Pyruvate provides cardioprotection in the experimental model of myocardial ischemic reperfusion injury

The present study was designed to evaluate the cardioprotective potential of pyruvate and to characterize the mechanism underlying the protection. Wistar albino rats were randomly divided into three groups. Two groups were administered saline orally (sham, ischemia-reperfusion (I-R) control group) and animals of third group received pyruvate (500 mg/kg) for 4 weeks. On the 29th day, animals of the I-R control and pyruvate treated groups underwent 45 min of occlusion of the left anterior descending (LAD) coronary artery and were thereafter reperfused for 60 min. In the I-R control group, a significant cardiac necrosis, depressed mean arterial pressure (MAP) and heart rate (HR), decline in myocardial antioxidant status and elevation in lipid peroxidation were observed as compared to sham control. Pyruvate treatment restored the myocardial antioxidant status and favorably modulated the altered MAP as compared to I-R control. Furthermore, I/R-induced lipid peroxidation was significantly inhibited by pyruvate treatment. These beneficial cardioprotective effects translated into significant improvement in MAP. Histopathological examination and restored specific myocardial injury marker CK-MB isoenzyme activity further confirmed protective effects of pyruvate. In conclusion, our study has demonstrated that the beneficial effect of pyruvate likely results from improved MAP and suppression of oxidative stress.     

Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.     

Rat testicular germ cell type(s) targeted by anti-spermatogenic agents in vivo and their recovery on withdrawal of treatment--a flow cytometric study

Spermatogenesis goes through very critically and precisely balanced ratios of germ cells with diverse DNA ploidies (1C, 2C and 4C). Antispermatogenic agents that reversibly interrupt spermatogenesis may have a contraceptive relevance. With a view to study the precise mechanism of action of antispermatogenic agents and identify the germ cell type(s) targeted by various agents in vivo, spermatogenic cells with diverse DNA ploidies were measured in rat testis during treatment and recovery with compounds CDRI-84/35, gossypol and estradiol, using Flow Cytometry. Rats were treated with either CDRI-84/35 (100mg/(kg day) for 15 days followed by 25mg/(kg day) for 55 days) or gossypol (20mg/(kg day) for 70 days) or estradiol benzoate (2.5microg/(rat day) for 70 days) and 3 rats from each group were sacrificed after 22, 41, 53 and 70 days of treatment to monitor the changes in population of 1C, 2C, S-phase and 4C germ cell types. Treatment with CDRI-84/35 resulted in a significant and rapid drop in 1C population with a concomitant and parallel rise in 2C population. In gossypol-treated animals 1C peak disappeared gradually and the arrest was seen predominantly at 2C stage and partially at 4C stage. At the end of the treatment most of the germ cells were arrested at 2C stage. Estradiol affected spermatogenesis differently with 1C population falling in complement to rise in both 2C and 4C peaks. Germ cells were mainly arrested at the 4C stage after the treatment. The data suggest that germ cells fail to enter meiosis in CDRI-84/35-treated rats. Few cells entering meiosis do not complete the cell division and remain arrested at 4C stage. However in case of estradiol and gossypol the meiotic 4C cells become incapable of further differentiation into haploid cells. After receiving 70 days of treatment a few rats were allowed to recover for 60, 90 and 120 days. The population of various germ cell types in the testis of recovery-group animals indicated that spermatogenesis resumes substantially in case of estradiol treatment and partially in case of treatment with the other two agents.     

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria

Successful treatment of human tuberculosis requires 6–9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms – organized communities of surface-attached cells – but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.     

Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.     

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.     

Developmentally regulated proteases in Allomyces arbuscula

A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula. The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca²⁺ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca²⁺ for its activity and is of the serine type.     

DNA nucleotide sequence homologies between some zoosporic fungi

Summary Representative species from the monoflagellate Blastocladiales and the biflagellate Saprolegniales were studied for their DNA base composition, heterogeneity, nucleotide sequence homology and divergence. Intergeneric, intrageneric and interstrain DNAs of Blastocladiales were heterogenous. The G+C values for their main component (average 64 percent) and two minor ones (average 52 and 44 percent) were found to be significantly higher than the corresponding values from the biflagellate Saprolegnia ferax (55, 46 and 36 percent respectively). In Allomyces species, the two hybrid, male and female strains were found to have closer homology with their parental types than these last between themselves. Among Blastocladiales, interspecific similarities between the epigynous A. macrogynus and the hypogynous A. arbuscula were higher (average 75 percent) than intergeneric similarities between Allomyces and Blastocladiella (average 58 percent). The biflagellate S. ferax was found to be distantly related to the uniflagellate Allomyces (average 48 percent similarity). The nucleotide sequence divergences obtained from thermal elution data correlated the hybridization values.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride