An endoplasmic reticulum-specific stress-activated caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells by respiratory syncytial virus

Respiratory syncytial virus (RSV) infection induced programmed cell death or apoptosis in the cultured lung epithelial cell line, A549. The apoptotic cells underwent multiple changes, including fragmentation and degradation of genomic DNA, consistent with the activation of the DNA fragmentation factor or caspase-activated DNase (DFF or CAD). The infection led to activation of FasL; however, a transdominant mutant of FAS-downstream death domain protein, FADD, did not inhibit apoptosis. Similarly, modest activation of cytoplasmic apoptotic caspases, caspase-3 and -8, were observed; however, only a specific inhibitor of caspases-3 inhibited apoptosis, while an inhibitor of caspase-8 had little effect. No activation of caspase-9 and -10, indicators of the mitochondrial apoptotic pathway, was observed. In contrast, RSV infection strongly activated caspase-12, an endoplasmic reticulum (ER) stress response caspase. Activation of the ER stress response was further evidenced by upregulation of ER chaperones BiP and calnexin. Antisense-mediated inhibition of caspase-12 inhibited apoptosis. Inhibitors of NF-kappa B had no effect on apoptosis. Thus, RSV-induced apoptosis appears to occur through an ER stress response that activates caspase-12, and is uncoupled from NF-kappa B activation.     

Plasmid-Mediated Sulfamethoxazole Resistance Encoded by the sul2 Gene in the Multidrug-Resistant Shigella flexneri 2a Isolated from Patients with Acute Diarrhea in Dhaka,Bangladesh

In this study, mechanisms of plasmid-mediated sulfamethoxazole resistances in the clinical strains of multi-drug resistant (MDR) Shigella flexneri 2a were elucidated for the first time in Bangladesh. From 2006 to 2011, a total of 200 S. flexneri 2a strains were randomly selected from the stock of the Enteric and Food Microbiology Laboratory of icddr,b. Antimicrobial susceptibility of the strains showed 73%, 98%, 93%, 58%, 98%, 64% and 4% resistance to trimethoprim-sulfamethoxazole, nalidixic acid, ampicillin, erythromycin, tetracycline, ciprofloxacin and ceftriaxone respectively. Plasmid profiling revealed heterogeneous patterns and interestingly, all the trimethoprim-sulfamethoxazole resistant (SXT^R) strains yielded a distinct 4.3 MDa plasmid compared to that of the trimethoprim-sulfamethoxazole susceptible (SXT^S) strains. Curing of this 4.3 MDa plasmid resulted in the susceptibility to sulfamethoxazole alone suggesting the involvement of this plasmid in the resistance of sulfamethoxazole. Moreover, PCR analysis showed the presence of sul2 gene in SXT^R strains which is absent in SXT^S strains as well as in the 4.3 MDa plasmid-cured derivatives, confirming the involvement of sul2 in the resistance of sulfamethoxazole. Furthermore, pulsed-field gel electrophoresis (PFGE) analysis revealed that both the SXT^R and SXT^S strains were clonal. This study will significantly contributes to the knowledge on acquired drug resistance of the mostly prevalent S. flexneri 2a and further warrants continuous monitoring of the prevalence and correlation of this resistance determinants amongst the clinical isolates of Shigella and other enteric pathogens around the world to provide effective clinical management of the disease.     

PepBind: A Comprehensive Database and Computational Tool for Analysis of Protein-peptide Interactions

Protein–peptide interactions, where one partner is a globular protein (domain) and the other is a flexible linear peptide, are key components of cellular processes predominantly in signaling and regulatory networks, hence are prime targets for drug design. To derive the details of the protein–peptide interaction mechanism is often a cumbersome task, though it can be made easier with the availability of specific databases and tools. The Peptide Binding Protein Database (PepBind) is a curated and searchable repository of the structures, sequences and experimental observations of 3100 protein–peptide complexes. The web interface contains a computational tool, protein inter-chain interaction (PICI), for computing several types of weak or strong interactions at the protein–peptide interaction interface and visualizing the identified interactions between residues in Jmol viewer. This initial database release focuses on providing protein–peptide interface information along with structure and sequence information for protein–peptide complexes deposited in the Protein Data Bank (PDB). Structures in PepBind are classified based on their cellular activity. More than 40% of the structures in the database are found to be involved in different regulatory pathways and nearly 20% in the immune system. These data indicate the importance of protein–peptide complexes in the regulation of cellular processes. PepBind is freely accessible at http://pepbind.bicpu.edu.in/.     

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin

Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.     

