C-2 Epimer Formation of Tetrose, Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg -1 min -1 from d -glucose, 0.56 mg mg -1 min -1 from d -arabinose and 3.0 mg mg -1 min -1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.     

Oxidative Stress EPR Measurement in Human Liver by Radical-probe Technique. Correlation with Etiology,Histology and Cell Proliferation

The role of reactive oxygen species (ROS) in liver disease is controversial. This mostly reflects the difficulties to quantify ROS in vivo, particularly in humans. We aimed to measure the presence of ROS in diseased human liver and identify possible relations between ROS levels and etiology, histology and hepatocyte proliferation. Liver biopsy specimens from 102 individuals: 18 healthy controls and 84 patients (42 HCV chronic hepatitis (CHC), 19 HBV chronic hepatitis (CHB), 7 PBC, 4 PSC, 4 HCV relapsing hepatitis after liver transplantation, 3 autoimmune hepatitis, 3 hepatocellular carcinoma, 2 alcoholic hepatitis) underwent analysis by radical-probe electron paramagnetic resonance (EPR). ROS in patients (median=5 &#50 10 &#109 6 mmol/mg) were higher than in controls (median=3 &#50 10 &#109 11 mmol/mg) ( p <0.001). Progressively increasing levels of ROS were recorded passing from control values to CHB (median=4 &#50 10 &#109 7 mmol/mg), CHC (median=3 &#50 10 &#109 6 mmol/mg) and PBC (median=2 &#50 10 &#109 5 mmol/mg), the differences being significant ( p <0.001). ROS in CHC positively correlated with histological disease activity ( r =0.92; p <0.001). No correlation was found between ROS and hepatocyte proliferation rate, presence/degree of steatosis, serum ferritin levels and aminotransferases. ROS overproduction in liver appears to be a common thread linking different pathologic conditions and seems to be influenced by diseases' etiologies.     

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log &#117 P value of the solvent and the activation free energy for denaturation ( &#106 G d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log &#117 P &#104 &#109 2.3 have protective effects, whereas solvents with log &#117 P &#83 &#109 1.8 are deleterious for penicillin acylase.     

Cyanidin-3- O -β-glucopyranoside Protects Myocardium and Erythrocytes from Oxygen Radical-mediated Damages

The cyanidin-3- O - &#103 -glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 &#119 M C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 &#119 M C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 &#119 M C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 &#119 M C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 &#119 M was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 &#119 M). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Protective Activity of (−)-Epicatechin 3- O -gallate against Peroxynitrite-mediated Renal Damage

The protective effect of ( &#109 )-epicatechin 3- O -gallate (ECg) against peroxynitrite (ONOO &#109 )-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level, an indicator of ONOO &#109 production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 &#119 moles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO &#109 . In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 &#119 M) prior to 3-morpholinosydnonimine (SIN-1, 800 &#119 M) exposure reduced ONOO &#109 formation and increased the viability of cultured renal epithelial (LLC-PK 1 ) cells in a dose-dependent manner. In particular, ECg inhibited ONOO &#109 -mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G 2 /M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO &#109 in cellular and in vivo systems.     

Teratogenic Actions of Thermally-stressed Culinary Oils in Rats

Lipid oxidation products (LOPs), generated in culinary oils during episodes of thermal stressing can give rise to cellular damage. The aims of this study were to determine whether orally-administered, LOP-containing thermally-stressed safflower oil exerts teratogenic actions in rats, and whether this effect could be prevented by co-administration of &#102 -tocopherol ( &#102 -TOH). Safflower oil was heated for a period of 20 min according to standard frying practices and stored at &#109 20°C under N 2 . Four experimental groups of pregnant Wistar rats were employed; two received 0.30 ml of pre-heated oil (HO), one of which was also supplemented with 150 mg of &#102 -TOH (HOE), and two served as controls, one treated with the non-heated oil (O) and the other without any treatment (C). The oil was administered daily by gavage from day 1 of pregnancy to day 11.5, when the animals were killed and the embryos examined. LOPs and &#102 -TOH were determined both in the heated and non-heated oils. The percentage of embryo malformations and reabsorptions were determined in the above four experimental groups. Heating the oil substantially increased its concentration of LOPs and decreased its &#102 -TOH content. The percentage of embryo malformations in the HO group was 21.73%, compared with 5.6 and 7% in the O and C groups, respectively. Supplementation of the pre-heated oil with &#102 -TOH was found to decrease the percentage of malformations to 7%. The results obtained from these investigations indicate that LOPs detectable at millimolar levels in the heated cooking oils administered (e.g. saturated and &#102, &#103 -unsaturated aldehydes, and/or their conjugated hydroperoxydiene precursors) exert potent teratogenic actions in experimental animals which are at least partially circumventable by co-administration of the chain-breaking antioxidant &#102 -TOH. Plausible mechanisms for these processes and their health relevance to humans regarding diet and methods of frying/cooking are discussed.     

