The effects of second messenger cAMP and its relative components on the contraction of uterine smooth muscle of rat.

OBJECTIVE: To investigate the effect of second messenger pathways on the uterine smooth muscle contraction and their associated mechanisms, and compare the evaluation methods. MATERIALS AND METHODS: Preparation of uterine smooth muscle strips from healthy pregnant 18-21 d SD and non-pregnant rats. When the contraction of muscle strips was stable, we conducted gradient administration: PDE4 inhibitors (Z90), prostaglandin PGE2, adenylate cyclase inhibitor (SQ 22,530), cAMP analogs (dbcAMP) and AMPK agonists (AICAR), solvent dimethyl sulfoxide (DMSO) as controlled. Gradient administration of acetylcholine (Ach) and oxytocin (oxytocin) induced the contraction of muscle strips. The tension transducer and biological information collecting system were applied to record the changes, including duration, dilation tension, contraction tension, peak height, and mean tension, before and after different administration. Principal components analysis was adopted to evaluate the five changes. RESULTS: SQ 22,530, DMSO, cAMP alone had no significant effect on the contraction of uterine smooth muscle; Z90 can inhibit the spontaneous contraction of pregnant uterine smooth muscle strips; dbcAMP and AICAR can antagonize acetylcholine and oxytocin-induced the contraction of pregnant uterine smooth muscle strips. Z90, SQ 22,530 + Z90, dbcAMP, AICAR can inhibit the uterine contraction peak, diastolic amplitude, average muscle tone and contraction duration of the pregnant uterine smooth muscle in a concentration-dependent manners. At the same time, we compared the parameters, which reflect the contraction of uterine smooth muscle, and conduct main components analysis to determine the effect of the drugs. CONCLUSIONS: The second messenger cAMP and its related components ATP, 5'- AMP, AC, PDE, PKA, and AMPK can affect the uterine smooth muscle contraction via related signaling pathway in rats, and principal components analysis can be adopted to evaluate the smooth muscle relaxant.

Continual feeding of two types of microalgal biomass affected protein digestion and metabolism in laying hens.

A 14-wk study was conducted to determine the nutritional efficacy and ssmetabolic impact of 2 types of microalgal biomass as alternative protein sources in laying hen diets. Shaver hens (total = 150 and 26 wk old) were fed 1 of 5 diets: a control or a defatted green microalgal biomass (DG; Desmodesmus spp.) at 25% and a full-fatted diatom biomass (FD; Staurosira spp.) at 11.7% inclusion with or without protease. This experiment consisted of 5 replicates per treatment and each replicate contained 6 hens individually reared in cages (1 hen for biochemical data/replicate). Despite decreased ADFI (P = 0.03), hens fed DG or FD had final BW, overall hen-day egg production, and egg quality similar to the controls. Feeding DG or FD did not alter plasma concentrations of insulin, glutamine, and uric acid or alkaline phosphatase activity at wk 8 or 14 but decreased plasma 3-methyhistine concentrations (P = 0.03) and tartrate-resistant acid phosphatase (TRAP) activities (P < 0.001) at wk 14 and improved (P = 0.002) ileal total AA digestibility. Although DG or FD exhibited moderate effects on intestinal brush border protease activities and mRNA levels of duodenal transporters Pept1, Lat1, and Cat1, both substantially enhanced (P < 0.05) phosphorylation of hepatic protein synthesis key regulator S6 ribosomal protein (S6) and the ratio of phospho-S6 to S6 in the liver of hens. However, DG and FD manifested with different impacts on weights of egg and egg albumen, proteolytic activity of jejunal digesta, plasma TRAP activity, ileal total AA digestibility, and several intestinal genes and hepatic proteins. Supplemental protease in the DG and FD diets produced mixed effects on a number of measures. In conclusion, our findings revealed the feasibility of including greater levels of microalgal biomass as a source of feed protein for laying hens and a novel potential of the biomass in improving dietary protein digestion and body protein metabolism than previously perceived.

