Mannitol oxidase and polyol dehydrogenases in the digestive gland of gastropods: Correlations with phylogeny and diet.

Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.

Proteomics of human aqueous humor.

The aqueous humor is a colorless, transparent fluid that fills the anterior chamber of the eye. It plays an important role in maintaining the intraocular pressure and providing nourishment to the lens and cornea. The constitution of the aqueous humor is controlled by the blood-aqueous barrier. Though this ocular fluid has been extensively studied, its role in ocular physiology is still not completely understood. In this study, aqueous humor samples were collected from 250 patients undergoing cataract surgery, subjected to multiple fractionation strategies and analyzed on a Fourier transform LTQ-Orbitrap Velos mass spectrometer. In all, we identified 763 proteins, of which 386 have been identified for the first time in this study. Sorbitol dehydrogenase (SORD), filensin (BFSP1), and phakinin (BFSP2) are some of the proteins that have not been previously reported in the aqueous humor. Gene Ontology analysis revealed 35% of the identified proteins to be extracellular, with a majority of them involved in cell communication and signal transduction. This study comprehensively reports 386 novel proteins that have important potential as biomarker candidates for future research into personalized medicine and diagnostics aimed towards improving visual health.

[Effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells].

OBJECTIVE: To explore the effects of glucose concentration fluctuation on function of cultured bovine arterial endothelial cells and underlying mechanism. METHODS: The thoracic aorta of newborn calf was used for primary endothelial cells culture. Cells were divided into 3 groups and cultured for 48 h: control group (C, 5.5 mmol/L), constant high glucose group (HG, 30 mmol/L) and glucose fluctuation (GF, three circles of 2 h 30 mmol/L followed by 3 h 5.5 mmol/L, 30 mmol/L overnight, repeat the whole procedure on the following day) groups. The membranes fluidity of endothelial cells was detected by fluorescence polarization method. The contents of sorbierite, aldose reductase (AR), sorbitol dehydrogenase (SDH) and advanced glycation end products (AGEs) were measured. RAGE, eNOS and ET-1 mRNA expressions were detected by semi-quantitative RT-PCR. RESULTS: The membranes fluidity of endothelial cells in HG or GF group were significantly decreased compared with the control group (all P < 0.01) and significantly lower in GF group than those in HG group (all P < 0.01). Sorbierite, AR and AGEs concentrations were significantly higher in HG and GF groups than those in control group (all P < 0.01) and AR and AGEs concentrations were significantly higher in GF group than that in HG group (all P < 0.01). SDH of endothelial cells in HG or GF group were decreased compared with the control group and lower in GF group than in HG group (all P < 0.05). In addition, the mRNA levels of RAGE, eNOS and ET-1 were significantly upregulated compared with the control group (all P < 0.01). CONCLUSIONS: Glucose concentration fluctuation can result in more severe bovine arterial endothelial cells dysfunction than high glucose via activating polyols metabolic pathways, upregulating the expression of AGEs, eNOS and ET-1. Therefore, glucose concentration fluctuation might play a crucial role on macrovascular complications of diabetes.

Nondenaturing two-dimensional electrophoresis enzyme profile involving activity and sequence structure of cytosol proteins from mouse liver.

After cytosol proteins in the mouse liver were separated by nondenaturing two-dimensional electrophoresis (2-DE), activities of several enzymes, such as fructose bisphosphatase, sorbitol dehydrogenase and malate dehydrogenase, transferase and sorbitol dehydrogenase, or several dehydrogenases, were analyzed on the same 2-D gel. Further, peptidase (or protease) activity can be examined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) when peptides such as angiotensin and adenocorticotropic hormone are incubated in the presence of the cytosol protein separated by nondenaturing 2-DE. Sequence structures of proteins on the 2-D gel were analyzed by peptide mass fingerprinting using MALDI-TOF-MS or by peptide sequencing using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). The combination of activity and sequence structure accurately verified the position and activity range of the separated enzymes on the nondenaturing 2-D gel. From these results, we created a nondenaturing 2-D enzyme profile involving activities and sequence structure of cytosol proteins from mouse liver. This profile can be used for checking whether activities of enzymes were specifically or nonspecifically inhibited by inhibitors.