Kinetics and Mechanism of the Reaction of a Nitroxide Radical (Tempol) With a Phenolic Antioxidant

In the absence of redox-active transition metal ions, the removal of Tempol by Trolox occurs by a simple bimolecular reaction that, most probably, involves a hydrogen transfer from phenol to nitroxide. The specific rate constant of the process is small (0.1 M &#109 1 s &#109 1 ). Metals can catalyze the process, as evidenced by the decrease in rate observed in the presence of diethylenetriaminepentaacetic acid (DTPA). Furthermore, addition of Fe(II) (20 &#119 M ferrous sulfate and 40 &#119 M EDTA) produces a noticeable increase in the rate of Tempol consumption.     

ENZYMIC AND CEREBRAL METABOLIC EFFECTS OF 2-DEOXY-d-GLUCOSE

—The time course of effects of 2-deoxy-d -glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d -glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d -glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d -glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d -glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d -glucose and 2-deoxy-d -glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d -[U-¹⁴C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d -glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d -glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d -glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d -glucose and its conversion to 2-deoxy-d -glucose-6-phosphate, the 2-deoxy-d -glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min. Measurement of enzyme activities in cerebral homogenates (using 1 mivs substrate concentration) showed that hexokinase (EC 2.7.1.1) was 40 per cent inhibited by 5 mm -deoxy-d -glucose (but not by 2-deoxy-d -glucose 6-P). Glucose 6-P dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and phosphoglucomutase (EC 2.7.5.1) were not affected by either 2-deoxy-d -glucose (5 mm ) or 2-deoxy-d -glucose 6-P (5 or 20 mm ). Hexose-phosphate isomerase (EC 5.3.1.9) was 70 per cent inhibited by 20 mm -d -deoxy-d -glucose 6-P. Phosphofructokinase (EC 2.7.1.11) was inhibited by 17 per cent by 2-deoxy-d -glucose 6-P (20 mm ). During the initial impairment of cerebral function by 2-deoxy-d -glucose there is competitive inhibition of glucose transport into the brain; later, glycolysis is more powerfully depressed by the inhibition of isomerase produced by the high intracerebral concentration of 2-deoxyglucose-6-phosphate.     

Monochloramine Inhibits the Expression of E-selectin and Intercellular Adhesion Molecule-1 Induced by TNF-α Through the Suppression of NF-κB Activation in Human Endothelial Cells

Reactive oxygen species have various effects on the expression of cell adhesion molecules induced by pro-inflammatory cytokines, such as tumor necrosis factor &#102 (TNF- &#102 ). We studied the effects of monochloramine (NH 2 Cl), a physiological oxidant derived from activated neutrophils, on the TNF- &#102 -induced expression of e-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). HUVEC were pretreated with or without NH 2 Cl (20-90 &#119 M for 20 min), then stimulated with TNF- &#102 (10 ng/ml), and the expression of e-selectin and ICAM-1 was measured. Without NH 2 Cl, TNF- &#102 induced marked expression of e-selectin and ICAM-1. Pretreatment with NH 2 Cl resulted in a significant, but transient inhibition of the expression of adhesion molecules. Higher dose of NH 2 Cl showed more pronounced inhibition, and the inhibitory effect lasted for 8 h when 70 &#119 M of NH 2 Cl was added. TNF- &#102 stimulation also induced marked activation of nuclear factor &#115 B (NF- &#115 B). Notably, NH 2 Cl also inhibited this NF- &#115 B activation in a dose- and time-dependent manner, which was similar to the inhibition of e-selectin and ICAM-1 expression. In addition, I &#115 B- &#102 phosphorylation and degradation were also inhibited by NH 2 Cl pretreatment. These observations indicated that NH 2 Cl inhibited TNF- &#102 -induced expression of e-selectin and ICAM-1 through the inhibition of NF- &#115 B activation. We speculate that neutrophil-derived chloramines may have a regulatory role in the recruitment of leukocytes.     

