Polymorphism and divergence at a Drosophila pseudogene locus

The larval cuticle protein (Lcp) cluster in Drosophila melanogaster contains four functional genes and a closely related pseudogene. A 630-bp fragment including the larval cuticle pseudogene locus (Lcp psi) was nucleotide sequenced in 10 strains of D. melanogaster and a 458-bp Lcp psi fragment from D. simulans was also sequenced. We used these data to test the hypotheses that the rates of synonymous and nonsynonymous substitution are equal, that the absolute levels of variation are higher than in functional genes, and that intraspecific polymorphism is correlated with interspecific divergence. As predicted, synonymous and nonsynonymous substitution rates were equivalent, and overall nucleotide divergence between D. melanogaster and D. simulans (Jukes-Cantor distance = 0.149 +/- 0.150) was extremely high. However, within-species DNA sequence comparisons at Lcp psi revealed lower levels of polymorphism (theta = 0.001 +/- 0.001) than at many functional loci in D. melanogaster. Using the HUDSON, KREITMAN, and AGUADE (HKA) test, we show that the level of polymorphism in Lcp psi within D. melanogaster is lower than expected given the amount of divergence between D. melanogaster and D. simulans when the pseudogene data are compared to the Adh 5' flanking region. Because the Lcp psi lies in a region of relatively infrequent recombination, we suggest that the low level of within-species polymorphism is the result of background selection.     

Peroxynitrite: a biologically significant oxidant

A prolongation of the intracellular acidosis after myocardial ischemia can protect the myocardium against reperfusion injury. In isolated hearts, this was achieved by prolongation of the extracellular acidosis. The aim of this study was to investigate whether regional reperfusion with acidotic blood after coronary artery occlusion can reduce infarct size and improve myocardial function in vivo. Anesthetized open-chest dogs were instrumented for measurement of regional myocardial function, assessed by sonomicrometry as systolic wall thickening (sWT). Infarct size was determined by triphenyltetrazolium staining after 3 h of reperfusion. The left anterior descending coronary artery (LAD) was perfused through a bypass from the left carotid artery. The animals underwent 1 h of LAD occlusion and subsequent bypass-reperfusion with normal blood (control, n = 6) or blood equilibrated to pH = 6.8 by using 0.1 mM HCl during the first 30 min of reperfusion (HCl, n = 5). Regional collateral blood flow (RCBF) at 30-min occlusion was measured by using colored microspheres. There was no difference in recovery of sWT in the LAD-perfused area between the two groups at the end of the experiments [-2.8+/-1.2% (HCl) vs. -4.4+/-2.5% (control); mean +/- SEM; p = NS]. RCBF was comparable in both groups. Infarct size (percentage of area at risk) was reduced in the treatment group (12.8+/-2.8%) compared with the control group (26.2+/-4.8%; p < 0.05). These results indicate that reperfusion injury after coronary artery occlusion can be reduced by a prolonged local extracellular acidosis in vivo.     

Modulation of the catalytic activity of brain succinic semialdehyde reductase by reaction with pyridoxal 5'-phosphate

An NADPH-dependent succinic semialdehyde reductase from bovine brain was inactivated by pyridoxal 5'-phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After sodium borohydride reduction of the inactivated enzyme, it was observed that 1 mol phosphopyridoxyl residue was incorporated/mol enzyme monomer. The coenzyme, NADPH, protected the enzyme against inactivation by pyridoxal 5'-phosphate. After tryptic digestion of the enzyme modified with pyridoxal 5'-phosphate in the presence and absence of NADPH followed by [1H]NaBH4 reduction, a radioactive peptide absorbing at 310 nm was isolated by reverse-phase HPLC. The amino acid sequence of the peptide identified a portion of the pyridoxal-5'-phosphate-binding site as the region containing the sequence I-L-E-N-I-Q-V-F-X-K, where X indicates that the phenylthiohydantoin amino acid could not be assigned. The missing residue, however, can be designated as a phosphopyridoxyl lysine as interpreted from the result of amino acid composition of the peptide. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of pyridoxal 5'-phosphate to a specific lysyl residue at or near the coenzyme-binding site of the protein.     

