Precisely adjusting the hepatic clearance of highly extracted drugs using the modified well-stirred model.

Hepatic clearance has been widely studied for over 50 yr. Many models have been developed using either theoretical or empirical tests to predict drug metabolism. The well-stirred, parallel-tube, and dispersion metabolic models have been extensively discussed. However, to our knowledge, these models cannot fully describe all relevant scenarios in hepatic clearance. We addressed this issue using the isolated perfused rat liver technique with minor modifications. Diazepam was selected to illustrate different levels of drug plasma-protein binding by changing the added concentration of human serum albumin. The free fractions of diazepam at different albumin concentrations were assayed by rapid equilibrium dialysis. The experimental data provide new insights concerning an accepted formula used to describe hepatic clearance. Regarding drug concentrations passing through the liver, the driving force concentration (CH,ss) in terms of Cin (influx in the liver) or Cout (efflux from the liver) needs to be carefully considered when determining drug hepatic and intrinsic clearances. The newly established model, termed the modified well-stirred model, which was derived from the original formula, successfully estimated hepatic drug metabolism. Using the modified well-stirred model, a theoretical driving force concentration of diazepam passing through the liver was evaluated. The model was further used to assess the predictability of in vitro to in vivo extrapolation. This study was not intended to refute the existing models, but rather to augment them using experimental data. The results stress the importance of proper calculation of dose when the drug clearance deviates from the prediction of the well-stirred model.

Exosomes: Potential Therapies for Disease via Regulating TLRs.

Exosomes are small membrane vesicles that retain various substances such as proteins, nucleic acids, and small RNAs. Exosomes play crucial roles in many physiological and pathological processes, including innate immunity. Innate immunity is an important process that protects the organism through activating pattern recognition receptors (PRRs), which then can induce inflammatory factors to resist pathogen invasion. Toll-like receptor (TLR) is one member of PRRs and is important in pathogen clearance and nervous disease development. Although exosomes and TLRs are two independent materials, abundant evidences imply exosomes can regulate innate immunity through integrating with TLRs. Herein, we review the most recent data regarding exosome regulation of TLR pathways. Specifically, exosome-containing materials can regulate TLR pathways through the interaction with TLRs. This is a new strategy regulating immunity to resist pathogens and therapy diseases, which provide a potential method to cure diseases.

Various Profiles of tet Genes Addition to tet(X) in Riemerella anatipestifer Isolates From Ducks in China.

To investigate tetracycline resistance and resistant genotype in Riemerella anatipestifer, the tetracycline susceptibility of 212 R. anatipestifer isolates from China between 2011 and 2017 was tested. The results showed that 192 of 212 (90.6%) R. anatipestifer isolates exhibited resistance to tetracycline (the MICs ranged from 4 to 256 µg/ml). The results of PCR detection showed that, 170 of 212 (80.2%) R. anatipestifer isolates possessed the tet(X) gene. Other genes, including tet(A), tet(M), tet(Q), tet(O), tet(B), and tet(O/W/32/O), were found at frequencies of 20.8, 4.7, 1.4, 0.9, 0.9, and 0.5%, respectively. However, tet(C), tet(E), tet(G), tet(K), and tet(W) were not detected in any isolate. In these tet gene positive strains, 31 (14.6%), 2 (0.9%), 5 (2.4%), 1 (0.5%), 3 (1.4%) were detected containing tet(A)/tet(X), tet(M)/tet(O), tet(M)/tet(X), tet(O)/tet(X), and tet(Q)/tet(X) simultaneously, respectively. One isolates, R131, unexpectedly contained three tet genes, i.e., tet(M), tet(O), and tet(X). Sequence analysis of the tet gene ORFs cloned from R. anatipestifer isolates confirmed that tet(A), tet(B), tet(M), tet(O), tet(Q) and an unusual mosaic tet gene tet(O/W/32/O) were present in R. anatipestifer. The MIC results of R. anatipestifer ATCC 11845 transconjugants carrying tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes exhibited tetracycline resistance with MIC values ranging from 4 to 64 µg/ml. Additionally, the tet(X) gene could transfer into susceptible strain via natural transformation (transformation frequencies of ~10-6). In conclusion, the tet(A), tet(B), tet(M), tet(O), tet(O/W/32/O), tet(Q), and tet(X) genes were found and conferred tetracycline resistance in R. anatipestifer isolates. Moreover, the tet(X) is the main mechanism of tetracycline resistance in R. anatipestifer isolates. To our knowledge, this is the first report of tet(A), tet(B), tet(M), tet(O), tet(Q), and mosaic gene tet(O/W/32/O) in R. anatipestifer.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli RosettaTM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2.