Prevention of Peroxynitrite-induced Renal Injury through Modulation of Peroxynitrite Production by the Chinese Prescription Wen-Pi-Tang

The effect of Wen-Pi-Tang extract on renal injury induced by peroxynitrite (ONOO &#109 ) production was investigated using rats subjected to intravenous lipopolysaccharide (LPS) injection and then renal ischemia followed by reperfusion. The plasma level of 3-nitrotyrosine, a marker of cytotoxic ONOO &#109 formation in vivo, was enhanced markedly in control rats subjected to LPS plus ischemia-reperfusion, but was significantly reduced by the oral administration of Wen-Pi-Tang extract, at doses of 62.5 and 125 mg/kg body weight/day, for 30 days prior to LPS plus ischemia-reperfusion. The activities of inducible nitric oxide synthase (iNOS) and xanthine oxidase (XOD) in renal tissue of control and Wen-Pi-Tang extract-treated rats did not change significantly, while those of the antioxidant enzymes, superoxide dismutase, catalase and gluta-thione peroxidase, were significantly increased by the administration of Wen-Pi-Tang extract, indicating that Wen-Pi-Tang improved the defense system by scavenging free radicals, not by directly inhibiting nitric oxide and superoxide production by iNOS and XOD. In addition, the levels of the hydroxylated products, m - and p -tyrosine, declined, whereas that of phenylalanine increased, after oral administration of Wen-Pi-Tang extract. Furthermore, the elevated plasma urea nitrogen and creatinine levels resulting from LPS plus ischemia-reperfusion process were significantly reduced by Wen-Pi-Tang extract, implying amelioration of renal impairment. The present study indicates that Wen-Pi-Tang extract contributes to the regulation of ONOO &#109 formation and plays a beneficial role against ONOO &#109 -induced oxidative injury and renal dysfunction in vivo .     

Pseudomonas rhodesiae PF1: A New and Efficient Biocatalyst for Production of Isonovalal from α-pinene Oxide

A new bacterial strain, identified as Pseudomonas rhodesiae PF1 and deposited under the accession number CIP 107491, is presented. It is very active for the production of the acyclic compound Z-2-methyl-5-isopropyl-hexa-2,5-dien-1-al (isonovalal) from &#102 -pinene oxide. Enzyme synthesis is induced by culturing cells on &#102 -pinene; growing bacteria also have the ability to synthesize the epoxide derivative of &#102 -pinene. Isonovalal production was performed without aeration and with concentrated resting cells previously frozen at &#109 20°C, and subsequently thawed in a water-organic solvent, two-phase system. The organic layer was hexadecane, the volume ratio being 1:1. The best results achieved allowed recovery of c.a. 60 g/l organic solvent of isonovalal in 2.5 h operation, which is the most efficient process to date in the area of terpene biotransformations.     

Characterization of a glucose 3-dehydrogenase from the cultivated mushroom (Agaricus bisporus )

An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of ¹H-NMR spectra in D₂O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k _cat/K _m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus. Received: 19 June 1998 / Received revision: 15 September 1998 / Accepted: 17 September 1998     

Scavenging of Benzylperoxyl Radicals by Carotenoids

Carotenoids scavenge simple lipid-like alkylperoxyl radicals. However, the rate constant is too low to be determined directly and the mechanism is likewise not known with certainty [Mortensen, A. and Skibsted, L.H. (1998) FEBS Lett . 426 , 392-396]. It is demonstrated that carotenoids react with peroxyl radicals only slightly more reactive than lipidperoxyl radicals neither by electron transfer nor by hydrogen atom donation, but by adduct formation. Benzylperoxyl radicals are scavenged by the carotenoids &#103 -carotene and canthaxanthin with a second-order rate constant of at least 1 &#117 &#50 &#117 10 6 &#117 M &#109 1 &#117 s &#109 1 by formation of an adduct which decays in a first-order reaction.     