Nonruminant Nutrition Symposium: Potential of defatted microalgae from the biofuel industry as an ingredient to replace corn and soybean meal in swine and poultry diets.

While feeding food-producing animals with microalgae was investigated several decades ago, this research has been reactivated by the recent exploration of microalgae as the third generation of feedstocks for biofuel production. Because the resultant defatted biomass contains high levels of protein and other nutrients, it may replace a portion of corn and soybean meal in animal diets. Our laboratory has acquired 4 types of full-fat and defatted microalgal biomass from biofuel production research (Cellana, Kailua-Kona, HI) that contain 13.9 to 38.2% crude protein and 1.5 to 9.3% crude fat. This review summarizes the safety and efficacy of supplementing 2 types of the biomass at 7.5 to 15% in the diets of weanling pigs, broiler chicks, and laying hens. Based on their responses of growth performance, egg production and quality, plasma and tissue biochemical indicators, and/or fecal chemical composition, all 3 types of animals were able to tolerate the microalgal biomass incorporation into their diets at 7.5% (on as-fed basis). Holistic analysis is also provided to explore the global potential of using the defatted microalgal biomass as a new feed ingredient in offsetting the biofuel production cost, reducing the dependence on staple crops such as corn and soybeans, decreasing greenhouse gas production of animal agriculture, and developing health value-added animal products.

Molecular mechanisms for hyperinsulinaemia induced by overproduction of selenium-dependent glutathione peroxidase-1 in mice.

AIMS/HYPOTHESIS: We previously observed hyperglycaemia, hyperinsulinaemia, insulin resistance and obesity in Gpx1-overexpressing mice (OE). Here we determined whether these phenotypes were eliminated by diet restriction, subsequently testing whether hyperinsulinaemia was a primary effect of Gpx1 overexpression and caused by dysregulation of pancreatic duodenal homeobox 1 (PDX1) and uncoupling protein-2 (UCP2) in islets. METHODS: First, 24 male OE and wild-type (WT) mice (2 months old) were given 3 g (diet-restricted) or 5 g (full-fed) feed per day for 4 months to compare their glucose metabolism. Thereafter, several mechanistic experiments were conducted with pancreas and islets of the two genotypes (2 or 6 months old) to assay for beta cell mass, reactive oxygen species (ROS) levels, mitochondrial membrane potential (Deltapsi(m)) and expression profiles of regulatory proteins. A functional assay of islets was also performed. RESULTS: Diet restriction eliminated obesity but not hyperinsulinaemia in OE mice. These mice had greater pancreatic beta cell mass (more than twofold) and pancreatic insulin content (40%) than the WT, along with an enhanced Deltapsi(m) and glucose-stimulated insulin secretion in islets. With diminished ROS production, the OE islets displayed hyperacetylation of H3 and H4 histone in the Pdx1 promoter, elevated PDX1 and decreased UCP2. CONCLUSIONS/INTERPRETATION: Overproduction of the major antioxidant enzyme, glutathione peroxidase 1, caused seemingly beneficial changes in pancreatic PDX1 and UCP2, but eventually led to chronic hyperinsulinaemia by dysregulating islet insulin production and secretion.

Distribution of supplemental Escherichia coli AppA2 phytase activity in digesta of various gastrointestinal segments of young pigs.