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer.

Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).

The suppression of apoptosis by α-herpesvirus.

Apoptosis, an important innate immune mechanism that eliminates pathogen-infected cells, is primarily triggered by two signalling pathways: the death receptor pathway and the mitochondria-mediated pathway. However, many viruses have evolved various strategies to suppress apoptosis by encoding anti-apoptotic factors or regulating apoptotic signalling pathways, which promote viral propagation and evasion of the host defence. During its life cycle, α-herpesvirus utilizes an elegant multifarious anti-apoptotic strategy to suppress programmed cell death. This progress article primarily focuses on the current understanding of the apoptosis-inhibition mechanisms of α-herpesvirus anti-apoptotic genes and their expression products and discusses future directions, including how the anti-apoptotic function of herpesvirus could be targeted therapeutically.

Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China.

Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 µg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs.

Evaluation of KIM-1 and NGAL as Early Indicators for Assessment of Gentamycin-Induced Nephrotoxicity In Vivo and In Vitro.

BACKGROUND/AIMS: The aminolycoside Gentamicin is a widely used antibiotic, applied in equine medicine. Despite its clinical use, concerns remain regarding the potential toxic side-effects, such as nephrotoxicity. Early detection of renal damage is critical in preclinical drug development. This study was aimed to determine whether kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) may be early indicators in the assessment of Gentamycin-induced nephrotoxicity. METHODS: In our study, a model of Gentamicin-induced nephrotoxicity in male Sprague Dawley rats treated for up to 7 days at 50 or 100mg/kg/day was used to monitor the expressions of novel biomarkers of renal toxicity during the progression of acute kidney injury (AKI). Additionally, biomarkers were assessed in human kidney proximal epithelial cells (HK-2) treated with Gentamicin for 2, 6, 12, 24, 36 or 48h in vitro. RESULTS: Repeated administration of Gentamicin to rats for 1, 3, or 7 days resulted in a dose- and time-dependent increase in the expression of KIM-1 and NGAL. The expressions of the two biomarkers changed prior to renal tubule damage and increases in serum creatinine (SCr) and blood urea nitrogen (BUN) levels, suggesting their usefulness for predicting Gentamicin-induced acute kidney injury (AKI) in vivo. CONCLUSIONS: In contrast, no significant increase in the expression of the biomarker genes and proteins were evident in HK-2 cells after treated by Gentamycin for up to 48h, suggesting that they may not be suitable endpoints for sensitive detection of nephrotoxic effects in vitro.

Complete Genome Sequence of Mycobacterium avium, Isolated from Commercial Domestic Pekin Ducks (Anas platyrhynchos domestica), Determined Using PacBio Single-Molecule Real-Time Technology.

Mycobacterium avium is an important pathogenic bacterium in birds and has never, to our knowledge, reported to be isolated from domestic ducks. We present here the complete genome sequence of a virulent strain of Mycobacterium avium, isolated from domestic Pekin ducks for the first time, which was determined by PacBio single-molecule real-time technology.

Outbreak of Avian Tuberculosis in Commercial Domestic Pekin Ducks ( Anas platyrhynchos domestica).

