Structural and Biophysical Characterization of the Interactions between the Death Domain of Fas Receptor and Calmodulin

The extrinsic apoptotic pathway is initiated by cell surface death receptors such as Fas. Engagement of Fas by Fas ligand triggers a conformational change that allows Fas to interact with adaptor protein Fas-associated death domain (FADD) via the death domain, which recruits downstream signaling proteins to form the death-inducing signaling complex (DISC). Previous studies have shown that calmodulin (CaM) is recruited into the DISC in cholangiocarcinoma cells, suggesting a novel role of CaM in Fas-mediated signaling. CaM antagonists induce apoptosis through a Fas-related mechanism in cholangiocarcinoma and other cancer cell lines possibly by inhibiting Fas-CaM interactions. The structural determinants of Fas-CaM interaction and the underlying molecular mechanisms of inhibition, however, are unknown. Here we employed NMR and biophysical techniques to elucidate these mechanisms. Our data show that CaM binds to the death domain of Fas (FasDD) with an apparent dissociation constant (K_d) of ∼2 μm and 2:1 CaM:FasDD stoichiometry. The interactions between FasDD and CaM are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. We also show that both the N- and C-terminal lobes of CaM are important for binding. NMR and surface plasmon resonance data show that three CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, tamoxifen, and trifluoperazine) greatly inhibit Fas-CaM interactions by blocking the Fas-binding site on CaM. Our findings provide the first structural evidence for Fas-CaM interactions and mechanism of inhibition and provide new insight into the molecular basis for a novel role of CaM in regulating Fas-mediated apoptosis.     

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.     

In vitro toxicity and antidiabetic activity of a newly developed polyherbal formulation (MAC-ST/001) in streptozotocin-induced diabetic Wistar rats

The present study was designed to investigate the hypoglycemic effect of an aqueous extract of MAC-ST/001 (a new polyherbal formulation) which was given once daily to rats at different doses. The animals were divided into diabetic and nondiabetic control groups. The duration of each experiment lasted from 1 week to 1 month, and the results were compared with that of the standard hypoglycemic drug glibenclamide (10 mg/kg), which was given once daily. In this study, biochemical and histopathological parameters were studied in streptozotacin (STZ) (single intraperitoneal injection of 55 mg/kg)-induced diabetic rats. The diabetic rats showed a significant (p?<?0.05 and p?<?0.01) decrease in their body weight and serum amylase with marked elevation in blood glucose, serum cholesterol, blood urea nitrogen, creatinine, alkaline phosphatase, and serum transaminases (AST and ALT) after 1 week till the 28th day of diabetes. Cytotoxicity of MAC-ST/001 formulation was also studied on C2C12, 3T3-L1, and HepG2 cells through MTT assay. Histological examination of the liver and pancreas of normal control, diabetic control, and drug-treated rats revealed significant results. Finally, it was concluded that administration of this MAC-ST/001 extract reversed most blood and tissue changes caused by STZ-induced diabetes in rats.     

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26–57%) and beat frequencies (by 8–16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.     

Basic Fibroblast Growth Factor Attenuates Bisphosphonate-Induced Oxidative Injury but Decreases Zinc and Copper Levels in Oral Epithelium of Rat

Recent studies have reported oxidative damage due to bisphosphonate (BP) in various cancer tissues and neurons, although basic fibroblast growth factor (bFGF) induced antioxidant effects in the cells. The bFGF may modulate the BP-induced oxidative stress in oral epithelium of rats. This study was undertaken to explore possible beneficial antioxidant effects of bFGF on oxidative stress induced by BP in oral epithelium of rats. Twenty-eight rats were equally divided into four groups. The first group was used as control. The second, third and fourth groups intraperitoneally received BP (zoledronic acid), bFGF and BP + bFGF. At the end of 10 weeks, the rats were sacrificed, and oral epithelium samples were taken for analyses. In BP group, the lipid peroxidation levels were increased in the oral epithelium, while the activities of glutathione peroxidase (GSH-Px) and the concentrations of total antioxidant status (TAS) were decreased. In rats treated with bFGF, lipid peroxidation levels decreased, and the activities of GSH-Px and concentrations of TAS improved in the oral epithelium. However, zinc and copper levels were decreased in the oral epithelium by BP and bFGF treatments. Concentrations of vitamin E and reduced glutathione in the samples did not change in the groups. In conclusion, treatment with bFGF modulated the antioxidant redox system and reduced the oral epithelium oxidative stress induced by BP. However, zinc and copper levels were decreased by BP and bFGF treatments.     