Comparison of the susceptibility of wild-type and CYP2E1 knockout mice to the hepatotoxic and pneumotoxic effects of styrene and styrene oxide.

Styrene causes both liver and lung damage in non-Swiss albino, CD-1, and other strains of mice. This is considered to be due to the bioactivation of styrene to styrene oxide by cytochromes P450, principally CYP2E1 and CYP2F2. If so, one would expect CYP2E1 knockout mice to be less susceptible to styrene-induced toxicity than wild-type mice. However, previous in vitro and in vivo studies demonstrated little difference in the metabolism of styrene to styrene oxide between wild-type and CYP2E1 knockout mice. These findings would suggest that there should be no difference in the toxic responses to styrene between these two strains. To determine which of these possibilities was correct, styrene (600 mg/kg) or styrene oxide (300 mg/kg) was administered i.p. 24 h prior to measurement of serum sorbitol dehydrogenase as a biomarker of hepatotoxicity or lactate dehydrogenase activity, protein, and cells in bronchoalveolar lavage fluid as biomarkers for pneumotoxicity. Styrene was more hepatotoxic in the wild-type mice than in the knockout mice suggesting CYP2E1 activity is important. Strain differences were not observed with styrene oxide indicating no difference in intrinsic susceptibility. For lung, the response was similar in both strains to both styrene and styrene oxide supporting the idea that CYP2F2 is important in the bioactivation of styrene in this tissue and that there is no strain difference in susceptibility to the active metabolite.

Pink snapper (Pagrus auratus) as a bioindicator of aquatic. Environmental health in Western Australia.

Pink snapper (Pagrus auratus), an endemic Western Australian fish species, was tested for its potential as a bioindicator of aquatic environmental health. Mixed function oxygenase (MFO) induction (as a biomarker of exposure), and sorbitol dehydrogenase (SDH) activity (as a biomarker of liver damage) were of special interest to the study as these biochemical tools have not been validated for any Western Australian fish species. Juvenile pink snapper were injected intraperitoneally (i.p.) with 0, 10, 100, 500, and 1000 micrograms PCB-126 per kilogram. Fish were sacrificed 10 days postinjection, and livers and blood samples were collected for MFO and SDH analysis, respectively. Doses of 10 and 100 micrograms PCB-126 per kilogram caused the highest MFO induction, while doses of 0 and 1000 micrograms PCB-126 per kilogram did not result in higher MFO activity relative to carrier-injected control fish. SDH activities were not significantly different among treatments indicating that hepatocellular damage was not responsible for the reduced MFO activity at the highest dose. The result of the study demonstrates that pink snapper may potentially be used as a bioindicator species in Western Australia when MFO is used as a biomarker.

Enzyme activity in parotid and mandibular saliva from red kangaroos, Macropus rufus.

Parotid and mandibular saliva was obtained from red kangaroos by concurrent acetylcholine isoprenaline stimulation. Salivary proteins were separated by horizontal electrophoresis on either cellulose acetate or starch gels and assessed by specific staining techniques for 23 enzymes commonly found in mammalian tissues and body fluids. Parotid saliva was positive for acid phosphatase, alpha-amylase, carbonic anhydrase, glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase and superoxide dismutase activities. Mandibular saliva was positive for alcohol dehydrogenase in addition to the above six enzymes. The kangaroo salivas lacked activity for alkaline phosphatase, beta-galactosidase and non-specific esterase which occur in saliva from some mammalian species.

Expression of Na+/Cl-/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney.

It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region.

[Hepatocyte cytolysis enzymes as the markers of operative trauma in open cholecystectomy]. / Fermenty tsitoliza gepatotsitov v otsenke operatsionnoi travmy pri otkrytoi kholetsistektomii.