The objective of this study was to determine the functional location and disappearance of activity of a supplemental Escherichia coli AppA2 phytase and its impact on digesta P and Ca concentrations in the gastrointestinal tract of pigs. In Exp. 1, 18 pigs (8.3 +/- 0.2 kg of BW) were allotted to 3 groups (n = 6 each) and fed a low-P (0.4%) corn-soybean meal, basal diet (BD), BD + phytase [500 units (U)/kg of feed], or BD + inorganic P (iP, 0.1%) for 4 wk. In Exp. 2, 30 pigs (14.5 +/- 0.2 kg of BW) were allotted to 3 groups (n = 10 each) and fed BD, BD + 500 U of phytase/kg of feed, or BD + 2,000 U of phytase/kg of feed for 2 wk. Five or six pigs from each treatment group were killed at the end of both experiments to assay for digesta phytase activity and soluble P concentration in 6 segments of the digestive tract and digesta total P and Ca concentrations in stomach and colon. Compared with pigs fed BD, pigs fed BD + 500 U of phytase/kg of feed in Exp. 1 and BD + 2,000 U of phytase/kg of feed in Exp. 2 had greater (P < 0.05) phytase activities in the digesta of the stomach and upper jejunum (2 m aborally from the duodenum). No phytase activity was detected in the digesta of the lower jejunum (2.12 m cranial to the ileocecal junction) or ileum from any of the treatment groups in either trial. Concentrations of digesta-soluble P peaked in the upper jejunum of pigs fed BD in Exp. 1 and 2, but showed gradual decreases between the stomach and the upper jejunum of pigs fed BD + phytase or BD + iP. In both experiments, pigs fed only BD had greater (P < 0.05) colonic digesta phytase activity and soluble P concentrations than those fed phytase. In Exp. 2, total colonic digesta P or Ca concentrations, or both, of pigs displayed a phytase-dose-dependent reduction (P < 0.05). In conclusion, supplemental dietary AppA2 mainly functioned in the stomach and was associated with a reduced phytase activity in colonic digesta of weanling pigs.

An improved method for a rapid determination of phytase activity in animal feed.

The current direct colorimetric assay for phytase activity in feeds has interference from high P background and other factors. Our objective was to develop a rapid and reliable spin column method to accurately determine phytase activity in feed ingredients or complete diets. After the feed sample was extracted by stirring in 0.2 M citrate buffer, pH 5.5, for 30 min at room temperature, the oily layer of the supernatant fraction was removed by passing through an acrodisc syringe filter (0.45-microm HT Tuffryn membrane, Gelman Laboratory, Ann Arbor, MI). The filtrate was then loaded onto a spin column (MW cutoff 30,000, Millipore, Bedford, MA) to remove free phosphate before the phytase activity assay. Compared with the direct assay, this new procedure improved both accuracy and reproducibility. When diets contained phytase at 0 to 1,500 U/kg (as fed), the CV for multiple assays of the same samples (n = 6) by the new method ranged from 1 to 6% compared with 28 to 39% by the direct method. A linear relationship was found between the added phytase activity in practical diets and the analyzed activity by the new method (r2 = 0.99; P < 0.01). In conclusion, the spin column method is an improved assay for phytase activity in animal feed, and may be used for quality control of phytase supplementation.

Effects of combining three fungal phytases with a bacterial phytase on plasma phosphorus status of weanling pigs fed a corn-soy diet.

The objective of this study was to determine possible synergistic effects of supplementing one of three fungal phytases: Aspergillus fumitagus PhyA (AFP),A. niger PhyA (ANP), or Peniophora lyci phytase (PLP) with an Escherichia coli AppA phytase (EP) in diets for pigs. Three experiments, each lasting for 4 wk, were conducted with a total of 106 weanling pigs (5 wk old). The corn-soybean meal basal diet (BD) contained no supplemental inorganic P. In Exp. 1, 35 pigs (8.6 +/- 1.0 kg BW) were fed (as-fed basis) BD + AFP at 750 U/ kg of feed, BD + inorganic P (0.2% P), or BD + PLP at 500, 750, or 1,000 U/kg feed. Pigs fed BD + AFP or BD + 0.2% P had higher (P < 0.05) plasma inorganic P concentrations than those fed BD + PLP at the end of the trial (wk 4). In Exp. 2, 35 pigs (8.1 +/- 0.9 kg BW) were fed BD + AFP, EP, PLP, a 1:1 mix of AFP:EP, or a 1:1 mix of PLP:EP at 500 U/kg. Pigs fed the AFP:EP mixture had growth performance and plasma measures similar to those fed either enzyme alone. Pigs fed the PLP:EP mixture had lower (P < 0.05) plasma alkaline phosphatase activity than those fed BD + PLP. Pigs fed BD + PLP had lower (P < 0.05) plasma inorganic P concentrations than pigs fed BD + EP, and higher (P < 0.05) plasma alkaline phosphatase activity than all other groups at wk 4. In Exp. 3, 36 pigs (9.1 +/- 1.2 kg BW) were fed BD + ANP, EP, or a 1:1 mix of ANP:EP at 500 U/kg feed. Pigs fed the two enzymes together had lower (P < 0.05) plasma inorganic P concentration than those fed BD + EP and lower (P < 0.05) plasma alkaline phosphatase activity than pigs fed BD + ANP at wk 4. In conclusion, although the four phytases showed different effects on plasma P status of weanling pigs, there was no synergistic effect between any of the three fungal phytases and the bacterial phytase on the plasma measures or growth performance under the conditions of the present study.

