Osmotic adjustments support growth of poplar cultured cells under high concentrations of carbohydrates.

KEY MESSAGE: Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500 mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from - 1 to - 10% after 21 days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l-1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

Potassium Salt of Fullerenylpenta-N-Dihydroxytyrosine Effects on Type 2 Diabetes Mellitus Therapeutic Targets.

It was shown for the first time that pentaamino acid derivative of fullerene C60 (potassium salt of fullerenylpenta-N-dihydroxytyrosine) affects three targets of type 2 diabetes mellitus. It competitively inhibits the enzymes aldose reductase and sorbitol dehydrogenase and also has an antiglycation effect on bovine serum albumin. The inhibition constants for these enzymes were calculated.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose.

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L-1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

Reproductive toxicity of melamine against male mice and the related mechanism.

Melamine is a nitrogen-containing heterocyclic organic compound with a triazine skeleton, which has been widely applied in industrial and chemical fields. Previous toxicity studies of melamine mainly focused on renal toxicity and hepatic pathological changes, but its toxicity against the reproductive system has seldom been assessed. We investigated the effects of melamine on the reproductive system of male mice. Forty healthy male Kunming mice were randomly divided into a normal saline negative control group, a low-dose melamine group, a medium-dose melamine group and a high-dose melamine group (n = 10). The mice were administered for five consecutive days and killed on the 35th day after first administration. In melamine administration groups, seminiferous tubules had disordered, loose arrangement, and spermatogenic cells at all levels obviously decreased. The sperm count and motility decreased significantly, and the sperm deformity rate increased significantly. Melamine induced apoptosis of testicular spermatogenic cells. To further explore the mechanism, we detected metabolism-related enzymes sorbitol dehydrogenase (SDH) and lactate dehydrogenase (LDH) as well as oxidative stress indices superoxide dismutase (SOD) and malondialdehyde (MDA). The activities of SDH, LDH and SOD in melamine treatment groups decreased significantly, and the MDA level increased obviously. The expressions of apoptosis-related proteins Bcl-2, Bax and caspase-3 were detected by immunohistochemistry. The expression of Bcl-2 significantly increased, but those of Bax and caspase-3 significantly reduced (p < 0.05). In conclusion, melamine damaged the reproductive system of mice via the oxidative stress pathway and by inducing cell apoptosis.

Influence of the natural Rio Negro water on the toxicological effects of a crude oil and its chemical dispersion to the Amazonian fish Colossoma macropomum.

The increment in crude oil exploitation over the last decades has considerably increased the risk of polycyclic aromatic hydrocarbon (PAH) contamination to Amazonian aquatic environments, especially for the black water environments such as the Rio Negro. The present work was designed to evaluate the acute toxicity of the Urucu crude oil (CO), the chemically dispersed Urucu crude oil (CO + D), and the dispersant alone (D) to the Amazonian fish Colossoma macropomum. Acute toxicity tests were performed, using a more realistic approach, where fish were acclimated to both groundwater (GW), used as internal control, and natural Rio Negro water (RNW) and exposed to CO, CO + D and D. Then, biomarkers such as ethoxyresorufin-O-deethylase (EROD), superoxide dismutase (SOD), lipid peroxidation (LPO), serum sorbitol dehydrogenase (s-SDH) in liver, DNA damage in blood cells, and the presence of the benzo[a]pyrene-type, pyrene-type, and naphthalene-type metabolites in fish bile were assessed. Fish exposed to CO and CO + D, at both water types tested, presented increased biomarker responses and higher PAH-type metabolites in the bile. However, fish exposed to these treatments after the acclimation to RNW increased the levels of LPO, s-SDH (hepatotoxicity), DNA damage in blood cells (genotoxicity), and benzo[a]pyrene-type metabolites when compared to fish in GW. Our data suggests that some physicochemical properties of Rio Negro water (i.e., presence of natural organic matter (NOM)) might cause mild chemical stress responses in fish, which can make it more susceptible to oxidative stress following exposure to crude oil, particularly to those chemically dispersed.

Evaluation of ameliorative effect of curcumin on imidacloprid-induced male reproductive toxicity in wistar rats.