Surgical and narcotic aggression lead to certain injuries of hepatocytes. The authors followed up the time course of liver injury marker enzymes in two groups of patients subjected to open cholecystectomy (n = 62): succinate dehydrogenase (SDH), glutamate dehydrogenase (GIDH), SGPT, and SGOT. In group 1 the operative trauma was minimal, in group 2 traumatism was higher because of technological difficulties. A high diagnostic significance of SDH and GIDH is worthy of note: their activities increase in proportion with the severity of surgical trauma. Comparison of laboratory data helps objectively assess the severity of operative injury in open cholecystectomy.

Hepatotoxicity of N, N-dimethylformamide in rats following intraperitoneal or inhalation routes of administration.

Male Sprague-Dawley rats were administered a single intraperitoneal injection of N, N-dimethylformamide (DMF, 0.01-1.5 g kg-1) or were exposed for 4 h to DMF vapours (75-900 ppm). The serum activities of the enzymes sorbitol deshydrogenase and glutamate deshydrogenase were used as indicators of liver damage, and were determined at 24, 48 or 72 h post-treatment. Following either route of administration DMF caused concentration-dependent elevations in enzyme activities, the maxima of which occurred later after administration of higher concentrations of DMF than after lower concentrations.

Study of the relationship between the hepatotoxicity and free radical induced by 1,1,2-trichloroethane and 1,1,1-trichloroethane in rat.

The hepatotoxicity and the relationship between the hepatotoxicity and free radical induced by 1,1,2-trichloroethane (1,1,2-TCE) and 1,1,1-trichloroethane (1,1,1-TCE) were studied by whole animals test and the isolated perfused rat liver test. Enzymatic parameters measured during the test included the assay of levels of glutamic pyruvic transaminase (GPT), sorbital dehydrogenase (SDH) and glutamate dehydrogenase (GDH). The observation of the pathologic changes of the liver by the light microscope and the measurement of the relative total free radical concentration in the liver were also made. The results showed that 1,1,2-TCE caused definite pathologic changes of rat liver. It led to much higher values for GPT, SDH and GDH both in serum and perfusate than 1,1,1-TCE did (P < 0.01). The concentration of perfusate K+ caused by 1,1,2-TCE was higher than that by 1,1,1-TCE (P < 0.01). The value of the relative total free radical concentration induced by 1,1,2-TCE was also greater than that by 1,1,1-TCE (P < 0.05). The results suggested that the hepatotoxicity of 1,1,2-TCE was stronger than that of 1,1,1-TCE. The free radical concentration was increased proportionally to the increase of the hepatotoxicity of 1,1,2-TCE and 1,1,1-TCE. It appeared that free radical may play an important role in the mechanism of the hepatic injury induced by 1,1,2-TCE.

Variation in sorbitol accumulation and polyol-pathway activity in cultured human proximal tubule cells.

The polyol pathway is present in tissues of several organs where its activation may participate in the development of diabetic complications. We measured the accumulation of polyol-pathway intermediates in HPT cells isolated from 21 different human kidneys from nondiabetic individuals. When exposed to 27.5 mM glucose in the growth media, cells isolated from approximately 75% of individuals (accumulators) accumulated sorbitol within 1-4 days, whereas 25% (nonaccumulators) accumulated only negligible amounts, even when the period of exposure was extended to 2 wk. Surprisingly, measurement of the activities of the polyol-pathway enzymes showed no difference in the levels of either AR or SDH between accumulators and nonaccumulators, even when the conversion of galactose to galactitol was used to measure AR activity in intact cells independently of SDH. Measurement of sorbitol in the growth media indicated that nonaccumulators were not releasing sorbitol into the growth media. Fructose levels in the conditioned growth media were 4 times higher in the sorbitol-accumulating cells. Together, these results indicate that the tendency of cells from an individual to accumulate significant amounts of sorbitol may reflect the cells' ability to metabolize sorbitol in steps subsequent to the polyol pathway.

