Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Effects of cage beddings on microsomal oxidative enzymes in rat liver

The purpose of the present studies was to evaluate the effects of some commercially available cage beddings on rat liver microsomal cytochrome P-450-dependent drug-metabolizing enzyme, ethylmorphine N-demethylase, and the carcinogen-metabolizing enzyme, benzo(a)pyrene hydroxylase. Sprague-Dawley rats were housed in cages containing cedar chip, corncob or heat-treated pinewood bedding for 3 weeks. Control rats were housed in cages on wire bottom floors containing no bedding material. Rats housed in cages containing cedar chip showed 18, 46 and 49% increases in liver cytochrome P-450 content, ethylmorphine N-demethylase and benzo(a)pyrene hydroxylase activities, respectively. The liver enzyme activities of rats housed in cages containing corncob bedding were similar to those obtained with control rats. In contrast, the pinewood-bedded rats showed a 21% decrease in ethylmorphine N-demethylase activity without affecting cytochrome P-450 content and benzo(a)pyrene hydroxylase activity. Hexobarbital-induced sleep times of the variously bedded rats were similar to those of control animals. These data suggest that the commercial bedding materials differ in their abilities to affect liver microsomal enzymes. Thus, interlaboratory variability in basal enzyme activities reported in the literature may be partly due to bedding materials used in the animal's cages.     

Integration and expression of theEscherichia coli xylose isomerase gene inSchizosaccharomyces pombe

Summary The xyclose isomerase gene inEscherichia coli was cloned complementarily into a Leu2-negativeSchizosaccharomyces pombe mutant (ATCC 38399). The subsequent integration of the plasmid into the chromosomal DNA of the host yeast was verified by using the dot blot and southern blot techniques. The expressed xylose isomerase showed activity on a nondenaturing polyacrylamide gel. The expression of xylose isomerase gene was influenced by the concentration of nutrients in the fermentation broth. The yeast possessed a xylose isomerase activity of 20 nmol/min/mg by growing in an enriched medium containing yeast extract-malt extract-peptone (YMP) andd-xylose. The conversion ofd-xylose tod-xylulose catalyzed by xylose isomerase in the transformed yeast cells makes it possible to fermentd-xylose with ethanol as a major product. When the fermentation broth contained YMP and 5% (w/v)d-xylose, the maximal ethanol yield and productivity reached 0.42 g/g and 0.19 g/l/h, respectively.     

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.     

Enzymatic and Microbial Preparation of d-Xylulose from d-Xylose

A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized.     

Differential effects of carboxyfullerene on MPP+/MPTP-induced neurotoxicity

The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.     

Determinants for Achieving the LDL-C Target of Lipid Control for Secondary Prevention of Cardiovascular Events in Taiwan

Background

Epidemiological and clinical studies have clearly established the link between low-density lipoprotein cholesterol (LDL-C) and atherosclerosis-related cardiovascular consequences. Although it has been a common practice for physicians to prescribe lipid-lowering therapy for patients with dyslipidemia, the achievement rate is still not satisfied in Taiwan. Therefore, the determinants for achieving the LDL-C target needed to be clarified for better healthcare of the patients with dyslipidemia.

Method

This registry-type prospective observational study enrolled the patients with cardiovascular diseases (coronary artery disease (CAD) and cerebrovascular disease (CVD)) from 18 medical centers across Taiwan, and clinically followed them for five years. At every clinical visit, vital signs, clinical endpoints, adverse events, concurrent medications and laboratory specimens were obtained as thoroughly as possible. The lipid profile (total cholesterol, high-density lipoprotein cholesterol, LDL-C, triglyceride), liver enzymes, and creatinine phosphokinase were evaluated at baseline, and every year thereafter. The cross sectional observational data was analyzed for this report.

Result

Among the 3,486 registered patients, 54% had their LDL-C < 100 mg/dL. By univariate analysis, the patients achieving the LDL-C target were associated with older age, more male sex, taller height, lower blood pressure, more under lipid-lowering therapy, more smoking cessation, more history of CAD, DM, physical activity, but less history of CVD. The multivariate analysis showed statin therapy was the most significant independent determinant for achieving the treatment target, followed by age, history of CAD, diabetes, blood pressure, and sex. However, most patients were on regimens of very-low to low equipotent doses of statins.

Conclusion

Although the lipid treatment guideline adherence is improving in recent years, only 54% of the patients with cardiovascular diseases have achieved their LDL-C target in Taiwan, and the most significant determinant for this was statin therapy.     

Effects of asphyxia on arterial blood pressure, formation of nitric oxide in medulla and blood parameters in the cat

The rostral ventrolateral medulla (RVLM) plays an important role in the integration of cardiovascular functions. We examined the effect of asphyxia on cardiovascular responses, on sympathetic vertebral nerve activity (VNA) and nitric oxide (NO) formation in the RVLM, on hemodynamics, and on plasma concentrations of catecholamines, blood gas partial pressures and carbohydrate metabolites. Using 16 anesthetized cats we found that the systemic arterial pressure (SAP), VNA, NO formation and the release of plasma catecholamine components of norepinephrine and epinephrine were increased during asphyxia. The onset of NO production was significantly earlier than that of SAP and VNA. The venous partial pressure of O2 decreased, while the partial pressure of CO2 increased. Furthermore, metabolism of glucose and lactate increased, as did the blood concentrations of white and red blood cells, hemoglobin and platelets. Thus, asphyxia increased SAP, VNA and NO formation. It increased the plasma catecholamines, blood gases, carbohydrate metabolites and blood cells.     

Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.     

Ethanol production from pentoses and sugar-cane bagasse hemicellulose hydrolysate by Mucor and Fusarium species

The fermentation of carbohydrates and hemicellulose hydrolysate by Mucor and Fusarium species has been investigated, with the following results. Both Mucor and Fusarium species are able to ferment various sugars and alditols, including d-glucose, pentoses and xylitol, to ethanol. Mucor is able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. Fusarium F5 is not able to ferment sugar-cane bagasse hemicellulose hydrolysate to ethanol. During fermentation of hemicellulose hydrolysates, d-glucose was utilized first, followed by d-xylose and l-arabinose. Small amounts of xylitol were produced by Mucor from d-xylose through oxidoreduction reactions, presumably mediated by the enzyme aldose reductase¹ (alditol: NADP⁺ 1-oxidoreductase, EC 1.1.1.21). For pentose fermentation, d-xylose was the preferred substrate. Only small amounts of ethanol were produced from l-arabinose and d-arabitol. No ethanol was produced from l-xylose, d-arabinose or l-arabitol.