Effects of hyperbaric oxygen on the Nrf2 signaling pathway in secondary injury following traumatic brain injury.

We investigated the effects of hyperbaric oxygen treatment on the Nrf2 signaling pathway in secondary injury following traumatic brain injury, using a rat model. An improved Feeney freefall method was used to establish the rat traumatic brain injury model. Sixty rats were randomly divided into three groups: a sham surgery group, a traumatic brain injury group, and a group receiving hyperbaric oxygen treatment after traumatic brain injury. Neurological function scores were assessed at 12 and 24 h after injury. The expression levels of Nrf2, heme oxygenase 1 (HO-1), and quinine oxidoreductase 1 (NQO-1) in the cortex surrounding the brain lesion were detected by western blotting 24 h after the injury. Additionally, the TUNEL method was used to detect apoptosis of nerve cells 24 h after traumatic injury and Nissl staining was used to detect the number of whole neurons. Hyperbaric oxygen treatment significantly increased the expression of nuclear Nrf2 protein (P < 0.05), HO-1, and NQO-1 in the brain tissues surrounding the lesion after a traumatic brain injury (P < 0.05) and also significantly reduced the number of apoptotic and injured nerve cells. The neurological function scores also improved with hyperbaric oxygen treatment (P < 0.05). Therefore, hyperbaric oxygen has a neuroprotective role in traumatic brain injury, which is mediated by up-regulation of the Nrf2 signaling pathway.

Activity of esterases from different tissues of freshwater fish and responses of their isoenzymes to inhibitors.

Activity of nonspecific esterase from different tissues (i.e., liver, gallbladder, heart, intestine, and muscle) of five species of freshwater fish, namely, topmouth gudgeon (Pseudorasbora parva), goldfish (Carassius auratus), nile tilapia (Tilapia nilotica), mosquitofish (Gambusia affinis), and rainbow trout (Salmo gairdneri) was tested using alpha-naphthyl acetate as substrate. The results indicated that activity of the enzyme was mainly concentrated in the digestive system (i.e., intestine, liver, bile). The overall activity was highest in nile tilapia, followed by mosquitofish, topmouth gudgeon, goldfish, and lowest in rainbow trout. Electrophoresis and the following in vitro treatment of the isoenzymes with triphenol phosphate (TPP, an inhibitor of carboxylesterase) indicated the TPP-sensitive esterase was mainly distributed in liver of the five species. The enzyme was not found in the other five tissues (including gill) except in gallbladder of topmouth gudgeon and goldfish. The correlation was obviously improved between susceptibility and detoxification capacity if activity of the TPP-sensitive esterase was employed instead of that of the nonspecific esterase to make the comparison. In vitro treatment of nonspecific esterase in liver with malaoxon proved that the active metabolite of malathion inhibited a different isoenzyme from the TPP-sensitive one.

Gas chromatographic determination of chlorothalonil in leaves and roots of Scrophularia and in soil.

A gas chromatographic method is described for determination of chlorothalonil residues in leaves and roots of Scrophularia and in soil. Samples were extracted with acetone and cleaned up on a Florisil column. Chlorothalonil residues are chromatographed directly on a glass column of 1.5% OV-17 and 2% QF-1 coated on 80-100 mesh Chromosorb W (HP) support and measured with a 63Ni electron capture detector. Detection limits are 0.001 ppm for leaf, 0.005 ppm for root, and 0.001 ppm for soil.

Gas chromatographic determination of systemic fungicide tricyclazole in soil and water.

A method is described for determination of tricyclazole residues in soil and water. Tricyclazole is extracted from soil by refluxing with ethyl acetate-acetone (80 + 20 v/v) and from water by partitioning into dichloromethane. The soil extract is purified by coagulation. The compound is detected and measured by gas chromatography using a flame photometer operated in the sulfur mode. Detection limits are 8 ppb for soil and 0.8 ppb for water. Recoveries for control samples fortified with tricyclazole at 0.1-5.0 ppm averaged 97.1% for soil and 108.1% for water.

