Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by <Emphasis Type="Italic">N</Emphasis>-acetyl cysteine

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψ_mt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.     

Investigation of Optical Spectroscopic and Computational Binding Mode of Bovine Serum Albumin with 1, 4‐Bis ((4‐((4‐Heptylpiperazin‐1‐yl) Methyl)‐1H‐1, 2, 3‐Triazol‐1‐yl) Methyl) Benzene

A newly synthesized 1, 4‐bis ((4‐((4‐heptylpiperazin‐1‐yl) methyl)‐1H‐1, 2, 3‐triazol‐1‐yl) methyl) benzene from the family of piperazine derivative has good anticancer activity, antibacterial and low toxic nature; its binding characteristics are therefore of huge interest for understanding pharmacokinetic mechanism of the drug. The binding of piperazine derivative to bovine serum albumin (BSA) was investigated using fluorescence spectroscopy. The molecular distance r between the donor (BSA) and acceptor (piperazine derivative) was estimated according to Forster's theory of nonradiative energy transfer. The physicochemical properties of piperazine derivative, which induced structural changes in BSA, have been studied by circular dichroism and those chemical environmental changes were probed using Raman spectroscopic analysis. Further, the binding dynamics was expounded by synchronous fluorescence spectroscopy and molecular modeling studies explored the hydrophobic interaction and hydrogen bonding results, which stabilize the interaction.     

An integrative approach to unravel the Ceratitis FAR (Diptera,Tephritidae) cryptic species complex: a review

This paper reviews all information gathered from different disciplines and studies to resolve the species status within the Ceratitis FAR (Ceratitis fasciventris, Ceratitis anonae, Ceratitis rosa) complex, a group of polyphagous fruit fly pest species (Diptera, Tephritidae) from Africa. It includes information on larval and adult morphology, wing morphometrics, cuticular hydrocarbons, pheromones, microsatellites, developmental physiology and geographic distribution. The general consensus is that the FAR complex comprises Ceratitis anonae, two species within Ceratitis rosa (so-called R1 and R2) and two putatitve species under Ceratitis fasciventris. The information regarding the latter is, however, too limited to draw final conclusions on specific status. Evidence for this recognition is discussed with reference to publications providing further details.     

Comparative analysis of development and survival of two Natal fruit fly Ceratitis rosa Karsch (Diptera,Tephritidae) populations from Kenya and South Africa

Comparative analysis of development and survivorship of two geographically divergent populations of the Natal fruit fly Ceratitis rosa Karsch designated as Ceratitis rosa R1 and Ceratitis rosa R2 from Kenya and South Africa were studied at seven constant temperatures (10, 15, 20, 25, 30, 33, 35 °C). Temperature range for development and survival of both populations was 15–35 °C. The developmental duration was found to significantly decrease with increasing temperature for Ceratitis rosa R1 and Ceratitis rosa R2 from both countries. Survivorship of all the immature stages of Ceratitis rosa R1 and Ceratitis rosa R2 from Kenya was highest over the range of 20–30 °C (87–95%) and lowest at 15 and 35 °C (61–76%). Survivorship of larvae of Ceratitis rosa R1 and Ceratitis rosa R2 from South Africa was lowest at 35 °C (22%) and 33 °C (0.33%), respectively. Results from temperature summation models showed that Ceratitis rosa R2 (egg, larva and pupa) from both countries were better adapted to low temperatures than R1, based on lower developmental threshold. Minimum larval temperature threshold for Kenyan populations were 11.27 °C and 6.34 °C (R1 and R2, respectively) compared to 8.99 °C and 7.74 °C (R1 and R2, respectively) for the South African populations. Total degree-day (DD) accumulation for the Kenyan populations were estimated at 302.75 (Ceratitis rosa R1) and 413.53 (Ceratitis rosa R2) compared to 287.35 (Ceratitis rosa R1) and 344.3 (Ceratitis rosa R2) for the South African populations. These results demonstrate that Ceratitis rosa R1 and Ceratitis rosa R2 from both countries were physiologically distinct in their response to different temperature regimes and support the existence of two genetically distinct populations of Ceratitis rosa. It also suggests the need for taxonomic revision of Ceratitis rosa, however, additional information on morphological characterization of Ceratitis rosa R1 and Ceratitis rosa R2 is needed.     

Design, solvent free synthesis, and antimicrobial evaluation of 1,4 dihydropyridines

Here in, we report the usage of cellulose sulfuric acid as a heterogeneous eco friendly catalyst for the synthesis of 1,4 dihydropyridines under solvent free conditions via Hantzsch three component reaction of an aldehyde, ethyl acetoacetate and ammonium acetate at 100°C for 2-5h. In silico studies were performed on twenty two possible 1,4 dihydropyridines (DHPs) analogues against K(+) channel receptor (KcsA). In order to validate in silico studies, thirteen compounds were synthesized and evaluated as antibacterials against twenty seven ESBL isolates of Klebsiella pneumoniae and Escherichia coli.     

Prediction of the three-dimensional structure of aflatoxin of Aspergillus flavus by homology modelling

Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server. The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the 3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of inhibition of aflatoxin.     

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase

Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.     

Interleukin-21 enhances NK cell activation in response to antibody-coated targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.     

In vitro refolded napin-like protein of Momordica charantia expressed in Escherichia coli displays properties of native napin

Napins belong to the family of 2S albumin seed storage proteins and are shown to possess antifungal activity. Napins, in general, consist of two subunits (derived from single precursor) linked by disulphide bridges. Usually, reducing environment of the E. coli cytosol is not conducive for proper folding of heterodimeric proteins containing disulphide bridges. Present investigation reports for the first time expression of napin-like protein of Momordica charantia (rMcnapin) in E. coli and its in vitro refolding to produce biologically active protein. Full-length cDNA encoding napin-like protein (2S albumin) was isolated from M. charantia seeds by immunoscreening a cDNA expression library. The cDNA consisted of an open reading frame encoding a protein of 140 amino acid residues. The 36 amino acids at the N-terminus represent the signal and propeptide. The region encoding small and large chains of the M. charantia napin is separated by a linker of 8 amino acid residues. The region encoding napin (along with the linker) was PCR amplified, cloned into pQE-30 expression vector and expressed in E. coli. rMcnapin expressed as inclusion bodies was solubilized and purified by Ni2+-NTA affinity chromatography. The denatured and reduced rMcnapin was refolded by rapid dilution in an alkaline buffer containing glycerol and redox couple (GSH and GSSG). Refolded His-rMcnapin displayed similar spectroscopic properties as that of mature napin-like protein of M. charantia with 48.7% alpha-helical content. In addition, it also exhibited antifungal activity against T. hamatum with IC50 of 3 microg/ml. Refolded His-rMcnapin exhibited approximately 90% antifungal activity when compared with that of mature napin-like protein of M. charantia. Thus, a heterologous expression system and in vitro refolding conditions to obtain biologically active napin-like protein of M. charantia were established.