Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Effects of winter flooding on phosphorus dynamics in rice fields

Limnology - Controlling phosphorous (P) loads from rice fields is important for the conservation of aquatic ecosystems, in part because P is relatively concentrated at its sources. Recently, winter...     

Enzymatic desulfurization of dibenzothiophene by a cell-free system of Rhodococcus erythropolis D-1

Abstract Pseudomonas syringae cells were exposed to Cu²⁺ alone or in the precence of acetate, proline or cysteine, at concentrations that reduced free Cu²⁺ to 1/10 of the total copper. Ligand concentrations (designated as isoeffective) were determined experimentally using a Cu²⁺-selective electrode and confirmed by computer calculations using published stability constants. Exposure of P. syringae cells to Cu²⁺ alone resulted in rapid and pronounced cell death, and binding of most of the copper in solution. The addition of acetate, proline or cysteine, a few minutes after Cu²⁺ treatment, resulted in a significant reduction in cell death, and in the amount of copper bound to the cells. For short exposures to Cu²⁺, cysteine was more effective than acetate or proline, but after 60 min of treatment, similar results were observed with these ligands. The addition of ligands before Cu²⁺ resulted in even more reduced copper toxicity. The results showed that, at isoeffective concentrations, weak and moderate copper-ligands can effectively antagonize copper toxicity, and that this protective effect does not require previously equilibrated copper-ligand solutions and is not very dependent of the nature of the ligand.     

Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: a scanning and transmission electron microscopical study

We examined the morphodifferentiation of subneural apparatuses at neuromuscular junctions with scanning and transmission electron microscopy (SEM and TEM) in the sternothyroid muscle of postnatal rats. As evidenced with SEM, primitive synaptic troughs found at birth were smooth cup-like depressions 5-6 micron in diameter. At the 5th postnatal day, low sarcoplasmic ridges appeared in the depression which successively grew and upheaved to remodel the depression into anastomosed gutters during the next 10 days. Subneural apparatuses attained almost the adult form by the 30th day, though synaptic troughs were smaller in size and exhibited a less complex pattern. At birth, the depression contained a few mostly pit-like or elongated oval invaginations:incipient junctional folds. By the 15th day, junctional folds rapidly developed, resulting in about an 18-fold increase in number per endplate with the parallel differentiation of slit-like junctional folds of adult form. At the 30th day, junctional folds were mostly slit-like, though pits still coexisted in a small proportion. As a shape factor, we measured the ratio of the length of the folds to their maximum width (L/W); the folds with L/W less than 2 were defined as pits, those with 2 less than or equal to L/W less than 5 as short slits, and those with L/W greater than or equal to 5 as long slits. At birth, pits occupied about 67% of the total number of the folds per endplate, which decreased to about 14% at the 30th day. Concomitantly, long slits remarkably increased from about 3 to 38%. Short slits increased from about 30 to 50% during the first 10 days but remained almost unchanged thereafter. The maximum L/W ratio was 12 at the 15th day and exceeded 20 after the 30th day. These quantitative data and the finding that pits were often closely associated with each other and also with a slit in a serial fashion indicate that the adjacent pits may fuse to each other and to the preformed slits. With TEM, a few incipient junctional folds were found at the 5th day, which extended into the subneural sarcoplasm with a depth less than 0.4 micron. At the 15th day, junctional folds increased both in number and in the maximum depth of about 0.8 micron. There also occurred a number of basal lamina-containing vacuoles identical in many respects to the transversely sectioned profiles of incipient junctional folds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrops fetalis caused by intrauterine human parvovirus infection

During a large outbreak of erythema infectiosum in 1987 in Toyama prefecture, Japan, a 32-year-old woman acquired a mild rash on her arms and legs at 18 weeks of gestation. At 26 weeks and 4 days of gestation, the fetus died by hydrops fetalis and pregnancy was terminated. Histological studies of the fetus revealed degeneration of erythroblastic cells in the liver and bone marrow. Extensive extramedullary hematopoiesis and hemosiderin deposits were observed in the liver. Antibody response to human parvovirus B19 virus was demonstrated in maternal sera by ELISA. Furthermore, dot hybridization with the molecularly cloned DNA probe revealed the presence of human parvovirus DNA sequence in the fetal liver, spleen, lung, kidney and placenta. This report describes the first case in Japan of hydrops fetalis caused by human parvovirus B19 infection.     

Insulin-like growth factor I rapidly stimulates tyrosine phosphorylation of a Mr 185,000 protein in intact cells

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.     

Effect of insulin on the glucose utilization in isolated cardiac myocytes from adult rat

The acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 microM to 5.5 mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 microM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (greater than 100 microM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5 mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of apolipophorin-III by the fat body in locusts

Biosynthesis of locust apolipophorin-III (apo-III) was studied in vitro. Gel electrophoresis and immunoblotting analyses of the locust hemolymph demonstrated that apo-III first appears in the hemolymph on the day 3 of the adult stage after the final molt and its hemolymph concentration increases thereafter. When incubated in vitro in a medium containing radioactive amino acid, the fat body cells synthesized the radiolabeled apo-III and released it into the medium. The developmental change in the apo-III synthesizing activity in the fat body reflected that of the apo-III concentration in the hemolymph. RNA isolated from the adult fat body directed the synthesis of apo-III as a major translation product in a cell-free system. These results indicate that the fat body is the tissue responsible for the synthesis of the locust apo-III, and biosynthesis of apo-III is developmentally regulated at the level of mRNA in accordance with the flight activity of the locust.