Gas-liquid chromatographic determination of chlordimeform and its metabolites in cargo rice and husk.

A specific method is described for the alkali flame ionization gas-liquid chromatographic determination of chlordimeform (N'-(4-chloro-o-tolyl)-N,N-dimethylformamidine) and 3 potential metabolites in cargo rice and husk. Samples are extracted with absolute alcohol or hexane, and cleaned up on neutral alumina columns. Residues of chlordimeform and its metabolites are chromatographed directly on a column of 1% DEGS coated on 60-80 mesh 405 support (PEG 20M bonded phase). The detection limits for chlordimeform, 4-chloro-o-toluidine, 2,2'-dimethyl-4,4'-dichloroazobenzene, and N-formyl-(4-chloro-o-toluidine) are 0.03, 0.028, 0.11, and 0.43 ppm for cargo rice and 0.03, 0.028, 0.22 and 0.43 ppm for husk, respectively.

Studies of the human testis. VII. Conversion of pregnenolone to progesterone.

Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.

Daytime titers of testosterone, LH, estrone, estradiol, and testosterone-binding protein: acute effects of LH and LH-releasing hormone in men.

Four normal 18-20 yr-old men were studied on 3 occasions, from 0830 h to 1500 h. The baseline for each study consisted of 3 or 4 measurements of the respective hormone obtained between 0830 and 0900 h. In the control studies mean testosterone (T) fell by 43% (P less than 0.01) during the final 30 min. The fall was gradual throughout the day and was significant by 1100 h (P less than 0.05). Administration of LH and LH-releasing hormone (LHRH) at 0900 h resulted in 9-fold (5 min) and 3-fold (30 min) higher concentrations of LH respectively. LH declined more slowly after LHRH. Titers of T rose to the 0830-0900 h mean 130 min after LH but were never significantly elevated; the occurrence of a significant drop in mean T was delayed for 70 min. After LHRH there was a nonsignificant 24% increase of the mean T followed by a slow decline; however, T did not fall significantly below the mean baseline level. In contrast, in 2 of the 4 subjects LHRH resulted in rises in T levels (P less than 0.05) above the basal titers. Testosterone-binding globulin (TeBG) mean titers showed no diurnal rhythm in the control studies. There were statistically significant elevations of mean TeBG 150 min after LH and 340 to 370 min after LHRH, as well as sustained increases during the final 30 to 210 min of 1 or 2 individuals in each group. The reason for these increases in TeBG is not presently known. Estrogen analyses performed in all studies on 2 of the subjects revealed: 1) afternoon titers of estrone were lower than baseline in all 6 studies, 2) there was no diurnal rhythm for estradiol in control studies, and 3) estradiol increased during the final 2.5 to 3 h after LHRH (P less than 0.01), but after LH it was not altered in 1 man and was lower in the other.

Studies of the human testis. V. Properties of delta-5-3beta and 17beta-hydroxysteroid dehydrogenases in the biosynthesis of testosterone from dehydroepiandrosterone.

