of temsirolimus towards prefibrillar aggregates. Circular dichroism spectroscopy revealed α to ß transition upon prolonged incubation with temsirolimus suggesting the formation of prefibrillar aggregates as aggregates are known to possess high ß content. Scanning electron microscopy confirmed these non-native species to be prefibrillar aggregates evident by observed sheath-like structures. Comet assay was retorted to confirm genotoxic nature of these prefibrillar aggregates; DNA damage was observed for temsirolimus-incubated HSA confirming the genotoxic nature of prefibrillar aggregates. These prefibrillar aggregates are observed at heart of many pathological conditions, thus making our study clinically significant.

Oxadiargyl induced conformational transition of cystatin isolated from yellow mustard seeds: Biophysical and biochemical approach.

Phytocystatins are thiol proteinase inhibitors crucial due to their inhibitory activity in plants. These play important roles in improving crop yield, protection against insects and pathogens and modulation of apoptosis. In this chemical era, various pesticides are being used globally to increase the crop biomass. These pesticides accumulate in plant body and produce harmful effects on plants itself by interacting with essential proteins. In this present study, we have monitored the interaction of a herbicide; oxadiargyl, with phytocystatin isolated from yellow mustard seeds (YMP) by employing spectroscopic techniques viz. UV, fluorescence, FTIR and CD spectroscopy and Isothermal titration calorimetry (ITC). UV and fluorescence spectroscopy shows YMP transformation from native to non-native form apparent by decreased absorbance and decreased fluorescence. FTIR and CD spectroscopy further confirmed secondary structural disruption of YMP. Anti-papain activity assay was also carried out; a reduction in activity was observed in presence of oxadiargyl. Thermodynamic parameters obtained from ITC and stern-volmer plot shows affinity of oxadiargyl towards phytocystatin. Oxadiargyl was also found to induce ROS generation in YMP as evident by DNPH assay. Thus oxadiargyl binds to phytocystatin causing structural alterations reducing its physiological benefits and altering its functionality and ultimately leading to reduced crop yield.

Biochemical, immunological and kinetic characterization and partial sequence analysis of a thiol proteinase inhibitor from Bubalus bubalis kidney: An attempt targeting kidney disorders.

In the present study a thiol proteinase inhibitor was isolated from buffalo kidney making use of ammonium sulphate precipitation and gel filtration chromatography on Sephacryl S-100HR column. Purified inhibitor is homogeneous as it displayed a single band in gel electrophoresis both under reducing and non-reducing environment and is of 65KDa as revealed by gel filtration and SDS PAGE. Kinetic studies revealed the presence of reversible accompanied with competitive mode of inhibition; showing maximum efficacy against papain (Ki=2.90×10-4). It was maximally active at pH 8.0 and was stable for a period of 30, 60 and 90 days at 37, 4 and -20°C respectively. Immunological studies confirmed its purity of epitopes as a single precipitin line is obtained in immunodiffusion. N-terminal analysis revealed that it shared a good homology with mouse kidney cystatin as well as with Human Cys C and Cys E thereby advocating its use as a model for various human oriented studies which targets how the kidney cystatin level varies in accordance with various drugs that are currently being used as a target for variety of diseases.