Biotransformation of Digitoxin in Cell Cultures of Capsicum frutescens in the Presence of β-cyclodextrin

Cell suspension cultures of Capsicum frutescens accumulated digoxin, purpureaglycoside A and other unknown derivatives when digitoxin, a cardiac glycoside, was used as a precursor. The feeding of digitoxin complexed with &#103 -cyclodextrin increased the accumulation of digoxin, purpureaglycoside A and other unknown derivatives. Control cultures (without digitoxin) did not produce any of these metabolites. The growth of cells was affected by both digitoxin as well as digitoxin- &#103 -cyclodextrin. The accumulation of purpureaglycoside A and digoxin reached a maximum of 1241 and 374 &#119 g 100 ml &#109 1 culture on the 6th and 2nd day, respectively, which was 3.9 and 4.5 fold higher than cultures treated with digitoxin alone (sampled on the 13th day). The other unknown derivatives formed in digitoxin- &#103 -cyclodextrin fed cultures were 15 times higher than digitoxin alone fed C. frutescens cultures. The addition of glucose to digitoxin- &#103 -cyclodextrin treated cultures increased the accumulation of purpureaglycoside A which reached a maximum of 3589 &#119 g 100 ml &#109 1 culture after 12 h incubation, which was a 2.9 fold increase over cultures treated with digitoxin- &#103 -cyclodextrin alone.     

Identification of the Hydroxyl Radical and Other Reactive Oxygen Species in Human Neutrophil Granulocytes Exposed to a Fragment of the Amyloid Beta Peptide

A fragment of the amyloid beta protein, &#103 A(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that &#103 A(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, &#103 A(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by &#103 A(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to &#103 A(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 &#148 &#109 ) - and hydroxyl ( &#148 OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of &#148 OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of &#148 OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to &#103 A(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that &#103 A(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 &#148 &#109 can lead to HOCl and further formation of &#148 OH, which both have a cytotoxic potential.     

Effect of High Product Concentration in a Dual Fed-batch Asymmetric 3-oxo Ester Reduction by Baker's Yeast

Baker's-yeast-mediated asymmetric ethyl 3-oxobutanoate reduction using a fed-batch feeding strategy for both the 3-oxo ester and the electron donor, was explored as potential production system for enantiopure ethyl ( S )-3-hydroxybutanoate. The dual feed strategy was based on kinetic and stoichiometric data. One major aspect is the effect of high product concentrations on the progress of the reduction. According to initial rate experiments, product inhibition occurs at concentrations above 600 mM product causing a 10-fold decrease of the initial biomass-specific reduction rate. By using optimized feed rates and a biomass concentration of 43 g dw l &#109 1, a product concentration of 350 mM was reached within 80 h with a degree of conversion of 95%. The volumetric productivity was 0.58 g l &#109 1 h &#109 1, using 2.1 kg pressed yeast kg product &#109 1 and 0.52 kg glucose kg product &#109 1. During the fed-batch biotransformation the reduction rate continuously decreased and reduction ceased after 80 h, due to biocatalyst inactivation after prolonged use at increasing high product concentrations. The continuous decrease in reducing activity led to very high ethyl 3-oxobutanoate levels in the reactor resulting in an increase of the undesired specific ethyl ( R )-3-hydroxybutanoate production rate. Therefore, the enantiomeric excess of the product decreased, from initially 100 to ~75% at 80 h. It is concluded that the design of processes for efficient asymmetric bioreduction cannot solely be based on initial rate kinetics, but require detailed knowledge of the effects on activity and enantioselectivity upon long-term exposure to process conditions.     

SYNAPTOSOMAL PHOSPHORYLATION OF D-GLUCOSAMINE

Abstract— Synaptosomal fractions from rat brains can phosphorylate d -glucosamine once it is transported across the synaptosomal membrane. Intrasynaptosomal phosphorylation of d -glucosamine was rapid; up to 80% of the d -glucosamine was recovered as the phosphorylated amino sugar during 15 min of incubation with the substrate. Transport of d -glucosamine was the rate-limiting step in the uptake process since in the presence of cytochalasin B, a specific sugar transport inhibitor, the uptake of d -glucosamine was inhibited to the same extent as its phosphorylation. The pH optimum for phosphorylation of d -glucosamine by lysed synaptosomal preparations was between 7.5 and 9.0. The mean K _m and V _max values for this phosphorylation were 0.46 DIM and 676 nmol/mg protein per min, respectively. Phosphorylation of d -glucosamine was inhibited competitively by d -glucose, 2-deoxy- d -glucose and N -acetyl- d -glucosamine and uncompetitively by d -glucose-6-phosphate. The phosphorylation was strongly inhibited by several sulfhydryl reagents, but was insensitive to product inhibition, cytochalasin B, phloretin and phloridzin.     

Growth hormone control of glucose oxidation pathways in hypophysectomized rats.

