Effects of stress hormone cortisol on the mRNA expression of myogenenin,MyoD, Myf5, PAX3 and PAX7

The present investigation was carried out to evaluate the effect of stress hormone cortisol on the myogenic markers in the C2C12 cells co-cultured with 3T3-L1 preadipocytes. Co-culturing was achieved by transwell inserts with a 0.4 μm porous membrane. C2C12 and 3T3-L1 cells were grown independently on the transwell plates. After differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates for co-culturing. 10 μg/μl of cortisol was added to the medium. After 72 h of treatment, C2C12 cells which were in the lower well were harvested for analysis. RT-PCR analysis of myogenic markers such as of myogenin, MyoD, Myf5, PAX3 and PAX7 showed a significant reduction in the mRNA expression of these myogenic markers. In addition, cortisol increased calpain activity, which led to accelerated protein degradation, which in turn reduced the myogenic rate. In conclusion, cortisol treatment reduced mRNA expression of myogenic markers in the co-cultured C2C12 cells, which is quite distinct from one dimensional mono-cultured C2C12 cells.     

Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum.     

Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.     

Prevalence of Pulmonary Tuberculosis among Adults in a Rural Sub-District of South India

Background

We conducted a survey to estimate point prevalence of bacteriologically positive pulmonary TB (PTB) in a rural area in South India, implementing TB program DOTS strategy since 2002.

Methods

Survey was conducted among persons ≥15 years of age in fifteen clusters selected by simple random sampling; each consisting of 5–12 villages. Persons having symptoms suggestive of PTB or history of anti-TB treatment (ATT) were eligible for sputum examination by smear microscopy for Acid Fast Bacilli and culture for Mycobacterium tuberculosis; two sputum samples were collected from each eligible person.Persons with one or both sputum specimen positive on microscopy and/or culture were labeled suffering from PTB. Prevalence was estimated after imputing missing values to correct for bias introduced by incompleteness of data.In six clusters, registered persons were also screened by X-ray chest. Persons with any abnormal shadow on X-ray were eligible for sputum examination in addition to those with symptoms and ATT. Multiplication factor calculated as ratio of prevalence while using both screening tools to prevalence using symptoms screening alone was applied to entire study population to estimate prevalence corrected for non-screening by X-ray.

Results

Of 71,874 residents ≥15 years of age, 63,362 (88.2%) were screened for symptoms and ATT. Of them, 5120 (8.1%) - 4681 (7.4%) with symptoms and an additional 439 (0.7%) with ATT were eligible for sputum examination. Spot specimen were collected from 4850 (94.7%) and early morning sputum specimens from 4719 (92.2%). Using symptom screening alone, prevalence of smear, culture and bacteriologically positive PTB in persons ≥15 years of age was 83 (CI: 57–109), 152 (CI: 108–197) and 196 (CI :145–246) per 100,000 population respectively. Prevalence corrected for non-screening by X-ray was 108 (CI: 82–134), 198 (CI: 153–243) and 254 (CI: 204–301) respectively.

Conclusion

Observed prevalence suggests further strengthening of TB control program.     

Stereochemistry of α-aminoisobutyric acid peptides in solution: Conformations of decapeptides with a central triplet of contiguous L-amino acids

The decapeptides Boc-Aib-L -Val-Aib-Aib-(L -Val)₃-Aib-L -Val-Aib-OMe and Boc-Aib-L -Leu-Aib-Aib-(L -Leu)₃-Aib-L -Leu-Aib-OMe have been studied in CDCl₃ and (CD₃)₂SO solutions by 270-MHz¹ H-nmr. In CDCl₃, the presence of eight intramolecularly hydrogen-bonded NH groups has been established, consistent with a 3₁₀-helical conformation, for both decapeptides. In (CD₃)₂SO, only seven solvent-shielded NH groups are observed, supporting either an α-helical conformation or a partially unfolded 3₁₀-helix. Ir studies provided supporting evidence for intramolecularly hydrogen-bonded structures in CHCl₃, while CD studies suggest helical conformation in both decapeptides in various solvents. CD studies also support helical folding in the C-terminal hexapeptides. The central triplet of L-amino acids appears to destabilize 3₁₀-helical conformations in polar solvents like (CD₃)₂SO.     

Conformational transitions in a Pro-Pro peptide on dissolution of single crystals

The peptide t-butyloxycarbonyl-α-aminoisobutyryl-L-prolyl-L-prolyl-N-methylamide has been shown to adopt an extended structure in the solid state. The Pro-Pro segment occurs in the poly-proline II conformation. On dissolution of single crystals at ～ 233°K, a single species corresponding to the all $\text{trans}$ peptide backbone is observed by 270 MHz ¹H NMR. On warming, $\text{trans}$ to $\text{cis}$ isomerization about the Pro-Pro bond is facilitated. Both $\text{cis'}$ (ψ ～?50°) and $\text{trans'}$ (ψ ～ 130°) rotamers about the Pro³ C^αCO bond are detectable in the Pro-Pro $\text{cis}$ conformer, at low temperature. These observations demonstrate unambiguously the large differences in the solid state and solution conformations of a Pro-Pro sequence.     

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.     

Turritella attenuata (Kasinathan): as biological indicator of marine pollution--a trace metal analytical study

A study to monitor marine pollution with reference to trace elements (Fe, Zn, Mn and Cu) on T. attenuata, commonly called as screw shell over a period of one year on the whole body and various organs, viz. digestive diverticula, foot, mantle and ovary was conducted from the sandy beach of Porto Novo Coast (Lat 11 degrees 29' N Long: 79 degrees 46' E) of Peninsular India using Atomic Absorption Spectrophotometer (AAS). Higher concentration of all the four trace metals analysed were recorded in the digestive diverticula, whereas lower concentration of zinc and manganese were recorded in the ovary during the monsoon period. The higher level of trace metal concentration in the monsoon period may be due to the presence of these pollutants in large amounts in water. The accumulation of selected trace metals varies in different seasons according to the extent of pollution load in the marine environment.