Insight into the molecular basis of coagulation proteinase specificity by mutagenesis of the serpin antithrombin

Specific cleavage of factor V at several P1Arg sites is critical for maintenance of hemostasis. While cleavage by procoagulant proteinases fXa and thrombin activates the cofactor, its cleavage by the anticoagulant proteinase activated protein C (APC) inactivates it. Antithrombin (AT), a specific serpin inhibitor of both thrombin and factor Xa, but not APC, was used as a model system to investigate molecular determinants of APC specificity in the inactivation reaction. Two mutants were prepared in which the P2 or the P3-P3' residues of the reactive site loop of the serpin were replaced with the corresponding residues of the APC cleavage site in factor V spanning residues 504-509 (Asp(504)-Arg-Arg-Gly-Ile-Gln(509)). Kinetic analysis showed that the reactivities of mutants were impaired by approximately 2-3 orders of magnitude with both factor Xa and thrombin, but improved by approximately 2 orders of magnitude with APC. The saturable dependence of the observed first-order rate constants on the concentrations of AT in complex with approximately 70-saccharide high-affinity heparin revealed that changes in the reactivity of the 504-509 mutant with proteinases are primarily due to an effect in the second reaction step in which a noncovalent serpin-proteinase encounter complex is converted to a stable, covalent complex. These results suggest that the P3-P3' residues of the APC cleavage site in factor Va, particularly P2Arg, confer specificity for the anticoagulant proteinase by improving the reactivity of the catalytic pocket with the transition state of the substrate in the second step of the reaction.     

Mutagenesis studies toward understanding the mechanism of differential reactivity of factor Xa with the native and heparin-activated antithrombin

A unique pentasaccharide fragment of high-affinity heparin activates antithrombin (AT) to enhance its rate of complex formation with factor Xa (FXa) by 200-300-fold. Recent results have indicated that the activation of AT is associated with the exposure of a cryptic exosite on the serpin that is an interactive site for FXa in the complex. Previously, we identified Arg(150) on the autolysis loop of FXa as a candidate residue that may specifically interact with the heparin-activated AT. Three other surface loops on FXa including 39, 60, and the sodium-binding 220 loops have been implicated to be critical for the protease interaction with the activated AT. To determine the extent of the contribution of these loops to the specificity of the FXa interaction with activated AT, several loop mutants of the protease were prepared and their reactivity with AT was studied in both the absence and presence of pentasaccharide. Analysis of the inhibition kinetic data suggests that the residues of both 39 and 60 loop make a minor contribution to the recognition of AT in both the native and activated conformation of the serpin. On the other hand, the reactivity of AT with the sodium loop mutants of FXa in the absence of the cofactor was severely impaired. However, the extent of the rate-accelerating effect of pentasaccharide in the AT inhibition of the mutants was not affected. These results suggest that all three loops play a role in the specificity of the FXa-AT interaction; however, neither loop specifically interacts with the activated conformation of the serpin.     

Interaction between protein subunits of the type IV secretion system of Bartonella henselae

In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae. We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5.     

Homeostatic proliferation is a barrier to transplantation tolerance

Despite the ease of inhibiting immune responses by blockade of T-cell costimulation in naive rodent models, it is difficult to suppress those responses in animals with memory cells. Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that extensive T-cell proliferation can occur under conditions of lymphopenia. This process, termed homeostatic proliferation, may induce acquisition of functional memory T cells. Here, using clinically relevant mouse models of peripheral T-cell depletion, we show that residual nondepleted T cells undergo substantial homeostatic expansion. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo. These findings establish the importance of homeostatic proliferation in clinically relevant settings, demonstrate the barrier that homeostatic proliferation can present to the induction of transplantation tolerance, and have important implications for transplantation protocols that use partial or complete peripheral T-cell depletion.     

Transport phenomena in articular cartilage cryopreservation as predicted by the modified triphasic model and the effect of natural inhomogeneities

Knowledge of the spatial and temporal distribution of cryoprotective agent (CPA) is necessary for the cryopreservation of articular cartilage. Cartilage dehydration and shrinkage, as well as the change in extracellular osmolality, may have a significant impact on chondrocyte survival during and after CPA loading, freezing, and thawing, and during CPA unloading. In the literature, Fick's law of diffusion is commonly used to predict the spatial distribution and overall concentration of the CPA in the cartilage matrix, and the shrinkage and stress-strain in the cartilage matrix during CPA loading are neglected. In this study, we used a previously described biomechanical model to predict the spatial and temporal distributions of CPA during loading. We measured the intrinsic inhomogeneities in initial water and fixed charge densities in the cartilage using magnetic resonance imaging and introduced them into the model as initial conditions. We then compared the prediction results with the results obtained using uniform initial conditions. The simulation results in this study demonstrate the presence of a significant mechanical strain in the matrix of the cartilage, within all layers, during CPA loading. The osmotic response of the chondrocytes to the cartilage dehydration during CPA loading was also simulated. The results reveal that a transient shrinking occurs to different levels, and the chondrocytes experience a significant decrease in volume, particularly in the middle and deep zones of articular cartilage, during CPA loading.     

