Evaluation of RAPD-PCR and protein profile analysis to differentiate <Emphasis Type="Italic">Vibrio harveyi</Emphasis> strains prevalent along the southwest coast of India

Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

The formin DAD domain plays dual roles in autoinhibition and actin nucleation

Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.     

Reactions of reactive oxygen species (ROS) with curcumin analogues: Structure-activity relationship

Three curcumin analogues viz., bisdemethoxy curcumin, monodemethoxy curcumin, and dimethoxycurcumin that differ at the phenolic substitution were synthesized. These compounds have been subjected for free radical reactions with DPPH radicals, superoxide radicals (O(2)(?-)), singlet oxygen ((1)O(2)) and peroxyl radicals (CCl(3)O(2)(?)) and the bimolecular rate constants were determined. The DPPH radical reactions were followed by stopped-flow spectrometer, (1)O(2) reactions by transient luminescence spectrometer, and CCl(3)O(2)(?) reactions using pulse radiolysis technique. The rate constants indicate that the presence of o-methoxy phenolic OH increases its reactivity with DPPH and CCl(3)O(2)(?), while for molecules lacking phenolic OH, this reaction is very sluggish. Reaction of O(2)(?-) and (1)O(2) with curcumin analogues takes place preferably at β-diketone moiety. The studies thus suggested that both phenolic OH and the β-diketone moiety of curcumin are involved in neutralizing the free radicals and their relative scavenging ability depends on the nature of the free radicals.     

Structural characterization and study of immunoenhancing and antioxidant property of a novel polysaccharide isolated from the aqueous extract of a somatic hybrid mushroom of Pleurotus florida and Calocybe indica variety APK2

A water-soluble polysaccharide was isolated from the aqueous extract of the fruit bodies of somatic hybrid PCH9FB, obtained through intergeneric protoplast fusion between the strains Pleurotus florida and Calocybe indica var. On the basis of total acid hydrolysis, the polysaccharide was found to contain galactose, fucose, and glucose in a molar ratio of nearly 2:1:2. Methylation analysis and NMR experiments ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) showed that the structure of the repeating unit present in the polysaccharide was This molecule showed macrophage, splenocyte, thymocyte activation as well as antioxidant property.     

Behavioral response of female Helicoverpa armigera (Hübner) to different plant part extracts of African marigold Tagetes erecta (L.)

The flowering stage of African marigold Tagetes erecta (L.) was the most preferred for oviposition by Helicoverpa armigera (Hübner). The lower sides of leaf margins received more eggs compared to buds, stem tip and flowers. Olfactometer studies revealed that more female moths were attracted towards the flower extracts, followed by leaf, bud and stem tip extracts of African marigold. The thin layer chromatography analysis of these extracts showed a blend of compounds including careen, ocimene, methyl benzoate, phenyl acetaldehyde and phenyl ethanol. Attempts have been made to correlate insect behavior with the presence of these compounds.     

Interaction properties of biosynthesized cadmium sulphide quantum dots with human serum albumin: further investigation of antibacterial activities and sensing applications

The synthesis of small-sized quantum dots (QDs) (1–10 nm) via the green route has garnered great interest regarding their prospective use in many biological applications (diagnosis, drug delivery and in vivo sensing); this is difficult to achieve using chemical synthesis methods, which produce larger size QD particles and also require hazardous reagents. Here, we synthesized biogenic cadmium sulphide (CdS) QDs using green tea extract as the reducing agent to produce particles that were homogeneous and a smaller size of 2–4 nm. We also elucidated the (a) protein binding, (b) antibacterial use and (c) sensing applications of biogenic CdS QDs in this present work. The biosynthesized CdS QDs were found to have extensive antibacterial activity against both Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial strains. The introduction of QDs in biological medium can lead to the formation of protein–QD complexes; therefore we investigated the binding interaction of CdS QDs with the carrier protein human serum albumin (HSA) in vitro. The synthesized CdS QDs quenched the intrinsic fluorescence of HSA through a static quenching mechanism and the binding constant (K_b) was in the order of 10⁴ M⁻¹. It was also observed that the presence of biogenic CdS QDs affected the HSA–ligand interactions in vitro. The synthesized CdS made highly effective sensors for tetracycline, rifampicin, and bilirubin with limit of detection (LOD) values of 99, 141 and 29 ng/ml, respectively.     

Circular Dichroism Studies of the Structure of DNA Complex with Berberine

Abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs.

On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Effect of Portal Glucose Sensing on Systemic Glucose Levels in SD and ZDF Rats

BackgroundThe global epidemic of Type-2-Diabetes (T2D) highlights the need for novel therapeutic targets and agents. Roux-en-Y-Gastric-Bypass (RYGB) is the most effective treatment. Studies investigating the mechanisms of RYGB suggest a role for post-operative changes in portal glucose levels. We investigate the impact of stimulating portal glucose sensors on systemic glucose levels in health and T2D, and evaluated the role of sodium-glucose-cotransporter-3 (SGLT3) as the possible sensor.MethodsSystemic glucose and hormone responses to portal stimulation were measured. In Sprague-Dawley (SD) rats, post-prandial state was simulated by infusing glucose into the portal vein. The SGLT3 agonist, alpha-methyl-glucopyranoside (αMG), was then added to further stimulate the portal sensor. To elucidate the neural pathway, vagotomy or portal denervation was followed by αMG+glucose co-infusion. The therapeutic potential of portal glucose sensor stimulation was investigated by αMG-only infusion (vs. saline) in SD and Zucker-Diabetic-Fatty (ZDF) rats. Hepatic mRNA expression was also measured.ResultsαMG+glucose co-infusion reduced peak systemic glucose (vs. glucose alone), and lowered hepatic G6Pase expression. Portal denervation, but not vagotomy, abolished this effect. αMG-only infusion lowered systemic glucose levels. This glucose-lowering effect was more pronounced in ZDF rats, where portal αMG infusion increased insulin, C-peptide and GIP levels compared to saline infusions.ConclusionsThe portal vein is capable of sensing its glucose levels, and responds by altering hepatic glucose handling. The enhanced effect in T2D, mediated through increased GIP and insulin, highlights a therapeutic target that could be amenable to pharmacological modulation or minimally-invasive surgery.     

A switch from α‐helical to β‐strand conformation during co‐translational protein folding

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo‐EM structure determination to show that folding of a β‐barrel protein begins with formation of a dynamic α‐helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N‐terminal part of the nascent chain refolds to a β‐hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α‐helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl‐transferase center suggest that protein folding could modulate ribosome activity.