Avian tuberculosis is a contagious disease affecting various domestic and wild bird species, and is caused by Mycobacterium avium . It is reported extremely rarely in commercial poultry flocks and has not been reported in commercial domestic ducks to date, with domestic ducks reported to be moderately resistant to M. avium infection. Here, we report the outbreak of avian tuberculosis in commercial Pekin duck ( Anas platyrhynchos domestica) flocks. Postmortem and histopathologic findings included nodules presenting in the visceral organs of ducks, and granulomas with central caseous necrosis surrounded by infiltrating lymphocytes. The M. avium pathogen was isolated and further identified by Ziehl-Neelsen staining and PCR based on insert sequence IS901 and the 16S rRNA gene. We highlight that avian tuberculosis not only has economic significance for the duck industry, but also presents a potential zoonotic hazard to humans.

Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data. RESULTS: To identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster. CONCLUSIONS: Our data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.

Genome Sequence of Riemerella anatipestifer Strain RCAD0122, a Multidrug-Resistant Isolate from Ducks.

Riemerella anatipestifer is an important pathogenic bacterium in waterfowl and other avian species. We present here the genome sequence of R. anatipestifer RCAD0122, a multidrug-resistant strain isolated from infected ducks. The isolate contains at least nine types of antibiotic resistance-associated genes.

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine.

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.

KIM-1 and NGAL as biomarkers of nephrotoxicity induced by gentamicin in rats.

Gentamicin is a member of aminoglycosides, which has represented highly effective antimicrobial agents especially in Gram-negative infections despite their toxic effects in the kidney. Rapid diagnosis is vital to preserve renal function and to slow down renal injury. Owing to the poor sensitivity and specificity of serum creatinine (SCr) and blood urea nitrogen (BUN), new biomarkers for earlier and more accurate detection are needed. The aim of our study was to determine whether kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) may be useful biomarkers in the assessment of gentamicin-induced nephrotoxicity in rats. In this study, the two biomarkers of renal toxicity were assessed via ELISA, quantitative real-time PCR, and immunohistochemistry in rats treated with gentamicin for up to 7 days. Repeated administration of gentamicin to male SD rats for 1, 3, or 7 days resulted in a dose- and time-dependent increase in the expression of KIM-1 and NGAL. Changes in gene and protein expressions were found to correlate with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function. Both of the biomarkers are supported to be used as sensitive indicators of acute kidney injury caused by gentamicin.

Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.

Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bß and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.

Functional characterization of a slow and tight-binding inhibitor of plasmin isolated from Russell's viper venom.

BACKGROUND: Snake venoms are rich in Kunitz-type protease inhibitors that may have therapeutic applications. However, apart from trypsin or chymotrypsin inhibition, the functions of most of these inhibitors have not been elucidated. A detailed functional characterization of these inhibitors may lead to valuable drug candidates. METHODS: A Kunitz-type protease inhibitor, named DrKIn-II, was tested for its ability to inhibit plasmin using various approaches such as far western blotting, kinetic analyses, fibrin plate assay and euglobulin clot lysis assay. In addition, the antifibrinolytic activity of DrKIn-II was demonstrated in vivo. RESULTS: DrKIn-II potently decreased the amidolytic activity of plasmin in a dose-dependent manner, with a global inhibition constant of 0.2nM. Inhibition kinetics demonstrated that the initial binding of DrKIn-II causes the enzyme to isomerize, leading to the formation of a much tighter enzyme-inhibitor complex. DrKIn-II also demonstrated antifibrinolytic activity in fibrin plate assay and significantly prolonged the lysis of the euglobulin clot. Screening of DrKIn-II against a panel of serine proteases indicated that plasmin is the preferential target of DrKIn-II. Furthermore, DrKIn-II treatment prevented the increase of FDP in coagulation-stimulated mice and significantly reduced the bleeding time in a murine tail bleeding model. CONCLUSION: DrKIn-II is a potent, slow and tight-binding plasmin inhibitor that demonstrates antifibrinolytic activity both in vitro and in vivo. GENERAL SIGNIFICANCE: This is the first in-depth functional characterization of a plasmin inhibitor from a viperid snake. The potent antifibrinolytic activity of DrKIn-II makes it a potential candidate for the development of novel antifibrinolytic agents.