The properties of delta-5-3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase in the human testis were examined using cell-free homogenates with added cofactors. Michaelis constants of the delta-5-3beta-hydroxysteroid dehydrogenase enzyme at 37 C and pH 7.4 were 8.2 times 10 minus 7M for dehydroepiandrosterone and 2.9 times 10 minus 6M for androstenediol. The optimal pH for both substrates was approximately 8.15. Dehydroepiandrosterone and androstenediol are competitive substrates for the enzyme. When free and conjugated C19 steroids in the order of 10 minus 6 were added, androstenedione and testosterone inhibited the enzyme activity for dehydroepiandrosterone while the activity for androstenediol was inhibited by addition of dehydroepiandrosterone and its sulfate as well as by androstenedione and testosterone. 17beta-Hydroxysteroid dehydrogenase had two apparent Michaelis constants for dehydroepiandrosterone, 3.3 times 10 minus 6M at low substrate concentrations and 1 times 10 minus 5M at high substrate concentrations. The enzyme activities for dehydroepiandrosterone and androstenedione were found to be enhanced by addition of the 17beta-hydroxysteroids examined and slightly inhibited by addition of dehydroepiandrosterone-sulfate and androstenediol-3-monosulfate. Androstenedione caused an inhibition of the 17beta-hydroxysteroid dehydrogenase for dehydroepiandrosterone. The interconversion between androstenedione and testosterone by the enzyme favored testosterone formation. Following simultaneous incubation of 3H-dehydroepiandrosterone and 14C-androstenediol in equal amounts, initially more testosterone was produced from dehydroepiandrosterone than from androstenediol under the conditions employed, while subsequently with accumulation of androstenediol more testosterone was produced from androstenediol.

Studies on the human testis. VI. NADH-linked reactions of microsomal steroid 20alpha-and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase.

NADH-linked 20alpha- and 20beta-hydroxysteroid dehydrogenase and 17alpha-hydroxylase activities were demonstrated in the microsomal fraction of the human testis. The microsomal 20alpha-hydroxysteroid dehydrogenase showed substrate affinity to pregnenolone and progesterone and not to 17alpha-hydroxyprogesterone and preferred NADH to NADPH as a hydrogen donor. In the presence of NADH, the optimal pH for the enzyme was 7.7 and the apparent Michaelis constants of the enzyme for progesterone and pregnenolone at 37 C and pH 7.4 were 6.9-7.1 X 10-6M and in the order of 10-5M, respectively, 17alpha, 20beta-Dihydroxypregn-4-en-3-one was the only significant metabolite produced from 17alpha-hydroxyprogesterone by microsomal fraction of the human testis in the presence of NADH. The apparent Michaelis constant of microsomal 20beta-hydroxysteroid dehydrogenase for 17alpha-hydroxyprogesterone in the presence of NADH was in the order of 10-5M at 37 C and pH 7.4. The microsomal 17alpha-hydroxylase catalyzed the metabolism of pregnenolone and progesterone at a similar rate in the presence of NADH. The optimal pH and the apparent Michaelis constant at 37 C and pH 7.4 of the NADH-linked reaction of 17alpha-hydroxylase for progesterone were 7.7 and 5.3-5.4 X 10-7M, resepctively. The NADH-linked enzyme activity for progesterone was competitively inhibited by both pregn-5-ene-3beta, 20alpha-diol (inhibition constant: 1.7 X 10-7M) and 20alpha-hydroxypregn-4-en-3 one (inhibition constant: 6.6 X 10-7M), and was resistant to poor oxygen supply during incubation. The results indicate that the microsomal 20alpha-hydroxysteroid dehydrogenase is a different enzyme from the one in the soluble fraction of the human testis and that microsomal 17alpha-hydroxylase in the human testis is activated by NADH as well as NADPH.

Nucleoside diphosphate-sugar 4-epimerases. II. Uridine diphosphate arabinose 4-epimerase of wheat germ.

Uridine diphosphate (UDP)-arabinose 4-epimerase (EC 5.1.3.5) has been purified at least 20-fold from wheat germ by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, dialysis, and Sephadex and diethylaminoethyl cellulose column chromatography. The enzyme has no action on UDP-d-glucose, UDP-d-glucuronic acid, or TDP-d-glucose. The pH optimum is 8.0. Km values are 1.5 mM for UDP-d-xylose and 0.5 mm for UDP-l-arabinose. The equilibrium constant, K, for the reaction UDP-l-arabinose left arrow over right arrow UDP-d-xylose is 1.25. The enzyme is neither activated by nicotinamide adenine dinucleotide nor inhibited by reduced nicotinamide adenine dinucleotide. It is completely inhibited by p-chloromercuri-phenylsulfonate; the inhibition is reversed by cysteine.