Metastatic mouse melanoma cells release collagen-gelatin degrading metalloproteinases as components of shed membrane vesicles

The purpose of this study has been to compare collagen-gelatin degrading enzymes isolated from cancer cell organelles and cytosol to the metalloproteinases released by cancer cells. To this end, metastatic mouse melanoma cell organelles were isolated by sucrose density gradient centrifugation and metalloproteinases were assayed using native and denatured [methyl-3H]collagen substrates. Solubilized proteinases were purified by ammonium sulfate precipitation, anion exchange, concanavalin A affinity and gel-filtration column chromatographic procedures and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The conclusions were as follows: malignant melanoma cells have a metalloproteinase (Mr = 59,000) which is shed from cells into conditioned medium as a component of intact membrane vesicles rather than as a soluble enzyme; storage of tumor-conditioned medium leads to the generation of autoactivated soluble metalloproteinases of lower molecular weight; purification of these metalloproteinase species yielded variant collagenases that have considerable gelatinolytic activity and a cleavage preference site for the Gly-Ile bond in a collagen-like synthetic octapeptide substrate which is typical for collagenase-type metalloproteinases. It is proposed that localization of potent proteinases to the surface of cancer cells facilitates the local breakdown of connective tissues during the invasive process.     

The effect of experimental diabetes on the molecular characteristics of soluble rat-tail tendon collagen

Acid soluble rat-tail tendon collagen was prepared from animals rendered diabetic by treatment with either streptozotocin or alloxan and from matched controls. In comparison to the normal, the diabetic collagens consistently demonstrated decreased solubility of reconstituted fibrils, marked increase in intrinsic viscosity and a decreased ratio of alpha to beta components. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed a marked decrease in migration of alpha1, alpha2, and beta components from both types of diabetic collagen. These data indicate that diabetic collagens are larger than normal and are capable of higher degrees of polymerization due to increased intra- and inter-molecular interactions. These changes could explain, in part, the altered response of diabetic connective tissues to inflammation and trauma.     

Crystal engineering principles applied to solution photochemistry: controlling the photodimerization of stilbazolium salts within gamma-cyclodextrin and cucurbit[8]uril in water.

Borrowing concepts from crystal engineering techniques we have been able to steer the photodimerization of stilbazolium salts included in gamma-cyclodextrin towards a desired dimer.     

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     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.     

Increasing productivity of Spirulina platensis in photobioreactors using artificial neural network modeling

Although production of microalgae in open ponds is conventionally practiced due to its economy, exposure of the algae to uncontrollable elements impedes achievement of quality and it is desirable to develop closed reactor cultivation methods for the production of high value products. Nevertheless, there are several constraints which affect growth of in closed reactors, some of which this study aims to address for the production of Spirulina. Periodic introduction of fresh medium resulted in increased trichome numbers and improved algal growth compared to growth in medium that was older than 4 weeks in 20 L polycarbonate bottles. Mixing of the cultures by bubbling air and use of draft tube reduced the damage to the growing cells and permitted increased growth. However, there was better growth in inclined cylindrical reactors mixed with bubbling air. The oxygen production rates were very similar irrespective differences in the maintained cultures densities. The uniformity in oxygen production rate suggested a tendency towards homeostasis in Spirulina cultures. The frequency of biomass harvest on the productivity of Spirulina showed that maintenance of moderate culture density between 0.16 and 0.32 g/L resulted in about 14% more productivity than maintaining the cell density between 0.16 and 0.53 g/L or 48% more than by daily harvest above 0.16 g/L. An artificial neural network based predictive model was developed, and the variables useful for predicting biomass output were identified. The model could predict the growth of Spirulina up to 3 days in advance with a coefficient of determination >0.94.     

Anti-inflammatory,anti-oxidant effect and cytotoxicity of ocimum sanctum intra oral gel for combating periodontal diseases

It is of interest to evaluate the anti-inflammatory, anti-oxidant effect and cytotoxicity of Ocimum sanctum (an Indian herb, Thulsi) intra oral gel in combating periodontal diseases. Hence, 2% of O. sanctum gel was prepared with Carbopol940 soaked in purified water containing 0.2% w/v sodium benzoate overnight. Hydroxy proplyl methyl cellulose (HPMC) solution was mixed with propylene glycol using using tissue homogenizer. Anti-oxidant effect was analyzed using DPPH radical assay and anti-inflammatory effect was assessed using the inhibition of albumin denaturation assay. Ocimum sanctum gel with various dilutions from10 micro litres to 50 micro litres showed exponential increase in percentage of inhibition from 60.9 to 72.2 exhibiting antioxidant activity. The anti-inflammatory effect of Ocimum sanctum gel showed comparatively equivalent effect with standard diclofenac gel with values ranging from 76.6 for 50 micro liters of Ocimum sanctum gel and 89.6 for standard gel at 50 micro liters. Ocimum sanctum showed less toxicity towards brine shrimp nauplii. Thus we show that Ocimum sanctum gel showed potent anti-oxidant and anti-inflammatory effect and less toxic to brine shrimp nauplii as a promising agent for the treatment of periodontal diseases.     

Photophysics of ruthenium(II) complexes carrying amino acids in the ligand 2,2′-bipyridine and intramolecular electron transfer from methionine to photogenerated Ru(III)

New ruthenium(II) complexes carrying methionine and phenylalanine in the bipyridine ligand, [Ru(bpy)₂(4-Me-4′-(CONH-l-methionine methyl ester)-2,2′-bipyridine)](PF₆)₂ (IV) and [Ru(bpy)₂(4-Me-4′-(CONH-l-phenylalanine ethyl ester)-2,2′-bpy)](PF₆)₂(V) have been synthesized and characterized and their photophysical properties studied. Flash photolysis measurements of complex IV, in the presence of an electron acceptor, methyl viologen (MV²⁺) show that an intermolecular electron transfer from the excited state of Ru(II) in complex IV, to MV²⁺ takes place, forming Ru(III) and the methyl viologen cation radical, MV⁺. The formation of MV⁺ in this system is confirmed using time-resolved transient absorption spectroscopy. This intermolecular electron transfer is followed by intramolecular electron transfer from the thioether moiety (methionine) to the photogenerated Ru(III), regenerating Ru(II).