Recombinant cathepsin E has no proteolytic activity at neutral pH

Cathepsin E (CatE) is a major intracellular aspartic protease reported to be involved in cellular protein degradation and several pathological processes. Distinct cleavage specificities of CatE at neutral and acidic pH have been reported previously in studies using CatE purified from human gastric mucosa. Here, in contrast, we have analyzed the proteolytic activity of recombinant CatE at acidic and neutral pH using two separate approaches, RP-HPLC and FRET-based proteinase assays. Our data clearly indicate that recombinant CatE does not possess any proteolytic activity at all at neutral pH and was unable to cleave the peptides glucagon, neurotensin, and dynorphin A that were previously reported to be cleaved by CatE at neutral pH. Even in the presence of ATP, which is known to stabilize CatE, no proteolytic activity was observed. These discrepant results might be due to some contaminating factor present in the enzyme preparations used in previous studies or may reflect differences between recombinant CatE and the native enzyme.     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.     

H. pylori‐Induced Apoptosis in Human Gastric Cancer Cells Mediated via the Release of Apoptosis‐Inducing Factor from Mitochondria

Background and Aim: Our previous study of Helicobacter pylori‐induced apoptosis showed the involvement of Bcl‐2 family proteins and cytochrome c release from mitochondria. Here, we examine the release of other factors from mitochondria, such as apoptosis‐inducing factor (AIF), and upstream events involving caspase‐8 and Bid. Methods: Human gastric adenocarcinoma (AGS) cells were incubated with a cagA‐positive H. pylori strain for 0, 3, 6, and 24 hours and either total protein or cytoplasmic, nuclear, and mitochondrial membrane fractions were collected. Results: Proteins were immunoblotted for AIF, Bid, polyadenosine ribose polymerase (PARP), caspase‐8, and β‐catenin. H. pylori activated caspase‐8, caused PARP cleavage, and attenuated mitochondrial membrane potential. A time‐dependent decrease in β‐catenin protein expression was detected in cytoplasmic and nuclear extracts, coupled with a decrease in β‐actin. An increase in the cytoplasmic pool of AIF was seen as early as 3 hours after H. pylori exposure, and a concomitant increase was seen in nuclear AIF levels up to 6 hours. A band corresponding to full‐length Bid was seen in both the cytoplasmic and the nuclear fractions of controls, but not after H. pylori exposure. Active AIF staining was markedly increased in gastric mucosa from infected persons, compared to uninfected controls. Conclusion: H. pylori might trigger apoptosis in AGS cells via interaction with death receptors in the plasma membrane, leading to the cleavage of procaspase‐8, release of cytochrome c and AIF from mitochondria, and activation of subsequent downstream apoptotic events, as reported previously for chlorophyllin. This is consistent with AIF activation that was found in the gastric mucosa of humans infected with H. pylori. Hence, the balance between apoptosis and proliferation in these cells may be altered in response to injury caused by H. pylori infection, leading to an increased risk of cancer.     

Comparative in-vitro biodegradation studies of epoxy and its silicone blend by selected microbial consortia

A total of six bacterial isolates were developed into two consortia and tested for utilization of epoxy silicone blends (ESBs; % w/w: 3.0) and epoxy as the sole carbon source. In-vitro biodegradation studies in minimal broth revealed that higher biomass and more sustained growth of consortia were obtained in the presence of epoxy and/or ESBs when these were incubated under aerobic conditions for 15 days. Treated samples were analyzed by Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric–differential thermogravimetry–differential thermal analysis (TG–DTG–DTA), which indicated the breakage and formation of bonds in the polymer backbone. Moreover, a weight loss of 34.17 and 36.9% was found in epoxy and ESBs, respectively after 15 days of treatment with consortium-1. Further, in-vitro growth statistics study revealed more CFU count at mid-logarithmic phase in the presence of epoxy/ESBs unlikely to the absence of the polymers. However, the generation time was not affected. In the present study, consortium-1, comprising of Microbacterium sp., Pseudomonas putida and Bacterium Te 68R showed better biodegradation in comparison to consortium-2, wherein, P. putida and Pseudomonas aeruginosa were present. Overall, these results suggest that epoxy/ESBs polymers could be degraded by a biologically mediated process if a suitable consortium is used.     

A molecular evaluation of dengue virus pathogenesis and its latest vaccine strategies

More than one third of the world’s population living in tropical and subtropical areas of the world is at risk of dengue infections and as many as 100 million people are yearly infected. This disease has reemerged during the past 20 years in the form of an epidemic. Dengue is caused by one of four related serotypes of dengue virus and often leads to severe forms of the disease, resulting commonly from secondary infections. Dengue virus is a mosquito borne virus, belongs to the family Flaviviridae and consists of a single stranded positive sense RNA genome. Like other RNA viruses it escapes defense mechanisms and neutralization attempts by mutations, which make it more resistant and adaptable to its environment. Antiviral strategies and vaccine development is thus impaired and hence to date there is no licensed vaccine available for dengue virus. Here we discuss various efforts made towards the identification of potential vaccine targets for dengue as well as various strategies employed by research groups/pharmaceutical companies towards the development of a successful dengue vaccine.     

Neural surveillance of skeletal homeostasis

Endowed with sympathetic and peptidergic nerves, the vertebrate skeleton is under constant surveillance by the nervous system. In addition to pituitary hormone secretion, centrally regulated sympathetic release, as elegantly demonstrated by Karsenty and colleagues, integrate to control both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption (Elefteriou et al., 2005).