Biochemical medicine and metabolic biology最新文献

The generation of C₂- and C₃-deuterated lactic acid produced by rat parotid cells exposed to [1-¹³C] glucose, [2-¹³C]glucose, and [6-¹³C]glucose in the presence of D₂O was assessed by ¹³C NMR, The results indicated that the escape from deuteration amounted to about 46% at the phosphoglucoisomerase level, 100% at the phosphomannoisomerase level, 65% in the reactions catalyzed by phosphofructoaldolase and triose phosphate isomerase, and 58% at the level of glutamate pyruvate transaminase. Such high values are considered to support a possible enzyme-to-enzyme tunneling of metabolic intermediates at selected sites in the glycolytic pathway.

Glycolytic enzymes are known to be controlled by reversible binding to cytoskeleton. Our previous experiments have shown that insulin, epidermal growth factor (EGF), and Ca²⁺ induce a rapid and transient stimulation of binding of glycolytic enzymes to muscle cytoskeleton. We show here that platelet-derived growth factor (PDGF) exerts a similar action. Incubation of rat diaphragm muscle in the presence of PDGF resulted in rapid and transient stimulation of binding of phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) to muscle cytoskeleton. The increase in cytoskeleton-bound glycolytic enzymes induced by PDGF was prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca²⁺-calmodulin is involved in this effect of PDGF. Similarly, we previously found that stimulation of cytoskeleton-bound glycolytic enzymes exerted by insulin, EGF, or Ca²⁺, was also calmodulin mediated. The present and previous results suggest that the rapid, Ca²⁺-calmodulin-mediated increase in cytoskeleton-bound glycolytic enzymes, may be a general mechanism in the cell, in signal transduction of insulin, growth factors, and other Ca²⁺-mobilizing hormones. The accelerated cytoskeletal glycolysis will provide local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following binding of growth factor or hormone to its receptor.

Liposomal hamycin was found to elicit enhanced microbicidal activity and reduced toxicity in experimental leishmaniasis in a hamster model under in vivo conditions. Mannose-coated liposomal hamycin was seen to produce increased therapeutic efficacy as judged from the lowering of spleen parasite load. At an equivalent dose of 0.5 mg/kg, every 3 days for a total of three doses in 7 days, the mannose-coated liposomal hamycin was found to be most effective compared to either of the liposomal hamycin or the free hamycin. Because of the reduced toxicity as judged from the blood pathology, tissue histology, and specific enzyme level related to normal liver function, mannose-coated liposomal hamycin resulted in 80 to 100% survival for a period of 15-18 days. Hamycin intercalated in sterol-rich liposomes showed reduced hemolytic activity but comparable therapeutic efficacy as was found with ordinary liposomes.

A simple nonradioactive method was developed for identification of the Pro-30-Leu and Val-281-Leu mutant alleles in the CYP21B gene. Not only does this approach improve mutation analysis for patients with the late onset form of 21-hydroxylase deficiency, but it also decreases problems with interference by the CYP21A pseudogene sequence.

We have investigated various micronutrients important to folate metabolism in women with two previous neural tube defect (NTD)-affected pregnancies. Results suggest the disposition of plasma 5-methyltetrahydrofolate (5CH₃-H₄PteGlu) with respect to dietary intake may differ from that of the control population. It appears that to achieve a given plasma level of 5CH₃-H₄PteGlu, the population with a history of NTD pregnancies needs to take in more dietary folate than controls. We discuss this in the context of a potential lesion at or upstream from 5,10-methylenetetrahydrofolate reductase (MTHFR). This metabolic axis, which is responsible for the multienzymic conversion of PteGlu to 5CH₃-H₄PteGlu, has been investigated in a rat model using liver homogenate. The anticonvulsant drug (ACD) carbamazepine was found to inhibit the reaction in terms of a reduced V_max and increased K_m. Inhibition approaching maximal was found to occur at therapeutic levels of ACD. Various potential inhibitory sites along the methylfolate axis are considered and possible relationships to congenital malformations discussed. We describe folate and one carbon metabolism in relation to potential NTD lesion sites, not only in the light of present findings, but with respect to the published findings of other workers. Based on our hypothesis that an NTD lesion exists upstream from MTHFR, we expound how pteroylmonoglutamate supplementation may protect against NTD (i) by reducing endotoxic homocysteine and (ii) through inhibiting MTHFR (as do dihydrofolates) and thus diverting one carbon units into DNA thymine.

Beta-2 sympathomimetics, such as terbutaline, hare been shown to alter erythropoietin (EPO) secretion in animal studies. More recently introduced sympathomimetics, such as ritodrine, have been extensively used to inhibit uterine contractions in preterm labor. It has not been determined what effect ritodrine may have on EPO production. We investigated the effect of RD administered to rats in the last day of gestation on the dam and the fetuses′ levels of EPO. Rats at the 20th day of gestation were given, under anesthesia, either 3000 μg/kg ritodrine over a 10-min period or a similar volume of saline as control (CTL). Fetuses were removed at 0 and 4 h after injection. Ritodrine administration produced a decrease of dam EPO (23.4 ± 4.2 to 12.0 ± 2.9 pmol/ml), while the CTL showed no changes. The fetuses from the ritodrine-injected dams exhibited a marked decline over the 4-h period, while the CTL fetuses did not change. In other experiments 500 μg terbutaline was administered daily during the last 5 days of rat gestation. The drug produced a significant decline in EPO at delivery time, but plasma EPO in the pups was unchanged. Blood hematocrits were comparable to those of controls. The data show that acute administration of ritodrine reduces fetal and dam plasma EPO, while long-term terbutaline treatment late in gestation alters maternal, but not fetal plasma EPO, indicating that neither drug has any direct regulatory effect on the erythropoietic ability of the fetuses.

The second most common mutation associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-hire episodes) in Japan is the 3271 mutation. This mutation was found in a Brazilian family of Portuguese and Italian descent, indicating that this mutation also exists in a race other than Japanese. The propositus had mild clinical manifestations atypical of MELAS, suggesting that patients with the 3271 mutation exhibit heterogeneous phenotypic expression as seen in the 3243 mutation.

The fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) and trimethylammonium-DPH in the membranes of intact human erythrocytes and ghost membranes was compared. The anisotropy of fluorophores is significantly higher in intact erythrocytes compared to that in ghost membranes. Perturbation of membranes by heating at 47°C and use of a rotating stirrer affected the anisotropy of fluorophores in intact erythrocytes only. These results suggest that: (a) spectrin has a significant modulating effect on membrane fluidity, and (b) the physical properties of the cell membrane are different between intact erythrocytes and erythrocyte ghost membranes.

To elucidate the effects of fatty acids on the uptake of glucose and insulin in the muscle, the effects of oleic and palmitic acids on the uptake of glucose and insulin were investigated in perfused hindquarters of rats. In the absence of insulin, glucose uptake in the hindquarter was slightly, but not significantly, decreased by the addition of oleic and palmitic acids. In the presence of 100 or 500 μU/ml insulin, glucose uptake in the hindquarter (243 ± 58 or 282 ± 65 μmol/30 min) was significantly decreased by the addition of 1000 μM oleic acid and 500 μM palmitic acid (175 ± 50 or 207 ± 47 μmol/30 min). The decrease in insulin uptake rate, although not significant at 500 μU/ml insulin, is of similar magnitude to the decrease in glucose uptake. In the presence of 1000 μU/ml insulin, glucose and insulin uptake was not significantly altered by the addition of fatty acids. These results indicate that fatty acids directly inhibit the muscular glucose uptake via the decrease in muscular insulin uptake at a physiological concentration of insulin.