Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,³J_{HN.C 2}H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the C^αH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu⁵]- and [Met⁵]-enkephalinamides.     

Validation of coupled bioassay for inhibin by pituitary cell culture assay

Studies on the characterization of inhibin and inhibin-like factors have depended for the most part on the classicalin vitro pituitary cell culture assay. A major drawback with this assay is the turn-around time which is in the order of two weeks and consequently slows down purification efforts. The 24 h bioassay for inhibin has been found to be sufficiently sensitive and also statistically valid. Unfortunately, based as it is on a secondary response, ambiguities arise in interpreting the results. By including a parallel assay in which the mice are primed with human menopausal gonadotropin rather than human chorionic gonadotropin, it was possible to device the coupled bioassay. This enables distinguishing inhibin-like factors acting to suppress pituitary follicle stimulating hormone output from those acting at the level of gonads. In this study the coupled assay for inhibin has been compared with the classical pituitary cell culture assay in order to assess its biological and statistical validity. The data validates the bioassay on both the above counts and when considered in conjunction with the short turn-around time suggests that this assay can be highly useful in studies on isolation of inhibin from various sources.     

Hypercholesterolemia and tissue-specific differential mRNA expression of type-1 5'-iodothyronine deiodinase under different selenium status in rats

Type-1 5'-iodothyronine deiodinase (5'-DI) is responsible for conversion of T4 to T3. Selenium (Se) is an integral part of this enzyme. Keeping in view the strong association between atherosclerosis and hypothyroidism, the present study examined the behavior of 5'-DI in liver, aorta and thyroid during hypercholesterolemia following different Se status, i.e., Se deficiency (0.02 ppm), adequate (0.2 ppm) and excess dose (1 ppm) in SD male rats. Animals were fed a control or high-cholesterol diet (2%) for 1 and 2 months. 5'-DI activity and mRNA expression was measured by RIA and RT-PCR respectively. In liver and aorta, 5'-DI expression significantly decreased with the Se-deficient and the high-cholesterol diet. The trend was opposite in thyroid, i.e., mRNA expression increased significantly during selenium deficiency and with a high-cholesterol feeding. But with 1 ppm Se supplementation, the 5'-DI expression increased in all the three tissues. The present study indicates that hypercholesterolemia along with selenium deficiency is co-responsible for differential regulation of 5'-DI enzyme in thyroidal vs. extrathyroidal tissues. Distinct regulation of 5'-DI in the thyroid reflects the clinical importance of this selenoprotein during hypercholesterolemia as this enzyme is essential for T3 production, which further has a vital role in the maintenance of lipid metabolism.     

Hypercholesterolemia and LDL receptor mRNA expression: modulation by selenium supplementation

Selenium (Se) status has been associated with cardiovascular disorders. Present study was aimed to elucidate the protective role of Se supplementation on LDL receptor (LDL-R) activity as well as mRNA expression during experimental hypercholesterolemia in SD male rats. Animals were fed 0.2 and 1 ppm Se supplemented control diet as well as 2% cholesterol supplemented diet for 3 months. LDL-R activity was measured in-vivo by injecting radiolabeled LDL to rats and decrease in counts per minute with time was taken as a measure of LDL clearance and in turn LDL-R activity. LDL-R mRNA expression was studied by RT-PCR. LDL-R activity and mRNA expression decreased significantly on 2% cholesterol supplemented diet feeding. On 1 ppm Se supplementation LDL-R activity as well as mRNA expression increased significantly. Present results demonstrate that Se supplementation upto 1 ppm is responsible for up regulation of LDL-R activity as well as mRNA expression, during hypercholesterolemia. These findings highlight the therapeutic potential of Se supplementation in lipid metabolism.     

An Arabidopsis Mutant with High Cyclic Electron Flow around Photosystem I (hcef) Involving the NADPH Dehydrogenase Complex

Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A–sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.     

Cloning and characterization of lin genes responsible for the degradation of Hexachlorocyclohexane isomers by Sphingomonas paucimobilis strain B90

Hexachlorocyclohexane (HCH) has been used extensively against agricultural pests and in public health programs for the control of mosquitoes. Commercial formulations of HCH consist of a mixture of four isomers, alpha, beta, gamma, and delta. While all these isomers pose serious environmental problems, beta-HCH is more problematic due to its longer persistence in the environment. We have studied the degradation of HCH isomers by Sphingomonas paucimobilis strain B90 and characterized the lin genes encoding enzymes from strain B90 responsible for the degradation of HCH isomers. Two nonidentical copies of the linA gene encoding HCH dehydrochlorinase, which were designated linA1 and linA2, were found in S. paucimobilis B90. The linA1 and linA2 genes could be expressed in Escherichia coli, leading to dehydrochlorination of alpha-, gamma-, and delta-HCH but not of beta-HCH, suggesting that S. paucimobilis B90 contains another pathway for the initial steps of beta-HCH degradation. The cloning and characterization of the halidohydrolase (linB), dehydrogenase (linC and linX), and reductive dechlorinase (linD) genes from S. paucimobilis B90 revealed that they share approximately 96 to 99% identical nucleotides with the corresponding genes of S. paucimobilis UT26. No evidence was found for the presence of a linE-like gene, coding for a ring cleavage dioxygenase, in strain B90. The gene structures around the linA1 and linA2 genes of strain B90, compared to those in strain UT26, are suggestive of a recombination between linA1 and linA2, which formed linA of strain UT26.