This study was undertaken to investigate the toxic effects of imidacloprid (IM) on male reproductive system and ameliorative effect of curcumin (CMN) in male Wistar rats. For this purpose, IM (45 and 90 mg/kg, body weight) and CMN (100 mg/kg, body weight) were administered orally to the rats either alone or in combinations for a period of 28 days. At the end of experiment, male reproductive toxicity parameters (total sperm count and sperm abnormalities), testosterone level, steroidal enzymatic activity [3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-HSD], and oxidative stress indicators were estimated in testis and plasma. IM treatments resulted in significant decrease (p < 0.05) in total epididymal sperm count, sperm motility, live sperm count, and increase (p < 0.05) in sperm abnormalities. Activities of gamma-glutamyl transpeptidase, lactate dehydrogenase-x, and sorbitol dehydrogenase were significantly increased (p < 0.05), while, 3ß-HSD and 17ß-HSD enzymatic activity along with testosterone concentration in testis and plasma were decreased significantly (p < 0.05) in IM-treated rats. IM exposure resulted in significant increase (p < 0.05) in LPO and decrease (p < 0.05) in GSH level along with decreased activities of CAT, SOD, GPx, and GST. IM-treated rats showed histopathological alterations in testis and epididymis. However, the reproductive toxicity parameters, oxidative stress indicators, and histopathological changes were minimized and functional restorations were noticed by co-administration of CMN in IM-treated rats. The results of this study suggest that IM-induced male reproductive toxic effects could be ameliorated by CMN supplementation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1250-1263, 2016.

Influence of Tribulus terrestris on testicular enzyme in fresh water ornamental fish Poecilia latipinna.

The influence of Tribulus terrestris on the activities of testicular enzyme in Poecilia latipinna was assessed in lieu of reproductive manipulation. Different concentrations of (100, 150, 200, 250, and 300 mg) Tribulus terrestris extract and of a control were tested for testicular activity of enzymes in Poecilia latipinna for 2 months. The testis and liver were homogenized separately in 0.1 mol/l potassium phosphate buffer (0.1 mol/l, pH 7.2). The crude homogenate was centrifuged, and supernatant obtained was used as an enzyme extract for determination of activities. The activities of testicular functional enzyme ALP, ACP, SDH, LDH, and G6PDH levels were changed to different extent in treated groups compared with that of the control. The total body weight and testis weight were increased with the Tribulus terrestris-treated fish (Poecilia latipinna). These results suggest that Tribulus terrestris induced the testicular enzyme activity that may aid in the male reproductive functions. It is discernible from the present study that Tribulus terrestris has the inducing effect on reproductive system of Poecilia latipinna.

Effect of Vitamin E supplementation on the enzymatic activity of selected markers in Aohan fine-wool sheep testis.

The aim of this study was to evaluate the effects of dietary Vitamin E supplementation on the testicular 'marker' enzyme activity and Vitamin E content in Aohan fine-wool sheep. Thirty male Aohan fine-wool sheep (5 months of age) with similar body weight were selected from the Aohan fine-wool sheep-breeding farm of Inner Mongolia Autonomous Region, China. The sheep were randomly divided into five groups and supplemented with 0, 20, 200, 1000 or 2400 IU sheep(-1)d(-1) Vitamin E for 12 months. Three sheep in each group were slaughtered at 17 months to collect a testis sample for testicular marker enzyme analysis. The results showed that, compared to Control, supplementing the diet with Vitamin E at 200 IU sheep(-1)d(-1) significantly increased the content of Vitamin E in testis and improved the activity of testicular mitochondrial ATPase (P<0.01), lactate dehydrogenase (LDH) (P<0.01), sorbitol dehydrogenase (SDH) (P<0.01), and alkaline phosphatase (ALP) (P<0.05). The present study demonstrated that supplementing Vitamin E can have a positive role in improving testicular marker enzyme activity and that the optimum range of dose appeared to be 100-200 IU sheep(-1)d(-1).

Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats.

PURPOSE: Advanced glycation end products (AGE) are associated in the development of several pathophysiologies including diabetic cataract. Earlier we have reported that some common dietary agents have antiglycating activity and ginger (Zingiber officinalis) was one of the few prominent agents that effectively prevented AGE formation in vitro. In this study we investigated the potential of ginger to prevent diabetic cataract in rats. METHODS: Diabetes was induced in Wistar-NIN rats by intraperitoneal injection of streptozotocin (35 mg/kg bodyweight) and the control rats received vehicle alone. While a set of diabetic animals received AIN-93 diet, another set received either 0.5 or 3% ginger in their diet for a period of two months. Cataract progression was monitored by slit-lamp biomicroscope. At the end of two months, the animals were sacrificed to evaluate non-enzymatic glycation and osmotic stress in the eye lens. RESULTS: Slit-lamp examination revealed that feeding of ginger not only delayed the onset but also the progression of cataract in rats. Molecular analyses indicated that feeding of ginger significantly inhibited the formation of various AGE products including carboxymethyl lysine in the eye lens. In addition, ginger also countered hyperglycemia-induced osmotic stress in the lens. CONCLUSIONS: The results indicated that ginger was effective against the development of diabetic cataract in rats mainly through its antiglycating potential and to a lesser extent by inhibition of the polyol pathway. Thus, ingredients of dietary sources, such as ginger, may be explored for the prevention or delay of diabetic complications.

Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina.

This study evaluated the effects of retinal ischemia-reperfusion (IR) injury and pre-treatment with the potent and specific aldose reductase inhibitor fidarestat on apoptosis, aldose reductase and sorbitol dehydrogenase expression, sorbitol pathway intermediate concentrations, and oxidative-nitrosative stress. Female Wistar rats were pre-treated with either vehicle (N-methyl-D-glucamine) or fidarestat, 32 mg kg(-1) d(-1) for both, in the right jugular vein, for 3 consecutive days. A group of vehicle- and fidarestat-treated rats were subjected to 45-min retinal ischemia followed by 24-h reperfusion. Ischemia was induced 30 min after the last vehicle or fidarestat administration. Retinal IR resulted in a remarkable increase in retinal cell death. The number of TUNEL-positive nuclei increased 48-fold in the IR group compared with non-ischemic controls (p<0.01), and this increase was partially prevented by fidarestat. AR expression (Western blot analysis) increased by 19% in the IR group (p<0.05), and this increase was prevented by fidarestat. Sorbitol dehydrogenase and nitrated protein expressions were similar among all experimental groups. Retinal sorbitol concentrations tended to increase in the IR group but the difference with non-ischemic controls did not achieve statistical significance (p=0.08). Retinal fructose concentrations were 2.2-fold greater in the IR group than in the non-ischemic controls (p<0.05). Fidarestat pre-treatment of rats subjected to IR reduced retinal sorbitol concentration to the levels in non-ischemic controls. Retinal fructose concentrations were reduced by 41% in fidarestat-pre-treated IR group vs. untreated ischemic controls (p=0.0517), but remained 30% higher than in the non-ischemic control group. In conclusion, IR injury to rat retina is associated with a dramatic increase in cell death, elevated AR expression and sorbitol pathway intermediate accumulation. These changes were prevented or alleviated by the AR inhibitor fidarestat. The results identify AR as an important therapeutic target for diseases involving IR injury, and provide the rationale for development of fidarestat and other AR inhibitors.

Effect of Palestinian honey on spermatogenesis in rats.

Treatment of male albino rats with 5% honey for 20 days had no significant effect on total body weight or on the relative weight of other organs like the testis, seminal vesicles, spleen, kidneys, liver, heart, or brain. The only significant change was a 17% increase in the relative weight of the epididymis (P < or = .01). The relative weight of all the other organs was similar to those in control animals treated for the same period with drinking water. Treatment of rats for the same period with the same concentration of 5% sucrose produced no significant changes in absolute or relative weight of tested organs compared to control animals. The same treatment with Palestinian honey increased significantly the epididymal sperm count by 37% (P < or = .05). The activity of testicular marker enzymes for spermatogenesis such as sorbitol dehydrogenase (SDH) was increased by 31% (P < or = .05), and lactate dehydrogenase (LDH) was reduced by 48% (P < or = .05), which indicates that treatment with honey induces spermatogenesis. Similar treatment with sucrose had no significant effect on any of the key enzymes or epididymal sperm count. In conclusion, our results show that ingestion of honey induces spermatogenesis in rats by increasing epididymal sperm count, increasing selectively the relative weight of the epididymis, and increasing SDH activity and reducing LDH activity.

Attenuation of streptozotocin-induced oxidative stress in hepatic and intestinal tissues of Wistar rat by methanolic-garlic extract.