Efficacy of a phytase derived from Escherichia coli and expressed in yeast on phosphorus utilization and bone mineralization in turkey poults.

A slope-response bioassay was conducted with male turkey poults to determine the sparing effect of P, based on improvements in bone mineralization in turkey poults, from 10 to 21 d of age when diets were supplemented with a novel phytase. Reference diets for calculation of the sparing effect of P contained 0.47, 0.55, 0.70, and 0.79% nonphytate phosphorus (NPP). Diets with varying dosages of a swine, Escherichia coli-derived AppA2 phytase (ECP) expressed in Pichia pastoris yeast (0, 250, 500, 750, and 1,000 U/kg) were added to the 0.47% NPP diet and improvements in bone mineralization determined the sparing effect of P supplied from ECP. Two additional reference diets were included that contained 500 U/kg from one of two commercial phytases (PA and PB) derived from Aspergillus and Peniophora. At 500 U/kg diet the ECP spared an additional 0.22% NPP (if calculated from tibia ash %), 0.18% NPP (if calculated from toe ash %), 0.24% NPP (if calculated from mg tibia ash), or 0.21% NPP (if calculated from mg toe ash). Phosphorus retention results validate bioassay results, in that 500 U ECP/kg resulted in 68.2% P being retained (0.49% of diet P retained) as compared with only 58.9% P being retained from the unsupplemented control diet (0.421% of diet P retained; P < 0.05).

Effectiveness of an experimental consensus phytase in improving dietary phytate-phosphorus utilization by weanling pigs.

Consensus phytase is a new biosynthetic, heat-stable enzyme derived from the sequences of multiple homologous phytases. Two experiments were conducted to determine its effectiveness, relative to inorganic P and a mutant enzyme of Escherichia coli phytase (Mutant-EP), in improving dietary phytate-P availability to pigs. In Exp. 1, 36 pigs (3 wk old, 7.00 +/- 0.24 kg of BW) were fed a low-P corn-soybean meal basal diet plus consensus phytase at 0, 250, 500, 750, 1,000, or 1,250 U/kg of feed for 5 wk. Plasma inorganic P concentration, plasma alkaline phosphatase activity, bone strength, and overall ADG and gain:feed ratio of pigs were improved (P < 0.05) by consensus phytase in both linear (R2 = 0.20 to 0.70) and quadratic (R2 = 0.30 to 0.70) dose-dependent fashions. In Exp. 2, 36 pigs (4 wk old, 9.61 +/- 0.52 kg BW) were fed the basal diet + inorganic P at 0.1 or 0.2%, consensus phytase at 750 or 450 U/kg of feed, Mutant-EP at 450 U/kg of feed, or 225 U consensus + 225 U Mutant-EP/kg of feed. Pigs fed 750 U of consensus phytase or 450 U of Mutant-EP/kg feed had plasma inorganic concentrations and bone strength that fell between those of pigs fed 0.1 or 0.2% inorganic P. These two measures were 16 to 29% lower (P < 0.05) in pigs fed 450 U of consensus phytase/kg of feed than those of pigs fed 0.2% inorganic P. Plasma inorganic P concentrations were 14 to 29% higher (P < 0.05) in pigs fed Mutant-EP vs. consensus phytase at 450 U/kg at wk 2 and 3. In conclusion, the experimental consensus phytase effectively releases phytate P from the corn-soy diet for weanling pigs. The inorganic P equivalent of 750 U of consensus phytase/kg of feed may fall between 0.1 and 0.2%, but this requires further determination.