Effects of simulated Fowler-Stephens orchiopexy on testicular structure and function in rats.

To determine the effects of the Fowler-Stephens orchiopexy (FSO) on testicular structure and function, young rats underwent simulated FSO and concurrent contralateral orchiectomy, unilateral orchiectomy, or no operation. Rats were killed at 1, 2, 4, 6, 8, and 10 weeks postoperation, and serum testosterone, as well as testicular concentrations of the enzymes LDH & SDH, protein markers of testicular germinal cell development, were measured at the time of death. Although LDH and SDH concentrations decreased by 45 per cent in testes after simulated FSO at 4 weeks, control testes showed a 5 per cent increase in these enzymes. Serum testosterone decreased to one-fourth the initial value in rats after FSO, whereas control rats showed a slight increase. Within 2 weeks after simulated FSO, spermatocytes and sperm were sparse and there was marked disruption of tubular morphology; after 4 weeks interstitial fibrosis became prominent. Only a rare testis with good collateral vessels and resultant good histologic appearance and enzyme profile survived the FSO procedure, and these testes were considerably smaller than the controls.

Cytochemical localization of sorbitol dehydrogenase in kidney and liver from Myiopsitta m. monachus (Boddaert, 1783), during embryonic development.

A histochemical study to determine the localization of sorbitol dehydrogenase (SDH) in kidney and liver from embryionic, young and adult Myiopsitta m. monachus was performed. The enzyme activity increased with age in both organs. In the kidney, the enzyme appeared at the proximal convoluted tubules, and increased in the basal cytoplasm of the tubular cells. In the liver the localization was diffuse in the lobule but more intense in the cytoplasm of hepatocytes, especially in the perinuclear areas. These studies indicate that the cytochemical enzyme localization differs in this species, which is more evolutioned than Gallus gallus, and would be related to ontogenetic and phylogenetic differentiation.

Cytochemical localization of sorbitol dehydrogenase in kidney and liver from Myiopsitta m. monachus (Boddaert, 1783), during embryonic development

A histochemical study to determine the localization of sorbitol dehydrogenase (SDH) in kidney and liver from embryionic, young and adult Myiopsitta m. monachus was performed. The enzyme activity increased with age in both organs. In the kidney, the enzyme appeared at the proximal convoluted tubules, and increased in the basal cytoplasm of the tubular cells. In the liver the localization was diffuse in the lobule but more intense in the cytoplasm of hepatocytes, especially in the perinuclear areas. These studies indicate that the cytochemical enzyme localization differs in this species, which is more evolutioned than Gallus gallus, and would be related to ontogenetic and phylogenetic differentiation.(Au)

Reconstitution of the ethanol oxidase respiratory chain in membranes of quinoprotein alcohol dehydrogenase-deficient Gluconobacter suboxydans subsp. alpha strains.

The ethanol oxidase respiratory chain of Gluconobacter suboxydan was characterized by using G. suboxydans subsp. alpha, a variant species of G. suboxydans incapable of oxidizing ethanol. The membranes of G. suboxydans subsp. alpha exhibited neither alcohol dehydrogenase, ethanol oxidase, nor glucose-ferricyanide oxidoreductase activity. Furthermore, the respiratory chain of the organism exhibited an extremely diminished amount of cytochrome c and an increased sensitivity of the respiratory activity for cyanide or azide when compared with G. suboxydans. The first-subunit quinohemoprotein and the second-subunit cytochrome c of alcohol dehydrogenase complex in the membranes of G. suboxydans subsp. alpha were shown to be reduced and deficient, respectively, by using heme-staining and immunoblotting methods. Ethanol oxidase activity, lacking in G. suboxydans subsp. alpha, was entirely restored by reconstituting alcohol dehydrogenase purified from G. suboxydans to the membranes of G. suboxydans subsp. alpha; this also led to restoration of the cyanide or azide insensitivity and the glucose-ferricyanide oxidoreductase activity in the respiratory chain without affecting other respiratory activities such as glucose and sorbitol oxidases. Ethanol oxidase activity was also reconstituted with only the second-subunit cytochrome c of the enzyme complex. The results indicate that the second-subunit cytochrome c of the alcohol dehydrogenase complex is essential in ethanol oxidase respiratory chain and may be involved in the cyanide- or azide-insensitive respiratory chain bypass of G. suboxydans.