Diabetes is a major socio-economical burden with serious health consequences. The reactive oxygen species generated in this pathology alters the internal milieu of the cellular systems paving way to metabolic disorders. In the present investigation garlic's dose-dependent protective action against streptozotocin (STZ)-induced oxidative stress in hepatic and intestinal tissues has been studied. Methanolic garlic extract scavenged the DPPH radical with an IC(50) of 424 +/- 4.4 microg/ml proving its efficient antioxidant property. Garlic administration at 250 and 500 mg/kg body wt. significantly normalized the blood glucose in the diabetic rats. Biochemical analysis revealed a pronounced oxidative stress in STZ-rats (G-II) consequent to hyperglycemia as seen by a significant (P < 0.05 and 0.01) rise in malondialdehyde, protein carbonyls; accumulation of glycation products; disintegration of protein integrity (tryptophan fluorescence) followed by a decrease in reduced glutathione, antioxidant (GPx and CAT) enzymes culminating in apoptosis. Garlic administration in a dose-dependent manner has been found to restore and normalize significantly the above changes and thus restoring a normal functional integrity. These beneficial effects are prominent with 500 mg/kg body wt. dosage of garlic in comparison with 250 mg/kg body wt. dosage. But, 500 mg/kg body wt. dosage is not totally free from side effects as the decrease in body weight and increased intestinal tissue apoptosis were also found in control rats administered with garlic extract at 500 mg/kg body wt. along with diabetic rats. Based on these findings it is suggested that consumption of garlic at a lower dose is beneficial in terms of defensive action against oxidative stress.

The influence of methionine, selenomethionine, and vitamin E on liver metabolic pathways and steatosis in high-cholesterol fed rabbits.

Significant disorders of liver metabolic pathways enzymes after high-cholesterol diet could give information on liver steatosis development. This process could probably also be inhibited by some compounds, as examined in rabbits. Forty-two male rabbits were served a high-cholesterol diet (2 g%) (0.67 g/kg b.m./24 h) with addition of d,l-methionine (70 mg/kg b.m./24 h) or seleno-d,l-methionine (12.5 microg/kg b.m./24 h) or alpha-tocopherol (10 mg/kg b.m./24 h) for 3 months to compare the protection effect of used compounds on liver metabolism and steatosis. At the beginning and every month, blood was taken. After the experiment was completed, livers were dissected for histological examinations. The concentration of total cholesterol (t-CH), triacylglycerol (TG), and the activities of aldolase (ALD), sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined. Plasma t-CH and TG concentrations were significantly higher in all experimental groups vs control group. Blood serum AST and ALT activities did not undergo change but there were observed not significant increase in the CH group vs control group. Activities of SDH, GLDH, and LDH increased in blood serum and decreased in the liver in all experimental groups. Activities of LDH and SDH increased in the liver in the CH+Met group vs CH group. ALD activity decreased in the liver only in the CH and CH+Se groups. This data support a lipotoxic model of cholesterol-mediated hepatic steatosis. Prolonged administration of high-cholesterol diet not only disturbs the structure of cell membranes, which is expressed by decreased activity of enzymes in the liver and the migration of those enzymes to plasma but as well leads to steatosis of the liver, which has been confirmed by histological examinations. The applied compounds appear to have a varying influence upon the activity of enzymes determined in serum and liver. Obtained results showed a beneficial influence of methionine and vitamin E supplementation on liver steatosis development.

Long-term effect of Trigonella foenum graecum and its combination with sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues.

Trigonella foenum graecum seed powder (TSP) and Sodium Orthovanadate (SOV) have been shown to demonstrate antidiabetic effects by stabilizing glucose homeostasis and carbohydrate metabolism in experimental type-1 diabetes. However their efficacy in controlling histopathological and biochemical abnormalities in ocular tissues associated with diabetic retinopathy is not known. The purpose of this study was to investigate the comparative efficacy of individual as well as combination therapy of TSP and SOV in 8 weeks diabetic rat lens and retina. Retinas and lenses were taken from control, alloxan-induced diabetic rats and diabetic rats treated separately with insulin, 5%TSP, SOV (0.6 mg/ml) and a combined dose of SOV (0.2 mg/ml) and 5%TSP for 60 days. Control and each experimental group had six rats. Alterations in the activities of enzymes HK (hexokinase), AR (aldose reductase), SDH (sorbitol dehydrogenase), G-6-PD (glucose-6-phosphate dehydrogenase), GPx (glutathione peroxidase), GR (glutathione reductase) and levels of metabolites like sorbitol, fructose, glucose, MDA (malondialdehyde) and GSH (reduced glutathione) were measured in the cytosolic fraction of lenses besides measuring blood glucose levels and glycosylated haemoglobin. Histopathological abnormalities were studied in the lens using photomicrography and retina using transmission electron microscopy. Blood glucose, glycosylated haemoglobin levels and polyol pathway enzymes AR and SDH increased significantly causing accumulation of sorbitol and fructose in the diabetic lens and treatment with SOV and TSP significantly (p < 0.05) decreased these to control levels. Similarly, SOV and TSP treatments modulated the activities of HK, G-6-PD, GPx and GR in the rat lens to control values. Ultrastructure of the diabetic retina revealed disintegration of the inner nuclear layer cells with reduction in rough endoplasmic reticulum and swelling of mitochondria in the bipolar cells; and these histopathological events were effectively restored to control state by SOV and TSP treatments. In this study SOV and TSP effectively controlled ocular histopathological and biochemical abnormalities associated with experimental type-1 diabetes, and a combination regimen of low dose of SOV with TSP demonstrated the most significant effect. In conclusion, the potential of SOV and TSP alone or in low dose combination may be considered as promising approaches for the prevention of diabetic retinopathy and other ocular disorders.