An insight into the ribonucleolytic and antiangiogenic activity of buffalo lactoferrin

Lactoferrin (LF) has several biological effects ranging from ribonuclease activity to antiangiogenic activity. It thus serves as a potential target protein for studies related to ribonucleolytic activity in association with its antiangiogenic activity. We have isolated buffalo LF and checked the ribonucleolytic activity via an agarose gel-based assay and precipitation assay. The ribonucleolytic activity of LF is lower compared to RNase A and the pH profile is a bell-shaped curve, with a pK₁ value of 5.43 and pK₂ of 7.65. The ribonuclease inhibitor that inhibits many ribonuclease-type proteins by forming a tight complex is unable to inhibit the ribonucleolytic property of LF. Fe(III) behaves as a noncompetitive inhibitor for the ribonucleolytic activity of protein. The superoxide-scavenging activity of the protein has also been measured. Histidine modification by diethylpyrocarbonate was monitored by UV–Vis spectroscopy at pH 7 and pH 8 and the effect towards the ribonucleolytic activity was determined. The antiangiogenic property of LF was investigated by the chorioallantoic membrane assay. Finally, the possible active site was analyzed via docking studies and correlated with the experimental study.     

Spatiotemporal variability in activity patterns of urban street cattle as function of environmental factors

ABSTRACT

In the current study, we studied behavioral patterns, such as foraging, activity, standing, and laying in a population of cattle wandering on streets of Raipur city. We also determined the effects of four environmental factors (temperature, humidity, light, and noise intensities) on these behaviors. We recorded all four behavioral variables at four time points each day over a period of three consecutive days at 10 randomly selected locations using on-site human observation method, digital video, and still camera. We used Cosinor rhythmometry to analyze the time series data on all four behavioral variables. One-way ANOVA was performed to determine the impact of the factor “time of the day” on all observed behavioral variables. Pearson correlation was also employed to evaluate the relationship between the environmental and behavioral variables. Cosinor analyses revealed statistically significant 24-h rhythms in all observed behavioral variables, at the group level, with peaks of foraging, activity, standing, and laying located at 11.54 h, 14.54 h, 18.66 h, and 23.88 h, respectively. Factor “time of the day” had statistically significant effect on all observed behavioral variables. Foraging and activity were found to be positively correlated with temperature, light, and noise intensities; and negatively correlated with humidity. The laying behavior was found to be negatively associated with temperature, light, and noise intensities; and positively correlated with humidity. The findings of this study might be helpful in the management of cattle menace that has become an annoying phenomenon on the streets and highways of almost all Indian cities.     

A Comparative Study of Interaction of Tetracycline with Several Proteins Using Time Resolved Anisotropy,Phosphorescence, Docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θ_c) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θ_c) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes.     

Normalization of Tumor Microenvironment by Neem Leaf Glycoprotein Potentiates Effector T Cell Functions and Therapeutically Intervenes in the Growth of Mouse Sarcoma

We have observed restriction of the murine sarcoma growth by therapeutic intervention of neem leaf glycoprotein (NLGP). In order to evaluate the mechanism of tumor growth restriction, here, we have analyzed tumor microenvironment (TME) from sarcoma bearing mice with NLGP therapy (NLGP-TME, in comparison to PBS-TME). Analysis of cytokine milieu within TME revealed IL-10, TGFβ, IL-6 rich type 2 characters was switched to type 1 microenvironment with dominance of IFNγ secretion within NLGP-TME. Proportion of CD8⁺ T cells was increased within NLGP-TME and these T cells were protected from TME-induced anergy by NLGP, as indicated by higher expression of pNFAT and inhibit related downstream signaling. Moreover, low expression of FasR⁺ cells within CD8⁺ T cell population denotes prevention from activation induced cell death. Using CFSE as a probe, better migration of T cells was noted within TME from NLGP treated mice than PBS cohort. CD8⁺ T cells isolated from NLGP-TME exhibited greater cytotoxicity to sarcoma cells in vitro and these cells show higher expression of cytotoxicity related molecules, perforin and granzyme B. Adoptive transfer of NLGP-TME exposed T cells, but not PBS-TME exposed cells in mice, is able to significantly inhibit the growth of sarcoma in vivo. Such tumor growth inhibition by NLGP-TME exposed T cells was not observed when mice were depleted for CD8⁺ T cells. Accumulated evidences strongly suggest NLGP mediated normalization of TME allows T cells to perform optimally to inhibit the tumor growth.     

Circadian and Ultradian Variations in the Plasma Level of Maturational Gonadotropin (GTH II) in the Indian Catfish,Clarias batrachus

Analysis of changes in the plasma levels of GTH II over a 24-h day/night cycle revealed statistically significant circadian or ultradian variation or both in its circulating level during the gonadally active months of the reproductive cycle. The 24-h average and amplitude of circadian or ultradian rhythm in GTH II increased with the advancement of ovarian development and maturation. In April, a single peak in GTH II level was noticed in the night at 20.6 h. In contrast, in May, June and July a biphasic pattern (ultradian rhythm) was noticed with two characteristic peaks, one in the photophase and the other in the scotophase. In addition, in May a statistically significant circadian rhythm in plasma GTH II was validated with a peak located at 20.1 h. These rhythms seem to have physiological significance. The ultradian rhythm with two peaks during the reproductively active phase appears to provide a suitable physiological milieu for the temporally different yet synchronous population of oocytes for the secretion of steroids. Thus, the observed temporal organization in GTH II may have physiological consequences leading to accomplishment of reproductive process at appropriate time of the year.