Identification of glutamate ABC-Transporter component in Clostridium perfringens as a putative drug target

Clostridium perfringens is an anaerobic pathogen known to cause vast number of diseases in mammals and birds. Various toxins and hydrolysing enzymes released by the organism are responsible for the necrosis of soft tissues. Due to serious safety issues associated with current vaccines against C. perfringens, there is a need for new drug or vaccine targets. C. perfringens is extremely dependent on its host for nutrition which can be targeted for vaccine development or drug design. Therefore, it is of interest to identify the unique transport systems used by C. perfringens involved in uptake of essential amino acids that are synthesized by the host, so that therapeutic agents can be designed to target the specific transport systems. Use of bioinformatics tools resulted in the identification of a protein component of the glutamate transport system that is not present in the host. Analysis of the conservation profile of the protein domain indicated it to be a glutamate binding protein which also stimulates the ATPase activity of ATP Binding Cassettes (ABC) transporters. Homology modelling of the protein showed two distinct lobes, which is a characteristic of substrate binding proteins. This suggests that the carboxylates of glutamate might be stabilized by electrostatic interactions with basic residues as is observed with other binding proteins. Hence, the homology model of this potential drug target can be employed for in silico docking studies by suitable inhibitors.     

Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes.     

Characterization of immune response elicited by the recombinant outer membrane protein OmpF of Aeromonas hydrophila,a potential vaccine candidate in murine model

Porins, the outer membrane proteins of gram negative bacteria, perform vital roles in bacterial survival and virulence, such as nutrient transportation across the membrane as well as adhesion to host cells during infection. The outer membrane proteins, OmpF and OmpC, are part of a two-component regulatory system, essential for the maintenance of solute concentrations in the cytoplasmic milieu of bacteria, and are thus considered vital for bacterial survival. Exposed on the surface of gram-negative bacteria, these channel proteins are highly immunogenic and can thus be exploited as vaccine candidates. In the present study, we have cloned, characterized, and expressed outer membrane protein OmpF of Aeromonas hydrophila, a major fish pathogen and also known to cause severe infections in humans. The cloned ompF gene of A. hydrophila consisting of an open reading frame corresponding to mature OmpF was expressed and purified from the heterologous host, E. coli. High level of expression resulted in recovery of ~120 mg/L of the purified rOmpF at shake flask level. Polyclonal antisera raised against the recombinant OmpF showed a very high endpoint titer (>1:80,000) and were able to specifically agglutinate live A. hydrophila. Further, anti-OmpF antisera cross-reacted with the cell lysates of various Aeromonas isolates, suggesting that anti-rOmpF antibodies can be used to identify different A. hydrophila isolates in infected conditions. Antibody isotyping, cytokine ELISA, and ELISPOT assay indicated predominantly Th1 type of immune response. The recombinant OmpF reported in the present study thus has the potential to be used as a vaccine candidate against A. hydrophila.     

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7

Background

Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses.

Methods

In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~ 30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD₅₀ doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM.

Results

This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis.

Conclusions

A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells.

General significance

CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.     

Design, synthesis, and evaluations of antifungal activity of novel phenyl(2H-tetrazol-5-yl)methanamine derivatives

Fungal infections pose a continuous and serious threat to human health and life. The intrinsic resistance has been observed in many genera of fungi. Many fungal infections are caused by opportunistic pathogens that may be endogenous (Candida infections) or acquired from the environment (Cryptococcus and Aspergillus infections). So, new therapeutic strategies are needed to combat various fungal infections. Fluconazole shows good antifungal activity with relatively low toxicity and is preferred as first line antifungal therapy, but it has suffered from severe drug resistance. So, there is a need to design novel analogues by modification of fluconazole-like structure. A novel series of phenyl(2H-tetrazol-5-yl)methanamine derivatives were synthesized by reaction of α-amino nitrile with sodium azide and ZnCl₂ in presence of isopropyl alcohol. They were evaluated for antifungal activity against Candida albicans and Aspergillus niger and subjected to docking study against 1EA1.     

ALDH1A1 Maintains Ovarian Cancer Stem Cell-Like Properties by Altered Regulation of Cell Cycle Checkpoint and DNA Repair Network Signaling

Objective

Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance.

Methods

Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators.

Results

ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDH^HIGH displayed significantly lower progression free survival than the patients with ALDH^LOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells.

Conclusion

This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.     

Dimensional analysis of MINMOD leads to definition of the disposition index of glucose regulation and improved simulation algorithm

Background  

Frequently Sampled Intravenous Glucose Tolerance Test (FSIVGTT) together with its mathematical model, the minimal model (MINMOD), have become important clinical tools to evaluate the metabolic control of glucose in humans. Dimensional analysis of the model is up to now not available.     

Status of some free radical scavenging enzymes in the blood of myocardial infarction patients

Pro-oxidant and anti-oxidant systems and their levels have significant roles in occlusive vascular diseases. In the present communication, we have measured the levels of some representative anti-oxidant enzymes in the blood of the patients of myocardial infarction after reperfusion and compared them to age and sex matched healthy persons. Our findings show that the activities of anti-oxidant enzymes (viz. SOD, catalase and glutathione reductase) are significantly decreased whereas there is significant increase in the levels of malonaldialdehyde (a marker of free radical-mediated damage) in the patients. The findings point out that ischemic myocardial disorders are associated with excessive free radical generation and free radical-mediated damage of lipids.     

Ketopiperazine-based renin inhibitors: optimization of the "C" ring

A systematic investigation of the S3 sub-pocket activity requirements was conducted. It was observed that linear and sterically small side chain substituents are preferred in the S3 sub-pocket for optimal renin inhibition. Polar groups in the S3-sub-pocket were not well tolerated and caused a reduction in renin inhibitory activity. Further, compounds with clog P's < or = 3 demonstrated a dramatic reduction in CYP3A4 inhibitory activity.