Restoration of ultrastructural and biochemical changes in alloxan-induced diabetic rat sciatic nerve on treatment with Na3VO4 and Trigonella--a promising antidiabetic agent.

Vanadium has been reported to have broad pharmacological activity both in vitro and in vivo. Vanadium compound, sodium orthovanadate, Na3VO4, is well known for its hypoglycaemic effects. However, Na3VO4 exerts these effects at relatively high doses (0.6 mg/ml) and exhibit several toxic effects. In the present study lower doses of Na3VO4 (0.2 mg/ml) are combined with Trigonella foenum graecum seed powder (TSP), another hypoglycaemic agent, to reduce its toxicity without compromising its antidiabetic potential. The efficacy of the lower doses of Na3VO4 has been investigated in restoring the altered glucose metabolism and histological structure in the sciatic nerves in 21 and 60 days alloxan diabetic rats. A portion of the glucose was found to be channelled from the normal glycolytic route to polyol pathway, evident by the reduced hexokinase activity and increased polyol pathway enzymes aldose reductase and sorbitol dehydrogenase activity causing accumulation of sorbitol and fructose in diabetic conditions. Ultrastructural observation of the sciatic nerve showed extensive demylination and axonal loss after eight weeks of diabetes induction. Blood glucose levels increased in diabetic rats were normalized with the lower dose of vanadium and Trigonella treatment. The treatment of the diabetic rats with vanadium and Trigonella prevented the activation of the polyol pathway and sugar accumulations. The sciatic nerves were also protected against the structural abnormalities found in diabetes with Trigonella foenum graecum as well as Na3VO4. Results suggest that lower doses of Na3VO4 may be used in combination with TSP as an efficient antidiabetic agent to effectively control the long-term complications of diabetes in tissues like peripheral nerve.

Water extract of 1:1 mixture of Phellodendron cortex and Aralia cortex has inhibitory effects on oxidative stress in kidney of diabetic rats.

Enhanced activity of the lipid peroxidation and oxidative damages have been implicated in the pathogenesis of diabetic kidney complications. We explored to determine whether these changes in diabetic kidney could be prevented by water extract of mixture of Phellodendron cortex and Aralia cortex (P55A). Greatly elevated content of thiobarbituric acid reactive substances (TBARS) and carbonylated protein in kidneys of diabetic rats were significantly reduced by P55A treatment. In addition, abnormally low ratio of GSH/GSSG in diabetic kidneys was elevated to almost normal levels by the treatment with P55A. These results suggest that P55A extracts exert antioxidant effect by reducing lipid peroxidation and protein carbonylation as well as by elevating the ratio of GSF/GSSG in diabetic kidney.

Mannitol-1-phosphate dehydrogenase from Cryptococcus neoformans is a zinc-containing long-chain alcohol/polyol dehydrogenase.