Efficacy of an E. coli phytase expressed in yeast for releasing phytate-bound phosphorus in young chicks and pigs.

Four chick trials and one pig trial were conducted to investigate the phosphorus-releasing efficacy oftwo commercial phytase enzymes (Natuphos and Ronozyme) and an experimental E. coli phytase enzyme (ECP) when added to corn-soybean meal diets containing no supplemental inorganic P (iP). In the 13- or 14-d chick trials, three or four graded levels of iP (0, 0.05,0.10,0.15%) from KH2PO4 were added to the basal diet to construct standard curves from which bioavailable P release could be calculated for the phytase treatments. In all cases, phytase supplementation levels were based on an assessment of phytase premix activity (i.e., P release from Na phytate at pH 5.5). Linear (P < 0.01) responses in tibia ash and weight gain resulted from iP supplementation in all assays. In the first chick trial, supplementation of 500 phytase units (FTU)/kg of ECP resulted in superior (P < 0.01) weight gain and tibia ash values compared with 500 FTU/kg of Natuphos. Results of the second chick trial revealed P-release values of 0.032 and 0.028% for 500 FTU/kg Natuphos and Ronozyme, respectively, and these were lower (P < 0.01) than the 0.125% P-release value for 500 FTU/kg of ECP. Tibia ash responded quadratically (P < 0.05) in response to graded levels of ECP up to 1,500 FTU/kg in the third chick trial. Combining Natuphos with either Ronozyme or ECP in Chick Trial 4 revealed no synergism between phytases with different initiation sites of P removal. The pig trial involved 10 individually fed weanling pigs per diet, and and phytase enzymes were supplemented to provide 400 FTU/kg in diets containing 0.60% Ca. Based on the linear regression of fibula ash on supplemental iP intake (r2 = 0.87), P-release values were 0.081% for Natuphos, 0.043% for Ronozyme, and 0.108% for ECP. These trials revealed an advantage of the E. coli phytase over the commercial phytases in young chicks.

Comparative impacts of glutathione peroxidase-1 gene knockout on oxidative stress induced by reactive oxygen and nitrogen species in mouse hepatocytes.

Selenium-dependent glutathione peroxidase-1 (GPX1) protects against reactive-oxygen-species (ROS)-induced oxidative stress in vivo, but its role in coping with reactive nitrogen species (RNS) is unclear. Our objective was to compare the protection of GPX1 against cytotoxicity of superoxide generator diquat (DQ), NO donor S-nitroso-N-acetyl-penicillamine (SNAP) and peroxynitrite generator 3-morpholinosydnonimine (SIN-1). Primary hepatocytes were isolated from GPX1-knockout (KO) and wild-type (WT) mice and cultured in complete Williams's medium E with various levels of these agents alone or in combination for up to 12 h. While the KO cells were more susceptible to cell death, DNA fragmentation and protein carbonyl formation induced by 0.25-1 mM DQ, these cells were as tolerant as the WT cells to cytotoxicity of 0.1-1 mM SNAP or 0.5-2 mM SIN-1. Treating cells with SNAP (0.1 or 0.25 mM) in addition to DQ produced synergistic cytotoxicity that minimized differences in apoptotic cell death and oxidative injuries between the KO and WT cells. Less protein nitrotyrosine was induced by 0.05-0.5 mM DQ+0.25 mM SNAP in the KO than in the WT cells. Total GPX activity in the WT cells was reduced by 65 and 25% by 0.5 mM DQ+0.1 mM SNAP and 0.5 mM DQ, respectively. Decreases in Cu,Zn-superoxide dismutase (SOD) activity and increases in Mn-SOD activity in response to DQ or DQ+SNAP were greater in the KO cells than in the WT cells. In conclusion, GPX1 was more effective in protecting hepatocytes against oxidative injuries mediated by ROS alone than by ROS and RNS together. Knockout of GPX1 did not enhance cell susceptibility to RNS-associated cytotoxicity. Instead, it attenuated protein nitration induced by DQ+SNAP.

