Nanda K. Menon

Nicotine and amyloid formation

The major protein constituents of amyloid deposits in Alzheimers disease (AD) are the 40-residue beta-amyloid (Abeta) (1-40) peptide and the 42-residue Abeta(1-42) peptide. The Abeta(1-42) is more pathogenic and produced in greater quantities in familial forms of AD. A major goal of research is to uncover a suitable inhibitor that either slows down or inhibits Abeta formation (beta-amyloidosis). During beta-amyloidosis, structural changes associated with the conversion of monomeric Abeta peptide building blocks into the aggregated fibrillar beta-sheet structures occur (alpha-helix-->beta-sheet or random, extended chain-->beta-sheet). In previous work, we and others established that nicotine, a major component of cigarette smoke, inhibits beta-amyloidosis of the Abeta(1-42), which may result from nicotine binding to the alpha-helical structure. These conclusions were based on solution nuclear magnetic resonance (NMR) spectroscopic studies with the nonnative 28-residue Abeta(1-28). This information suggests that, when administered therapeutically to AD patients, nicotine may not only affect cholinergic activation, but could also conceivably alter amyloid deposition. In this report, NMR studies were augmented with the naturally occurring Abeta(1-42), under conditions where the peptide folds into a predominantly alpha-helical or random, extended chain structure. The major result is that nicotine shows only modest binding to these conformations, indicating that the nicotine inhibition to beta-amyloidosis probably results from binding to a small, soluble beta-sheet aggregate that is NMR invisible.

Metabolic Responses to Intracerebroventricular Leptin and Restricted Feeding

Leptin is a hormone secreted by adipocytes, which plays an important role in the control of food intake and metabolic processes. In the current study, a dose-dependent relationship was shown between a bolus intracerebroventricular rat recombinant leptin administration and reductions in food intake and body weight in Sprague-Dawley rats. During the 24 h postinjection period, food intake was decreased by 24, 26, and 52% with 0.625, 2.5, and 10 microg of leptin, respectively. Body weight was reduced by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 microg, respectively. Furthermore, indirect calorimetry demonstrated that five daily i.c.v. injections of leptin resulted in an increase in heat production per unit of metabolic body size and fat oxidation by approximately 10 and 48%, respectively. In contrast, food-restricted rats that consumed the equivalent amount of food as leptin-treated rats for 5 days decreased their energy expenditure by 10%. Food restriction was found to decrease respiratory quotient in a similar pattern as the leptin administration. When ad lib feeding was resumed, food-restricted rats quickly recovered their normal food intakes, body weights, and metabolism. Conversely, leptin treatment has prolonged effects on body weight resulting from different metabolic responses than food restriction. Leptin not only suppresses food intake, but also enhances energy expenditure to reduce fat depots.

Carboxy-terminal processing of the large subunit of [NiFe] hydrogenases

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigas have been detected by Western analysis. The faster moving form co‐migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C‐terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C‐terminal cleavage between His536 and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582 and Val583, in the E. coli hydrogenase‐1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

IDENTIFICATION OF THREE CLASSES OF HYDROGENASE IN THE GENUS, DESULFOVIBRIO

A comparison of amino-terminal amino acid sequences from the large and small subunits of hydrogenases from Desulfovibrio reveals significant differences. These results, in conjunction with antibody analyses, clearly indicate that the iron, iron + nickel, and iron + nickel + selenium containing hydrogenases represent three distinct classes of hydrogenase in Desulfovibrio.

Cloning, sequencing and overexpression of the Desulfovibrio gigas ferredoxin gene in E. coli

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin‐labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M r = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe‐4S] cluster which is characteristic of native D. gigas ferredoxin II.