Cryptococcus neoformans, the causative agent of cryptococcosis, produces large amounts of mannitol in culture and in infected mammalian hosts. Although there is considerable indirect evidence that mannitol synthesis may be required for wild-type stress tolerance and virulence in C. neoformans, this hypothesis has not been tested directly. It has been proposed that mannitol-1-phosphate dehydrogenase (MPD) is required for fungal mannitol synthesis, but no MPD-deficient fungal mutants or cDNAs or genes encoding fungal MPDs have been described. Therefore, C. neoformans was purified from a 148 kDa homotetramer of 36 kDa subunits that catalysed the reaction mannitol1-phosphate+NAD--><--fructose 6-phosphate+NADH. Partial peptide sequences were used to isolate the corresponding cDNA and gene, and the deduced MPD protein was found to be homologous to the zinc-containing long-chain alcohol/polyol dehydrogenases. Lysates of Saccharomyces cerevisiae transformed with the cDNA of interest (but not vector-transformed controls) contained MPD catalytic activity. Lastly, Northern analyses demonstrated MPD mRNA in glucose- and mannitol-grown C. neoformans cells. Thus, MPD has been purified and characterized from C. neoformans, and the corresponding cDNA and gene (MPD1) cloned and sequenced. Availability of C. neoformans MPD1 should permit direct testing of the hypotheses that (i) MPD is required for mannitol biosynthesis and (ii) the ability to synthesize mannitol is essential for wild-type stress tolerance and virulence.

Protection of rat hepatocytes exposed to CCl4 in-vitro by cynandione A, a biacetophenone from Cynanchum wilfordii.

To identify hepatoprotective agents from plant sources we use primary cultures of rat hepatocytes injured by CCl4. The hepatoprotective agents are the compounds that mitigate the injury caused by CCl4. Using this system we have investigated the biochemical mechanisms involved in the hepatoprotective activity of cynandione A, a biacetopherone, isolated from the roots of Cynanchum wilfordii Hemsley (Asclepiadaceae). Cynandione A (50 microM) significantly reduced (approximately 50%) the release into the culture medium of glutamic pyruvic transaminase and sorbitol dehydrogenase from the primary cultures of rat hepatocytes exposed to CCl4. Glutathione, superoxide dismutase, catalase and glutathione reductase play important roles in the cellular defence against oxidative stress. Cynandione A appeared to protect primary cultured rat hepatocytes exposed to CCl4 from significant drops in the levels of each of these four specific markers. Cynandione A also ameliorated lipid peroxidation by up to 50% as demonstrated by a reduction in the production of malondialdehyde. These results suggest that cynandione A protected the hepatocytes from CCl4-injury by maintaining the level of glutathione and by inhibiting the production of malondialdehyde, due to its radical scavenging properties.

Antihepatotoxic activity of bergenin, the major constituent of Mallotus japonicus, on carbon tetrachloride-intoxicated hepatocytes.

To determine the antihepatotoxic activity of bergenin from Mallotus japonicus, carbon tetrachloride (CCl4)-induced cytotoxicity in primary cultured rat hepatocytes has been adopted as an assay system. Bergenin significantly reduced the activities of glutamic pyruvic transaminase and sorbitol dehydrogenase released from the CCl4-intoxicated hepatocytes. The antihepatotoxicity of bergenin was also evidenced by elevating the activities of glutathione S-transferase and glutathione reductase, and content of glutathione in the CCl4-intoxicated hepatocytes. From these results, it is assumed that bergenin exerted antihepatotoxicity against CCl4-induced cytotoxicity through glutathione-mediated detoxification as well as free radical suppressing activity.

EROD induction and biliary metabolite excretion following exposure to the water accommodated fraction of crude oil and to chemically dispersed crude oil.

Immature Atlantic salmon (Salmo salar) were exposed to water accommodated fraction (WAF) of Bass Strait crude oil or to Corexit 9527-dispersed crude oil for 6 days, followed by a depuration period of 29 days. Serum sorbitol dehydrogenase (SDH) levels, indicator of liver damages, remained low during the experiment. Hepatic EROD activity was induced within 2 days following the onset of the exposure in both treatments, and persisted for 2-4 and 4-6 days after transfer to clean sea water in the WAF and dispersed oil treatment, respectively. Naphthalene-type metabolites, determined by fixed-wavelength fluorescence detection, appeared in the bile of the fish with 2 days' delay compared to EROD induction. In both treatments, EROD activity induction and levels of naphthalene-type metabolites in the bile were significantly related. The biliary levels of naphthalene-type metabolites were over 15 times higher in fish exposed to dispersed crude oil relative to fish exposed to the WAF of Bass Strait crude oil. BaP-type metabolites appeared only in the bile of the fish exposed to the WAF, possibly due to BaP-type compounds remaining associated with the dispersant in the water column or to an inhibition of Phase II detoxification enzymes by the dispersant. Bile metabolites as determined by fixed-wavelength fluorescence and EROD induction appear to be sensitive and complementary biomarkers of exposure to PAH.