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During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI. [ABSTRACT FROM AUTHOR]

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of chronic kidney disease. Here, we identified recessive mutations in the gene encoding the actin-binding protein advillin (AVIL) in 3 unrelated families with SRNS. While all AVIL mutations resulted in a marked loss of its actin-bundling ability, truncation of AVIL also disrupted colocalization with F-actin, thereby leading to impaired actin binding and severing. Additionally, AVIL colocalized and interacted with the phospholipase enzyme PLCE1 and with the ARP2/3 actin-modulating complex. Knockdown of AVIL in human podocytes reduced actin stress fibers at the cell periphery, prevented recruitment of PLCE1 to the ARP3-rich lamellipodia, blocked EGF-induced generation of diacylglycerol (DAG) by PLCE1, and attenuated the podocyte migration rate (PMR). These effects were reversed by overexpression of WT AVIL but not by overexpression of any of the 3 patient-derived AVIL mutants. The PMR was increased by overexpression of WT Avilor PLCE1, or by EGF stimulation; however, this increased PMR was ameliorated by inhibition of the ARP2/3 complex, indicating that ARP-dependent lamellipodia formation occurs downstream of AVIL and PLCE1 function. Together, these results delineate a comprehensive pathogenic axis of SRNS that integrates loss of AVIL function with alterations in the action of PLCE1, an established SRNS protein., Introduction Nephrotic syndrome (NS) is characterized by proteinuria caused by disruption of the glomerular filtration barrier (1). It is the second most frequent cause of chronic kidney disease before the [...]

A study was conducted to examine chemically activated arginines as probes to determine microenvironmental influences in enzymes that support the identification of anionic substrates. The Shimadzu UV-265 was utilized to determine spectrometric measurements while chemical modifications were carried out at 27 degrees C. Results indicated that electrostatic and solvation effects were important factors supporting arginine's role as an anion anchor.

Abstract Background Plasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. Results Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. Conclusion We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C‐terminal region to give a novel protein, HCII‐N. MD simulations of HCII‐N and three‐dimensional structure showed a unique 51 amino acid sequence at the C‐terminal having unique RCL‐like structure. The HCII‐N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence‐based analysis revealed a more compact structure of HCII‐N that was in a more hydrophilic environment. The HCII‐N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII‐N may have alternate protease specificity or a non‐inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene. [ABSTRACT FROM AUTHOR]

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10−7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems. [ABSTRACT FROM AUTHOR]

Antithrombin III (AT) is the most important endogenous anticoagulant, and genetic variability in SERPINC1, gene encoding AT, is low. Mutations leading to AT deficiency and increased thrombotic risk are well known; however, only 2 studies have reported mutations in regulatory region of SERPINC1 gene till date. Aim of the present study was to identify genetic variations in SERPINC1 5′ untranslated region (UTR) in Indian patients with deep vein thrombosis (DVT) having AT deficiency. DNA sequencing was used to identify underlying genetic defects in SERPINC1 regulatory region. In silico tools TFBIND and PROMO were used to identify transcription factor binding sites in the promoter region. We have identified 2 novel polymorphisms, g.25G>A and g.−1A>T, and 2 known g.67G>A and rs3138521 5′ UTR polymorphisms in SERPINC1 regulatory region in Indian patients with DVT for the first time. In present study, allele frequencies of rs3138521 (S: 0.37 and F: 0.63) were similar to that reported in Western population and were not associated with low plasma AT levels (P value .5). This is the first report of regulatory region polymorphisms in SERPINC1 gene in Indian population. Our results strongly suggest that similar studies should be included when ever no mutation is detected in protein-coding region of AT gene. [ABSTRACT FROM AUTHOR]

Phenotypic resistance to activated protein C (APC) is a complex mechanism associated with increased thrombosis risk. Activated protein C resistance (APCR) is mainly influenced by FVLeiden mutation, and various other single nucleotide polymorphisms (SNPs) in FV gene are known to be associated with APCR. The aim of present study was to investigate the incidence and assess possible mechanisms of APCR in Indian patients with deep vein thrombosis (DVT). Three hundred and ten Doppler-proven patients with DVT were screened for APCR, and 50 APCR positive patients and 50 controls were typed for FVLeiden, Hong Kong, Cambridge, HR2 haplotype, Glu666Asp, Ala485Lys, and Liverpool using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific PCR. FVLeiden was commonest cause of APCR (50%) in Indian patients with DVT being statistically significant (P = .001) compared to controls. FV Liverpool, FV Glu666Asp and FV Ala485Lys were studied for the first time in Indian population. FV Liverpool, FV Glu666Asp, Hong Kong, and Cambridge were found to be absent. High frequency of Ala485Lys in patients shows that it might be a risk factor contributing to APCR in Indian patients with DVT. HR2 haplotype was not associated with APCR; however, presence of homozygous HR2 haplotype in patients only indicates the role it might play in Indian APCR population. In conclusion, contribution of FVLeiden causing APCR in Indian population is not as strong as previously reported in Western countries. The presence of other SNPs observed in the present study requires such studies on larger sample size to understand the molecular basis of defect. [ABSTRACT FROM AUTHOR]