Risk Factors for Diabetes Mellitus in Women with Primary Ovarian Insufficiency

Primary ovarian insufficiency (POI) is not only a gynecological problem but also has serious effects on women’s health such as changes in hormone levels that can trigger fluctuations in blood sugar level and inflammation status. The present study was designed to determine vitamin D, copper, zinc, metabolic parameters [insulin, homeostasis model of assessment-insulin resistance (HOMA-IR)], inflammation parameters such as procalcitonin and high sensitivity C reactive protein (hs-CRP), and lipid profile in POI patients and control subjects with normal menstrual cycles. A total of 43 patients with nondiabetic POI were studied in order to evaluate and compare the findings with those of the control group, which comprised 33 women with normal menstrual cycles. The women with POI had higher levels of serum copper, serum insulin, glucose, LDL-cholesterol, total cholesterol, HOMA-IR, hs-CRP, and procalcitonin, whereas serum vitamin D and zinc levels were lower compared with the healthy control group. Follicle-stimulating hormone (FSH) levels were positively correlated with insulin, glucose, HOMA-IR, hs-CRP, procalcitonin, and copper and negatively correlated with vitamin D and zinc levels. In multivariate statistic analyses with body mass index and FSH as dependent variables, FSH was positively associated with copper and HOMA-IR negatively with vitamin D levels. The present study demonstrated that women with POI have traditional risk factors for diabetes mellitus, including lower levels of vitamin D, whereas higher levels of copper and HOMA-IR.     

Effects of Antiepileptic Drugs on Antioxidant and Oxidant Molecular Pathways: Focus on Trace Elements

Current reports on trace elements, oxidative stress, and the effect of antiepileptic drugs are poor and controversial. We aimed to review effects of most common used antiepileptics on antioxidant, trace element, calcium ion (Ca²⁺) influx, and oxidant systems in human and experimental animal models. Observations of lower blood or tissue antioxidant levels in epileptic patients and animals compared to controls in recent publications may commonly support the proposed crucial role of antioxidants in the pathogenesis of epilepsy. Effects of old and new antiepileptics on reactive oxygen species (ROS) production in epilepsy are controversial. The old antiepileptic drugs like valproic acid, phenytoin, and carbamazepine induced ROS overproduction, while new epileptic drugs (e.g., topiramate and zonisamide) induced scavenger effects on over production of ROS in human and animals. Antioxidant trace element levels such as selenium, copper, and zinc were generally low in the blood of epileptic patients, indicating trace element deficiencies in the pathogenesis of epilepsy. Recent papers indicate that selenium with/without topiramate administration in human and animals decreased seizure levels, although antioxidant values were increased. Recent studies also reported that sustained depolarization of mitochondrial membranes, enhanced ROS production and Ca²⁺ influx may be modulated by topiramate. In conclusion, there is a large number of recent studies about the role of antioxidants or neuroprotectants in clinical and experimental models of epilepsy. New antiepileptic drugs are more prone to restore antioxidant redox systems in brain and neurons.     

Virulence variation of Puccinia striiformis Westend. f. sp. tritici in Pakistan

Yellow rust populations of Pakistan were characterised for their virulence pathotypes/races and pathogenetic variation using seedling evaluation of differential genotypes under glasshouse conditions in Murree (6000 feet above sea level). Differential genotypes comprised a world set, an European set, near isogenic lines and the universally susceptible bread wheat cultivar “Morocco”. Over the two-year study a total of 18 race groups were identified. Out of these 18 race groups, several (68E0, 64E0, 66E0, 70E0, 6E0, 71E0, 6E0, 2E0, 67E0, and 68E16) were found previously. The new race group 70E32 found probably evolved because of mutation from the previously existing 70E16. Virulence frequencies of yellow rust (Yr) resistance genes were also determined on near isogenic lines. The highest virulence frequencies (%) were found for Yr7 (88%), Yr9 (57%), Yr18 (70%), and Yr24 (69%). Virulence frequencies were low for Yr 1 (4%), Yr5 (7%), Yr10 (10%) and Yr15 (4%). Our studies indicated that virulence existed for almost all yr genes, necessitating regular monitoring of the yellow rust populations and intensifying efforts to identify new sources of resistance to this pathogen.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.