First report on natural infection of the Phlebotomus tobbi by Leishmania infantum in northwestern Iran

Visceral leishmaniasis (VL) is an important health problem in Ardebil, where it borders Azerbaijan in the northwestern Iran. In spite of the presence of both cutaneous and visceral leishmaniasis (CL and VL) in northwestern Iran, previous researches have consistently revealed the etiologic agent of VL in the region to be Leishmania infantum. This is the first report of natural infection of Phlebotomus tobbi with L. infantum in Bilesavar district in the northern part of Ardebil province bordering Azerbaijan. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA, and CPB genes of the parasite followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses revealed presence of L. infantum in six out of 433 tested female sand fly specimens. Although sand flies of P. tobbi were infrequent, two out of 32 (6.25%) females captured in the area were found infected with the parasite. Phlebotomus perfiliewi transcaucasicus, the known vector of VL in the area, were the most dominant species but only four out of 273 (1.47%) tested were infected with L. infantum. This study showed that P. tobbi similar to P. perfiliewi transcaucasicus could play a significant role in the transmission of the L. infantum. However more investigations are needed to demonstrate that L. infantum is the only species circulating in the focus.     

Scolicidal effects of Olea europaea and Satureja khuzestanica extracts on protoscolices of hydatid cysts

Treatment of hydatid disease is mainly surgical, with medical treatment being reserved as a coadjuvant treatment. Use of effective scolicidal agents during surgery of cystic echinococcosis is essential to reduce the recurrence rate. The goal of this study was to evaluate the in vitro scolicidal effects of hydroalcoholic extracts of Satureja khuzestanica leaves and aqueous extracts of Olea europaea leaves on hydatid cyst protoscolices. Echinococcus granulosus protoscolices were collected from the liver of sheep infected with the hydatid cyst. Various concentrations of plant extracts were used in different exposure times for viability assay of protoscolices. Among the olive leaf extracts tested, 0.1% and 0.01% concentrations had strong scolicidal effects in 120 min. S. khuzestanica 0.1% had very strong scolicidal effects in 30, 60, and 120 min of exposure times and the mortality rate decreased with the lower concentration. The finding have shown that the scolicidal activity of S. khuzestanica against cystic echinococosis protoscolices were more effective, while the O. europaea extract showed less effects.     

Amelioration of diet-induced nonalcoholic steatohepatitis in rats by Mn-salen complexes via reduction of oxidative stress

Background

Nonalcoholic steatohepatitis (NASH), a progressive stage of nonalcoholic fatty liver disease (NAFLD), is characterized by steatosis (accumulation of triacylglycerols within hepatocytes) along with inflammation and ballooning degeneration. It has been suggested that oxidative stress may play an important role in the progress of NAFLD to NASH. The aim of present study was to determine whether antioxidant supplementations using EUK-8, EUK-134 and vitamin C could improve the biochemical and histological abnormalities associated with diet-induced NASH in rats.

Methods

NASH was induced in male N-Mary rats by feeding a methionine - choline deficient (MCD) diet. The rats were fed either normal chow or MCD diet for 10 weeks. After NASH development, the MCD-fed rats were randomly divided into four groups of six: the NASH group that received MCD diet, the EUK-8 group which was fed MCD diet plus EUK-8, the EUK-134 group which was fed MCD diet plus EUK-134 and the vitamin C group which received MCD diet plus vitamin C. EUK-8, EUK-134 and vitamin C (30 mg/kg body weight/day) were administered by gavage for eight weeks.

Results

Treatment of MCD-fed rats with salens reduced the sera aminotransferases, cholesterol, low density lipoprotein contents, the extent of lipid peroxidation and protein carbonylation whereas the HDL-C cholesterol levels were significantly increased. In addition, EUK-8 and EUK-134 improved steatosis, ballooning degeneration and inflammation in liver of MCD-fed rats.

Conclusion

Antioxidant (EUK-8, EUK-134 and vitamin C) supplementation reduces NASH-induced biochemical and histological abnormalities, pointing out that antioxidant strategy could be beneficial in treatment of NASH.     

Association of genetic polymorphisms of glutathione-S-transferase genes (GSTT1, GSTM1, and GSTP1) and susceptibility to nonalcoholic fatty liver disease in Zahedan, Southeast Iran

Oxidative damage is thought to play a pivotal role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Glutathione-S-transferases (GSTs) are involved in cell protection against oxidative stress. We examined whether GSTM1, GSTT1, and GSTP1 polymorphisms are associated with NAFLD in a sample of the Iranian population. The current case-control study included 83 patients with NAFLD and 93 healthy subjects. The GSTM1 and GSTT1 polymorphisms were analyzed by multiplex polymerase chain reaction (PCR). The GSTP1 polymorphism was detected by tetra amplification refractory mutation system-PCR assay. The GSTM1-null genotype was significantly associated with the development of NAFLD (odds ratios [OR]=2.171, 95% confidence intervals [CI]=1.188-3.970, p=0.015). The GSTP1 Val allele was shown to be a risk factor for NAFLD (OR=1.739, 95% CI=1.089-2.777, p=0.024). The GSTT1 polymorphism was not significantly different between control and patient groups (p=0.221). This study showed that GSTM1 and GSTP1, but not GSTT1, genetic polymorphisms are associated with NAFLD in a sample of the Iranian population, and may be used to determine the risk of development of NAFLD.