The hepatotoxic action of chloroform: short-time dynamics of biochemical alterations and dose-effect relationships.

Chloroform was administered ip to Balb/c mice as a single dose ranging from 1/8 to 1 of the approximate lethal dose. At different time periods after administration, mice were sacrificed. Serum glutamate-pyruvate transaminase (SGPT) and sorbitol dehydrogenase (SDH) as well as glutathione (GSH) and malondialdehyde (MDA) levels in the liver were determined. Increased SGPT and SDH levels were found for all doses exceeding 1/8 of the approximate lethal dose. The depletion of GSH level was kept within 40% for all doses. A 2-4 fold increase of hepatic MDA level was found. The depletion of hepatic GSH and, to some extent the increase of serum SGPT and SDH, occurred in biphasic fashion. Dose-effect functions for these biochemical alterations could only be constructed for the second, delayed phase of action. It is postulated that the hepatotoxicity of chloroform is mainly dependent on radical formation in the course of biotransformation.

[Testicular morphological and biochemical changes after Fowler-Stephens orchiopexy in rats].

Fowler-Stephens orchiopexy is the most common method of treating high-positioned undescended testes. Its success rate has been reported to be as high as 50-70% based on palpation or in a few circumstances on biopsy. Although it is convenient to evaluate testicular function by palpation and/or biopsy, this method is very subjective and is not scientific enough. To determine testicular function more precisely and objectively, we performed the Fowler-Stephens orchiopexy on Sprague-Dawley rats and observed the morphological and biochemical changes, including assays of LDH, SDH and the testosterone level. In our morphological study, with the use of Fowler-Stephens orchiopexy, only 17% (3/18) of the rat tests could be salvaged. The others revealed either necrosis or fibrosis. Testicular LDH, checked at the 4th postoperative week, revealed 0.62 +/- 0.04 U/mg which was statistically different (p less than 0.05) from that of the control group and the hemicastrated group (0.77 +/- 0.05 U/mg and 0.76 +/- 0.07 U/mg, respectively). The SDH obtained from the testes also revealed significant differences between the study group and the control and hemicastrated groups. Values obtained were 2.67 +/- 0.15 mU/mg, 3.77 +/- 0.4 mU/mg and 3.77 +/- 0.33 mU/mg, respectively (p less than 0.05). Using electrophoresis, 3 out of 18 rats had typical X bands, which is the classical picture of a normal mature testis. In contrast, the others showed faint X bands at the 2nd postoperative week, which subsequently faded thereafter. During testicular ischemia, the Leydig cells are more resistant than the Sertoli cells and the germinal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary fishmeal versus soyabean meal: an assessment of serum and liver enzyme response in the chicken.

In a five-week feeding trial, 90 broiler chicks were used to study the response of serum and liver alanine aminotransferase (ALT) aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH) and alkaline phosphatase following the replacement of varying levels (0, 20, 40, 60, 80 and 100 per cent) of fishmeal protein by soyabean meal protein. The results showed that both serum ALT and AST activities increased significantly (P less than 0.01) with the increasing levels of substitution of soyabean meal for fishmeal. Regression analysis showed a significant (P less than 0.01) positive correlation between ALT and AST activities and the level of soyabean meal substitution with correlation coefficient, r, of 0.93 and 0.98, respectively. The liver ALT and AST tended to increase with the increasing proportion of soyabean meal although such increases were not significant (P greater than 0.01). Serum and liver SDH and alkaline phosphatase activities were not significantly influenced by diet.