Spectrofluorimetric method for the estimation of total lipids in Eremothecium ashbyii fungal filaments using Nile blue and avoiding interference of autofluorescent riboflavin

A rapid, simple and sensitive spectrofluorimetric technique was developed for monitoring total lipids in hyphae of the riboflavin-overproducing fungus Eremothecium ashbyii using the fluorescent probe Nile blue in an aqueous system, avoiding the interference due to autofluorescent riboflavin. The existing methodologies for lipid estimation are tedious, requiring large biomass, solvent extraction and gravimetry. E. ashbyii is a hemiascomycete fungus which accumulates lipids in its mycelia prior to flavinogenesis. This study defines the conditions (wavelength selection and sensitivity) for the spectrofluorimetric quantification of lipids in situ in the macerated mycelia of this fungus in the presence of intracellular autofluorescent riboflavin without the need to extract the lipids from the mycelia. The fluorescent intensity was linear with the lipid concentration of the mycelia (by gravimetry) under three different growth conditions using glucose, olive oil and sunflower oil as carbon sources. This spectrofluorimetic method of lipid estimation can be applied to other fungi and microorganisms.     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells

Summary Felal calf serum (FCS) is conventionally used for animal cell cultures due to its inherent growth-promoting activities. However animal welfare issues and stringent requirements for human transplantation studies demand a suitable alternative for FCS. With this view, we studied the effect of FCS, human AB serum (ABS), and human umbilical cord blood serum (UCBS) on murine islets of Langerhans and human bone marrow-derived mesenchymal-like cells (hBMCs). We found that there was no difference in morphology and functionality of mouse islets cultured in any of these three different serum supplements as indicated by insulin immunostaining. A comparative analysis of hBMCs maintained in each of these three different serum supplements demonstrated that UCBS supplemented media better supported proliferation of hBMCs. Moreover, a modification of adipogenic differentiation protocol using UCBS indicates that it can be used as a supplement to support differentiation of hBMCs into adipocytes. Our results demonstrate that UCBS not only is suitable for maintenance of murine pancreatic islets, but also supports attachment, propagation, and differentiation of hBMCs in vitro. We conclude that UCBS can serve as a better serum supplement for growth, maintenance, and differentiation of hBMCs, making it a more suitable supplement in cell systems that have therapeutic potential in human transplantation programs.     

α-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes

The effects of ₁ and ₂ receptor ligands on Ca²⁺/Mg²⁺-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca²⁺/Mg²⁺-ATPase, in a concentration-dependent fashion. IC₅₀ values for half-maximal inhibition for phenylephrine and clonidine were 29 M and 18 M, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca²⁺]. The present study provides biochemical evidence to support auto receptor adrenergic receptor regulation of neurotransmitter release.     

Ligand‐based pharmacophore modelling and screening of DNA minor groove binders targeting Staphylococcus aureus

The recognition of DNA by small molecules is of special importance in the design of new drugs. Many natural and synthetic compounds have the ability to interact with the minor groove of DNA. In the present study, identification of minor groove binding compounds was attained by the combined approach of pharmacophore modelling, virtual screening and molecular dynamics approach. Experimentally reported 32 minor groove binding compounds were used to develop the pharmacophore model. Based on the fitness score, best three pharmacophore hypotheses were selected and used as template for screening the compounds from drug bank database. This pharmacophore‐based screening provides many compounds with the same pharmacological properties. All these compounds were subjected to four phases of docking protocols with combined Glide‐quantum‐polarized ligand docking approach. Molecular dynamics results indicated that selected compounds are more active and showed good interaction in the binding site of DNA. Based on the scoring parameters and energy values, the best compounds were selected, and antibacterial activity of these compounds was identified using in vitro antimicrobial techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

Chromosomal translocations in cancer

Genetic alterations in DNA can lead to cancer when it is present in proto-oncogenes, tumor suppressor genes, DNA repair genes etc. Examples of such alterations include deletions, inversions and chromosomal translocations. Among these rearrangements chromosomal translocations are considered as the primary cause for many cancers including lymphoma, leukemia and some solid tumors. Chromosomal translocations in certain cases can result either in the fusion of genes or in bringing genes close to enhancer or promoter elements, hence leading to their altered expression. Moreover, chromosomal translocations are used as diagnostic markers for cancer and its therapeutics. In the first part of this review, we summarize the well-studied chromosomal translocations in cancer. Although the mechanism of formation of most of these translocations is still unclear, in the second part we discuss the recent advances in this area of research.     

Non‐Tuberculosis mycobacterium speciation using HPLC under Revised National TB Control Programme (RNTCP) in India

Aims

Non‐Tuberculous Mycobacteria (NTM) are ubiquitous in nature. The data on prevalence of NTM under the RNTCP is scarce. Many NTM species have clinical significance, and hence their identification and speciation are important.

Methods and Results

It is a cross‐sectional study conducted at the five RNTCP accredited culture and drug susceptibility testing (CDST) laboratory. The culture isolates from AFB positive but Immunochromatographic test negative samples were taken for identification and speciation using HPLC. Of the total 266 isolates only 164 isolates had a second sample received at the laboratory. The speciation was done using HPLC for those isolates. The type of species identified are: 26·8% (44) were Mycobacterium chelonae, 12·8% (21) were Mycobacterium fortuitum, 9% (15) were Mycobacterium gordonae, 9% (15) were Mycobacterium tuberculosis complex, 6·1% (10) were Mycobacterium kansasii, 4·9% (8) were Mycobacterium simiae, 2·4% (4) were Mycobacterium thermophile, 1·2% (2) were Mycobacterium gastri, 0·6% (1) were Mycobacterium scrofulaceum, 0·6% (1) were Mycobacterium avium and 4·9% (8) isolates had chromatogram which was un‐interpretable.

Conclusion

Identification and its speciation of NTM are not routinely done under TB control programme. Since HPLC could identify 95% of isolates belonging to 10 species, the speciation of NTM using HPLC should gain importance in the diagnosis of disease caused by NTM.

Significance and Impact of Study

NTM are emerging as important causative agents of pulmonary and extra pulmonary disease, the ability to recognize disease caused by NTM and subsequently treat such disease has become increasingly important. The identification of NTM up to its species level should gain importance in all TB reference Laboratories.