Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents.
The effect of alkyltrimethylammonium ions on the thermostability of natural and modified DNA duplexes has been investigated. We have shown that the use of tetramethylammonium ions TMA+along with the chemical modification of duplexes allow the fine adjustment of T m and the possibility of obtaining several duplex systems with varied isostabilizedtemperatures, some of which show greater stability than those of natural DNA. This approach could be very useful for DNA sequencing by hybridization. (+info)
Inhibition of glutathione synthesis with propargylglycine enhances N-acetylmethionine protection and methylation in bromobenzene-treated Syrian hamsters.
The finding that liver necrosis caused by the environmental glutathione (GSH)-depleting chemical, bromobenzene (BB) is associated with marked impairment in O- and S-methylation of BB metabolites in Syrian hamsters raises questions concerning the role of methyl deficiency in BB toxicity. N-Acetylmethionine (NAM) has proven to be an effective antidote against BB toxicity when given after liver GSH has been depleted extensively. The mechanism of protection by NAM may occur via a replacement of methyl donor and/or via an increase of GSH synthesis. If replacement of the methyl donor is an important process, then blocking the resynthesis of GSH in the methyl-repleted hamsters should not decrease NAM protection. This hypothesis was examined in this study. Propargylglycine (PPG), an irreversible inhibitor of cystathionase, was used to inhibit the utilization of NAM for GSH resynthesis. Two groups of hamsters were pretreated with an intraperitoneal (ip) dose of PPG (30 mg/kg) or saline 24 h before BB administration (800 mg/kg, ip). At 5 h after BB treatment, an ip dose of NAM (1200 mg/kg) was given. Light microscopic examinations of liver sections obtained 24 h after BB treatment indicated that NAM provided better protection (P < 0.05) in the PPG + BB + NAM group than in the BB + NAM group. Liver GSH content, however, was lower in the PPG + BB + NAM group than in the BB + NAM group. The Syrian hamster has a limited capability to N-deacetylated NAM. The substitution of NAM with methionine (Met; 450 mg/kg) resulted in a higher level of GSH in the BB + Met group than in the BB + NAM group (P < 0.05). The enhanced protection by PPG in the PPG + BB + NAM group was accompanied by higher (P < 0.05) urinary excretions of specificO- and S-methylated bromothiocatechols than in the BB + NAM group. The results suggest that NAM protection occurs primarily via a replacement of the methyl donor and that methyl deficiency occurring in response to GSH repletion plays a potential role in BB toxicity. (+info)
Inhibition of gene expression by anti-sense C-5 propyne oligonucleotides detected by a reporter enzyme.
Using a reporter plasmid containing the luciferase gene under the control of the insulin-like growth factor 1 (IGF-1) promoter region [including its 5' untranslated region (UTR)], we demonstrate that a 17-mer oligophosphorothioate containing C-5 propyne pyrimidines is able to inhibit luciferase gene expression in the nanomolar concentration range when the anti-sense oligonucleotide is targeted either to a coding sequence in the luciferase gene or to the 5' UTR of the gene for IGF-1. Inhibition was obtained independently of whether the plasmid and the anti-sense oligonucleotide were co-transfected or transfected separately into hepatocarcinoma cells. However, the efficiency of inhibition by the anti-sense oligonucleotides was 10-fold greater in the first case. The unmodified oligophosphorothioate targeted to the 5' UTR of IGF-1 did not inhibit luciferase gene expression at a 100-fold higher concentration unless its length was increased from 17 to 21 nt, in which case an inhibition of gene expression was obtained and an IC50 of 200 nM was observed. (+info)
Elevated expression of liver gamma-cystathionase is required for the maintenance of lactation in rats.
Liver gamma-cystathionase activity increases in rats during lactation; its inhibition due to propargylglycine is followed by a significant decrease in lactation. This is reversible by N-acetylcysteine administration. To study the role of liver gamma-cystathionase and the intertissue flux of glutathione during lactation, we used lactating and virgin rats fed liquid diets. Virgin rats were divided into two groups as follows: one group was fed daily a diet containing the same amount of protein that was consumed the previous day by lactating rats (high protein diet-fed rats); the other virgin group was fed the normal liquid diet (control). The expression and activity of liver gamma-cystathionase were significantly greater in lactating rats and in high protein diet-fed virgin rats compared with control rats. The total glutathione [reduced glutathione (GSH) + oxidized glutathione (GSSG)] released per gram of liver did not differ in lactating rats or in high protein diet-fed rats, but it was significantly higher in these two groups than in control virgin rats. Liver size and the GSH + GSSG released by total liver were significantly higher in lactating rats than in high protein diet-fed virgin rats, and this difference was similar to the amount of glutathione taken up by the mammary gland (454.2 +/- 36.0 nmol/min). The uptake of total glutathione by the lactating mammary gland was much higher than the uptakes of free L-cysteine and L-cystine, which were negligible. These data suggest that the intertissue flux of glutathione is an important mechanism of L-cysteine delivery to the lactating mammary gland, which lacks gamma-cystathionase activity. This emphasizes the physiologic importance of the increased expression and activity of liver gamma-cystathionase during lactation. (+info)
Guest exchange in an encapsulation complex: a supramolecular substitution reaction.