Glutathione peroxidase-1 gene knockout on body antioxidant defense in mice.

To determine the in vivo role of cellular glutathione peroxidase (E.C.1.11.1.9, GPX1), we challenged the GPX1 knockout [GPX1(-/-)], the GPX1 overexpressing [GPX1(+)], and their respective wild-type (WT) mice of different Se and vitamin E status with acute oxidative stress. After these mice were injected with pro-oxidants paraquat or diquat at 12 to 125 mg/kg of body weight, their survival rate and time were a function of their GPX1 activity levels. The GPX1 protection was associated with attenuation of NADPH and NADH oxidation, protein carbonyl and F(2)-isoprostanes formation, and alanine transaminase release in various tissues, and was irreplaceable by high levels of dietary vitamin E or other selenoproteins. The GPX1 expression was also protective against moderate oxidative stress induced by low levels of paraquat or diquat, particularly in the Se-deficient mice. Alteration of GPX1 expression showed no impact on the expression of other selenoproteins and antioxidant enzymes in unstressed mice. Total Se content in liver of the Se-adequate GPX1(-/-) mice was reduced by 60% the WT controls. In conclusion, normal expression of GPX1 is essential and overexpression of GPX1 is beneficial to protect mice against acute oxidative stress.

Opposite roles of selenium-dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxynitrite-induced apoptosis and signaling.

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1-/-) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1-0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1-/- cells and by PN in only WT cells. These two groups of cells shared similar cytochrome c release, caspase-3 activation, and p21(WAF1/CIP1) cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1-/- cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1-/- than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.

Lipopolysaccharide and interferon-gamma-induced nitric oxide production and protein oxidation in mouse peritoneal macrophages are affected by glutathione peroxidase-1 gene knockout.

This study investigated the role of glutathione peroxidase-1 (GPX1) in protein oxidation in peritoneal macrophages. Macrophages isolated from both wild-type (WT) and GPX1 knockout (KO) mice were activated by lipopolysaccharide (LPS, 1 microg/ml) and interferon-gamma (IFN, 10 U/ml for 24 or 48 h in the presence or absence of 1 microM diquat (DQ), 250 microM aminoguanidine (AG, an inhibitor of inducible nitric oxide synthase), and (or) 100 microM diethyldithiocarbamate (DETC, an inhibitor of Cu,Zn-SOD). In the KO macrophages, there was no protein band detected by Western blot with anti-GPX1 antibody and 98% reduction in total GPX activity compared with WT cells. Nitric oxide (NO) synthesis was greatly enhanced after 24 h by GPX1 knockout and DQ, but inhibited by AG or DETC. Protein carbonyl formation in total cell extract was clearly associated with NO synthesis as higher levels of protein carbonyl were detected in activated KO than WT macrophages, and DQ enhanced slightly while AG or DETC virtually blocked its formation. A similarly marginal effect of GPX1 KO was observed on protein nitration. The LPS/IFN/DQ-induced DNA fragmentation was blocked by AG, but not by DETC. Cell viability at 48 h was decreased by the LPS/IFN activation and further reduced by the addition of DQ, but restored by AG. In conclusion, GPX1 affects the NO production in activated peritoneal macrophages and protects these cells against NO-associated protein oxidation.

Biotechnological development of effective phytases for mineral nutrition and environmental protection.

Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate (myo-inositol hexakisphosphate), the major phosphorus (P) form in animal feeds of plant origin. These enzymes can be supplemented in diets for food animals to improve P nutrition and to reduce P pollution of animal excreta. This mini-review provides a synopsis of the concept of "ideal phytase" and the biotechnological approaches for developing such an enzyme. Examples of Escherichia coli AppA and Aspergillus fumigatus PhyA are presented to illustrate how new phytases are identified from microorganisms and developed by genetic engineering based on the gene sequences and protein structures of these enzymes. We also discuss the characteristics of different heterologous phytase expression systems, including those of plants, bacteria, fungi, and yeast.

Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris.

Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties. Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively. Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA. The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%). Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties. First, it was more active at pH 3.5-5.5. Second, it retained more (P < 0.01) phytase activity than that of r-AppA. Third, its specific activity of phytase was 54% higher. Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively. Based on the recently published E. coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein. This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.

Phytase activity in Aspergillus fumigatus isolates.

Extracellular phytase from Aspergillus fumigatus isolates was characterized and their genes were cloned and sequenced. Based on their banding pattern in SDS-PAGE all phytases were found to be glycosylated and have similar molecular mass. A correlation between lower optimum pH (4.0) and a higher optimum temperature (70 degrees C) was found in these enzymes. All enzymes characterized displayed a lower specific activity for phytic acid and were more susceptible to proteolytic degradation than the Aspergillus niger phytase that is now commercially available. DNA sequencing established almost no sequence variation in any of the genes and no correlation is evident between a specific amino acid sequence and any physicochemical and catalytic properties of the enzymes. Despite two of the isolates having identical deduced amino acid sequence, characterization of the enzymes encoded by these two identical genes revealed differences in both pH and temperature optimum. This suggests that differences in pH and temperature optimum in these four isolates of A. fumigatus may be due in part to subtle differences in posttranslational modification.

Biochemical characterization of cloned Aspergillus fumigatus phytase (phyA).

The gene for Aspergillus fumigatus phytase (phyA) was cloned and expressed in Pichia pastoris. The enzyme expressed was purified to near homogeneity using sequential ion-exchange chromatography and was characterized biochemically. Although A. fumigatus phytase shows 66.2% sequence homology with A. ficuum phytase, the most widely studied enzyme, the cloned phytase showed identical molecular weight and temperature optima profile to the benchmark phytase. The pH profile of activity and kinetic parameters, however, differed from A. ficuum phytase. The cloned enzyme contains the septapeptide RHGARYP motif, which is also identical to the active site motif of A. ficuum phytase. Chemical probing of the active site Arg residues using both cyclohexanedione and phenylglyoxal resulted in the inactivation of phytase. The cloned A. fumigatus phytase, however, was more resistant to phenylglyoxal-induced inactivation. Both cloned A. fumigatus and A. ficuum phytases were identically affected by cyclohexanedione. Both the thermal characterization data and kinetic parameters of cloned and expressed A. fumigatus phytase indicate that this biocatalyst is not superior to the benchmark enzyme. The sequence difference between A. fumigatus and A. ficuum phytase may explain why the former enzyme catalyzes poorly compared to the benchmark enzyme. In addition, differential sensitivity toward the Arg modifier, phenylglyoxal, indicates a different chemical environment at the active site for each of the phytases.

Preferential resistance of dopaminergic neurons to the toxicity of glutathione depletion is independent of cellular glutathione peroxidase and is mediated by tetrahydrobiopterin.

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD) and could initiate dopaminergic neuronal degeneration. Nevertheless, experimental glutathione depletion does not result in preferential toxicity to dopaminergic neurons either in vivo or in vitro. Moreover, dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione depletion, possibly owing to differences in cellular glutathione peroxidase (GPx1) function. However, mesencephalic cultures from GPx1-knockout and wild-type mice were equally susceptible to the toxicity of glutathione depletion, indicating that glutathione also has GPx1-independent functions in neuronal survival. In addition, dopaminergic neurons were more resistant to the toxicity of both glutathione depletion and treatment with peroxides than nondopaminergic neurons regardless of their GPx1 status. To explain this enhanced antioxidant capacity, we hypothesized that tetrahydrobiopterin (BH(4)) may function as an antioxidant in dopaminergic neurons. In agreement, inhibition of BH(4) synthesis increased the susceptibility of dopaminergic neurons to the toxicity of glutathione depletion, whereas increasing BH(4) levels completely protected nondopaminergic neurons against it. Our results suggest that BH(4) functions as a complementary antioxidant to the glutathione/glutathione peroxidase system and that changes in BH(4) levels may contribute to the pathogenesis of PD.