GLP-1 tablet in type 2 diabetes in fasting and postprandial conditions

OBJECTIVE: To examine the absorption of glucagon-like peptide (GLP)-1(7-36) amide from the buccal mucosa of type 2 diabetic patients. Previously, the effects of the peptide have been studied following intravenous and subcutaneous injection. Now, a mucoadhesive, biodegradable buccal GLP-1 tablet (9 mm) containing 119 nmol has been developed as a possible alternative to injection. RESEARCH DESIGN AND METHODS: A total of 10 type 2 diabetic patients received a single tablet under fasting conditions and before a standard meal in this randomized placebo-controlled study. RESULTS: The mean peak GLP-1 concentration was 125.1 pmol/l and occurred 30 min after application. The mean placebo-adjusted area under the curve was 5,334 min pmol/l, consistent with a relative bioavailability of 6% vs. intravenous injection and 42% vs. subcutaneous injection. The half-life of total peptide activity after buccal administration was 17 min. The placebo-adjusted glucose concentrations decreased by 1.4 mmol/l in fasting experiments and by 4.2 mmol/l after a standard mixed meal. In the fasting state at 30 min, plasma insulin increased by 185% and glucagon decreased by 20%, consistent with the increase in plasma GLP-1 concentrations. The peptide exerted a significant insulinotropic effect during meals (calculated as an insulinogenic index, 0-120 min; 84.1 vs. 45.7 in placebo experiments). CONCLUSIONS: Potentially therapeutic plasma levels of GLP-1 were achieved after administration of a single buccal tablet in type 2 diabetic patients. The peptide had a marked glucose-lowering effect during the first 2 h. This new GLP-1 tablet may become a feasible alternative treatment for type 2 diabetic patients, although a more prolonged pharmacokinetic profile is required.     

Cytotoxic effects of quinoxaline derivatives on human cancer cell lines

The cytotoxicities of 6,7-modified-5,8-quinoxalinedione derivatives and heterocyclic quinoxaline derivatives containing nitrogen, sulfur, and oxygen on human lung adenocarcinoma cell (PC 14), human gastric adenocarcinoma cell (MKN 45), and human colon adenocarcinoma cell (colon 205) were examined in vitro using MTT assay. Pyrido[1,2-a]imidazo[4,5-g]quinoxaline-6,11-dione (10) was markedly cytotoxic against MKN 45 compared with adriamycin and cis-platin used as anticancer drugs. The IC50 value of compound 10 was 0.073 microM while those of adriamycin and cis-platin were 0.12 microM and 2.67 microM, respectively.     

Effects of percutaneous balloon mitral valvuloplasty and exercise training on the kinetics of recovery oxygen consumption after exercise in patients with mitral stenosis

AIMS: Kinetics of recovery oxygen consumption after exercise plays an important role in determining exercise capacity. This study was performed to assess the kinetics of recovery oxygen consumption in mitral stenosis and evaluate the effects of percutaneous balloon mitral valvuloplasty and exercise training on the kinetics. METHODS AND RESULTS: Thirty patients with mitral stenosis (valve area < or =1.0 cm2) and same sized age- and size-matched healthy volunteers were included for this study. All subjects performed maximal upright graded bicycle exercise. Thirty consecutive patients who underwent successful percutaneous balloon mitral valvuloplasty (valve area > or =1.5 cm2 and mitral regurgitation grade < or =2), were randomized to an exercise training group or non-training group. The exercise group performed daily exercise training for 3 months. Half-recovery time of peak oxygen consumption was significantly delayed in mitral stenosis as compared to normal subjects (120+/-42 s vs 59+/-5, P<0.01). Peak oxygen consumption (ml x min(-1) x kg(-1)) was significantly increased in both the training (16.8+/-4.9 to 25.3+/-6.9) and non-training groups (16.3+/-5.1 to 19.6+/-6.0) 3 months after percutaneous balloon mitral valvuloplasty. Half-recovery time of peak oxygen consumption was significantly shortened in the training group (124+/-39 to 76+/-13, P<0.01), but not in the non-training group (114+/-46 to 109+/-44 s, P=0.12) at 3 months follow-up. The degrees of symptomatic improvement after percutaneous balloon mitral valvuloplasty were more closely correlated with the changes of the half-recovery time of peak oxygen consumption than those of peak oxygen consumption. CONCLUSION: Kinetics of recovery oxygen consumption was markedly delayed in mitral stenosis, which was improved after exercise training but not after percutaneous balloon mitral valvuloplasty alone. These results suggest that adjunctive exercise training may be useful for improvement of recovery kinetics and subjective symptoms after percutaneous balloon mitral valvuloplasty.