An in vivo response of glucose oxidation to growth hormone has been demonstrated. Hypophysectomized rats were found to oxidize glucose at rates significantly higher than normal rats. Treatment with growth hormone 1 h before injection of 14C-U-glucose, 14C-6-glucose, or 14C-1-glucose caused a return to a normal oxidation pattern. This acute response was independent of insulin action but clearly time-dependent since no change from untreated hypophysectomized rats appeared when growth hormone was given at various times prior to administration of labeled glucose. The response observed for 14C-6-glucose was comparable to that observed for 14C-1-glucose with regard to dynamics but differed with respect to total 14C recovered as 14CO2. The cumulative percent 14CO2 recovered from oxidation of 14C-6-glucose 1 h after growth hormone injection exceeded that recovered from oxidation of 14C-1-glucose. These results suggest a change in glucose oxidation by a route that cannot be explained solely by changes in either the hexose monophosphate or Embden-Meyerhof pathways.     

New Insight on the Relationship between LDL Composition,Associated Proteins,Oxidative Resistance and Preparation Procedure

Oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. It is generally thought that LDL is mainly oxidized in the intima of vessel walls, surrounded by hydrophilic antioxidants and proteins such as albumin. The aim of this study was to investigate the possible interrelationships between oxidation resistance of LDL and its protein and lipid moieties. Proteins and to a lesser extent lipids, appeared to be the major determinants in the LDL Cu 2+ -oxidation resistance, which in turn depend on the ultracentrifugation (UC) procedure used. Comparing high speed/short time (HS/ST, 4 &#117 h), high speed/long time (HS/LT, 6-16 &#117 h) and low speed/long time (LS/LT, 24 &#117 h) conditions of UC, HS with the shortest time (4 &#117 h) led to prepare LDL (named LDL·HS-4 &#117 h) with higher total protein and triglyceride contents, unchanged total cholesterol, phospholipids and Vitamin E, and higher Cu 2+ -oxidation resistance. Among proteins, only albumin allows to explain changes. PAF acetyl hydrolase appeared to be unaffected, whereas its pro-oxidant role was established and found only in the absence of albumin. In contrast the pro-oxidant role of caeruloplasmin took place regardless of the albumin content of LDL. The antioxidant effect of albumin (the oxidation lag time was doubled for 20 &#117 mol/mol albumin per LDL) is assumed to be due to its capacity at decreasing LDL affinity for Cu 2+ . Interestingly, the LDL·HS-4 &#117 h albumin content mirrored the intrinsic characteristics of LDL in the plasma and was not affected by added free albumin. Moreover, it has been verified that in 121 healthy subjects albumin was the best resistance predictor of the Cu 2+ -oxidation of LDL·HS-4 &#117 h, with a multiple regression equation: lag time (min)=62.1+0.67(HSA/apoB)+0.02 (TG/apoB) &#109 0.01(TC/apoB); r =0.54, P <0.0001. Accounted for by lag time, the oxidation resistance did not correlate with &#102 -tocopherol and ubiquinol contents of LDL. The mean albumin content was about 10 &#117 mol/mol, and highly variable (0-58 &#117 mol/mol) with subjects. The LDL·HS-4 &#117 h may account for the status of LDL in its natural environment more adequately than LDL resulting from other conditions of UC.     

Effects of deflazacort and the L-6485 metabolite on epiphyseal cartilage carbohydrate metabolism: comparison with prednisone

Male Sprague-Dawley rats were injected with 1 mg/100 g bw/day of prednisone, 1.25 mg/1--g bw/day of deflazacort, or its metabolite, for a period of 20 days. Epiphyseal cartilage slices were incubated in a modified Krebs Ringer bicarbonate buffer, at 37 degrees C for 60 min, with either 14C-1- or 14C-6-glucose to quantitate both the absolute and relative rates of pentose shunt versus aerobic and anaerobic glycolytic activity, respectively. Measurements of both total and radioactive glucose uptake, lactate production and 14CO2 generation were expressed as either mumoles or DPM/mg cellular DNA/hr, respectively. This study demonstrated: (1) chronic prednisone administration decreased anaerobic glycolysis (glucose uptake and lactate production) 3-fold (P less than 0.01); (2) prednisone on a chronic basis produced no measured alteration in either the pentose shunt or Kreb's cycle activity; (3) both deflazacort and the deflazacort metabolite significantly stimulated (P less than 0.02) anaerobic glycolytic activity in epiphyseal cartilage tissue. In contrast to prednisone, the administration of either deflazacort or its L-6485 metabolite did not inhibit the glycolytic pathway of metabolism so necessary for epiphyseal cartilage growth and mineralization.