Encapsulation complexes are reversibly formed assemblies in which small molecule guests are completely surrounded by large molecule hosts. The assemblies are held together by weak intermolecular forces and are dynamic: they form and dissipate on time scales ranging from milliseconds to days-long enough for many interactions, even reactions, to take place within them. Little information is available on the exchange process, how guests get in and out of these complexes. Here we report that these events can be slow enough for conventional kinetic studies, and reactive intermediates can be detected. Guest exchange has much in common with familiar chemical substitution reactions, but differs in some respects: no covalent bonds are made or broken, the substrate is an assembly rather than a single molecule, and at least four molecules are involved in multiple rate-determining steps. (+info)
Effects of a lipoxygenase inhibitor, panaxynol, on vascular contraction induced by angiotensin II.
We investigated whether a lipoxygenase inhibitor, panaxynol, affected the vascular contraction induced by angiotensin (Ang) II and the mean arterial pressure in spontaneously hypertensive rats (SHR). Panaxynol suppressed dose-dependently the contractile responses induced by 30 nM Ang II in isolated intact and endothelial cell-denuded aorta in the hamster. IC50 values in the intact and endothelial cell-denuded aorta were 23 and 20 microM, respectively. In SHR, the mean arterial pressure after injection of 30 and 60 mg/kg panaxynol was reduced, and the maximum hypotensive values were 23 and 48 mmHg, respectively. Thus, lipoxygenase products may affect the renin-angiotensin system. (+info)
Similarity and dissimilarity in mode and mechanism of action between YT-146, a selective adenosine receptor A2 agonist, and adenosine in isolated canine hearts.
To elucidate the differences in mode and mechanism of action between YT-146, a highly selective adenosine A2 receptor agonist, and adenosine, we compared their effects on coronary circulation and myocardium and modifications of these effects by glibenclamide, a blocker of ATP-sensitive potassium (K) channels, in three kinds of isolated, blood-perfused canine heart preparations. YT-146 and adenosine were injected i.a. In all preparations both YT-146 and adenosine increased coronary blood flow and in this respect YT-146 was about 5 times as potent as adenosine. The increase in blood flow caused by adenosine was transient, whereas that produced by YT-146 was biphasic; the transient increase was followed by a sustained one. In isolated, blood-perfused sinoatrial (SA) node preparations, YT-146 failed to affect sinus rate, whereas adenosine reduced sinus rate by about 38% at its maximum effect. In isolated, blood-perfused atrioventricular (AV) node preparations, when injected into the artery supplying the AV node, YT-146 exerted no effect on AV conduction time, whereas adenosine prolonged AV conduction time by about 17% at the maximum effect. In isolated, blood-perfused papillary muscle preparations, the force of contraction was affected by neither YT-146 nor adenosine. In the same preparations the effect of YT-146 in increasing coronary blood flow was antagonized by glibenclamide in such a manner that the maximum increase was suppressed, but that of adenosine was not. Reactive hyperemia induced by ischemia for 30 seconds was not affected by glibenclamide. These results suggest that although both YT-146 and adenosine produce an increase in coronary blood flow via adenosine A2 receptors, the opening of ATP- or glibenclamide-sensitive K channels is involved in the action of the former, but scarcely in the action of the latter. The opening of ATP- or glibenclamide-sensitive K-channels is less likely involved in reactive hyperemia. (+info)
NAD(+)-dependent (S)-specific secondary alcohol dehydrogenase involved in stereoinversion of 3-pentyn-2-ol catalyzed by Nocardia fusca AKU 2123.
An NAD(+)-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K(m) and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 mumol/min/mg, and 0.33 mM and 130 mumol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K(m) and Vmax for 2-hexanone reduction were 2.5 mM and 260 mumol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp. (+info)