A quantitating reagent for methanethiolation of protein sulfhydryl groups

p-Nitrophenoxycarbonyl methyl disulfide has been synthesized for use as a quantitating agent for methanethiolation of protein sulfhydryl groups. This reagent reacts specifically and quantitatively with cysteine residues of proteins to yield an unsymmetrical disulfide containing a CH₃S group and concomitantly releases the chromophore, p-nitrophenol. Titration of the sulfhydryl groups of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) with this reagent has been studied. Incorporation of CH₃S as measured by the release of p-nitrophenol paralleled the loss of sulfhydryl group dependent activity of the enzyme. The enzyme was found inactive on modification of four of the eight sulfhydryl groups present in the enzyme. Stability of p-nitrophenoxycarbonyl methyl disulfide has also been studied in different buffer systems. The rate of decomposition of the p-nitrophenyl ester due to hydrolysis was found negligible below a pH of 8.0 compared to its rate of reaction with free sulfhydryl groups.     

Anthocyanins in Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida fruits with health benefits

The anthocyanins in native Cornus alternifolia, Cornus controversa, Cornus kousa and Cornus florida were quantified by HPLC and characterized by spectroscopic methods. The analyses of C. alternifolia and C. controversa revealed that both contained , and , respectively. Similarly, C. florida and C. kousa showed identical anthocyanin profiles with major anthocyanins as and cyanidin 3-O-glucoside (6), respectively. The amount of anthocyanins , and in C. alternifolia and C. controversa were 8.21, 8.44 and 0.02 mg; and 7.74, 5.92, and 0.02 mg/g of fresh fruits, respectively. The anthocyanins and in C. kousa and C. florida were 0.02 and 0.16 mg; and 0.62 and 0.03 mg/g fresh fruits, respectively. Anthocyanins and were not studied earlier for their inhibition of lipid peroxidation, cyclooxygenase enzymes (COX-1 and COX-2), and tumor cell proliferation. At 50 microg/mL, anthocyanins and inhibited lipid peroxidation by 71% and 68%, respectively. Similarly, they inhibited COX-1 enzymes by 39% and 49% and COX-2 enzyme by 54% and 48%, respectively, at 100 microg/mL. Anthocyanin displayed 50% growth inhibition (IC(50)) at 21, 25, 50, 60, and 75 microg/mL, against HCT-116 (colon), MCF-7 (breast), NCI-H460 (lung), SF-268 (central nervous system CNS), and AGS (stomach) human tumor cell lines, respectively. Similarly, IC(50) values for anthocyanin were 38, 30, 76, 100, and 100 microg/mL against HCT-116, MCF-7, NCI H460, SF-268, and AGS, respectively. This is the first report of the quantification and biological activities of anthocyanins in C. alternifolia, C. kousa and C. florida in addition to the anthocyanins not previously quantified in C. controversa.     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.     

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.     

Observations on the incidence and seasonal fluctuations of certain crustacean larvae in the plankton of the South-West Coast of India

The seasonal fluctuations in the incidence of planktonic organisms and the larval forms of certain crustaceans such as crabs, barnacles and post larvae of prawns in the plankton of the south-west coast of India have been followed for a period of three years from 1963. South-west monsoon period is the least productive period for zooplankton in this area. Brachyuran zoeae, post larvae of prawns and barnacle nauplii occur in the plankton throughout the year with distinct peaks for different groups. The zoeae ofUca annulipes occur in the plankton from September to May with a peak during November–December. The zoeae ofPortunus pelagicus are present in the plankton from September to June and their abundance is in February–March. The post larvae ofMetapenaeus affinis are found in the inshore plankton from November to June with the peak in March. The nauplii ofBalanus amphitrite communis occur in the plankton in all the months of the year, the peak incidence has been during November January. The zoeae ofU. annulipes are found to tolerate better the medium saline conditions. Of the ecological factors, salinity of the ambient water and the availability of planktonic food for the larvae seem to influence the seasonal fluctuations of these crustacean larvae in this locality.     

Proteasomal dysfunction and ER stress triggers 2′-hydroxy-retrochalcone-induced paraptosis in cancer cells

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2′-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.     

Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies

Alkaline phosphatase (AP) was purified to over 90% homogeneity from rat osteosarcoma by acetone precipitation followed by chromatography on DEAE-cellulose, Sephacryl S-200, and hydroxyapatite. The purified enzyme had a specific activity of 759 units/mg protein at its optimal pH (10.5), and a Km of 0.8 mM for p-nitrophenylphosphate. The enzyme's apparent subunit molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82,000 Da. The heat-inactivation profile and homoarginine inhibition were characteristic of the bone-liver-kidney AP isoenzyme. Monoclonal and polyclonal anti-AP antibodies were prepared and characterized. Polyclonal rabbit antiserum quantitatively precipitated the activity from purified AP preparations and tissue extracts but did not inhibit AP catalytic activity. This antiserum was almost 10-fold less active against heat-inactivated enzyme when tested in a competition assay using 125I-AP. Two distinct monoclonal antibodies were each partly effective in immunoprecipitating AP when tested individually; however, together they precipitated over 90% of the AP activity.