Quantitative real-time PCR study of the expression and regulation of the tetracycline resistance gene in Riemerella anatipestifer.

Riemerella anatipestifer (RA) is one of the most important pathogens of 1- to 8-wk-old ducklings that severely affects the development of the duck industry in China. Every year, antibiotic medicines including tetracycline and doxycycline are used in the duck industry. Few reports compare the expression of multidrug-resistant genes in RA before and after addition of chemical drugs. With this in mind, the direct effects of gradient concentration of tetracyclines on the expression of tetracycline resistance genes (TETr) in RA at the cDNA level were studied by using a quantitative real-time PCR method. The expression of TETr, tetA, tetC, and tetM was investigated in ATCC11845 and in 30 RA isolated from different samples. Using a range of doxycycline concentrations up to 50% of the minimum inhibitory concentration (MIC), the optimal induction concentration of 0.0625 µg/mL was selected. Under the optimal inducible expression, concentrations of TETr, tetC, and tetM cDNA were detected in all isolates, and the highest mRNA expression level of TETr genes was shown. Additionally, the expression levels of 3 TETr genes in RA14 (tetA and tetC) and RA17 (tetM and tetC) were compared. Both tetC and tetA found in isolate RA14 was found to express both tetC and tetA, and tetC cDNA was detected in isolate RA17 at all doxycycline concentrations tested, whereas tetM cDNA was not detected at any concentration. We can conclude that resistance pump is the main mechanism of tetracycline antibiotic resistance, and under the action of drug resistance pump tetC, the expression of tetM was not activated in RA17. These data suggest that the mRNA expression level of TETr genes was correlated with the MIC values, indicating that the degree of drug resistance is determined by the expression levels of TETr genes. Also, the induction of TETr is the major tetracycline resistance mechanism in RA, especially the resistance pump. However, lower concentrations of doxycycline induced higher TETr expression, and higher concentrations inhibited TETr expression. Maybe that is the reason for selection mutation to make tolerated bacteria survive.

Genetic diversity and population structure in wild Sichuan rhesus macaques.

Because wild rhesus macaque (Macaca mulatta) populations have suffered major declines, there is a growing need to characterize their genetic and population structure in order to protect the genetic integrity of this species. In this study, we genotyped a sample comprising 120 wild rhesus macaques from six sites in Sichuan Province for 30 nuclear microsatellite (STR) loci using an ABI 3130xl genetic analyzer. Bayesian analyses and PCA clearly differentiated monkeys from Heishui from those at other sites. The samples from all six sites exhibited high gene diversity suggesting that the Sichuan wild rhesus macaque populations are not threatened by a lack of genetic diversity. Deviation from Hardy-Weinberg equilibrium was more frequent in the Danba and Heishui populations. This may be due to the more fragmented habitat and less disturbance by humans in this area that foster greater subpopulation structuring than occurs in eastern China. We suggest that this population subdivision is the result of both long-term geographic barriers and human activity.

Glycan structures and intrageneric variations of venom acidic phospholipases A(2) from Tropidolaemus pitvipers.

Most of the phospholipases A(2) (PLA(2) ; EC3.1.1.4) variants isolated so far from snake venoms are nonglycosylated enzymes. In the present study, we purified an active glycosylated PLA(2) and an inactive nonglycosylated Lys49-like PLA(2) from two geographical venom samples of Tropidolaemus. The PLA(2) variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (Tropidolaemus subannulatus and Tropidolaemus wagleri). The active PLA(2) s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, as a result of the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6 stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both PLA(2) s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to comprise complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA(2) to be solved. Furthermore, the enzymatic removal of glycans from both PLA(2) s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as that of other homologous PLA(2) s. The presence of these oligosaccharides in PLA(2) s warrants further analyses, which may provide useful insights into the functional regulation of these biomolecules.

A novel heparin-dependent inhibitor of activated protein C that potentiates consumptive coagulopathy in Russell's viper envenomation.

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.