Aerobic growth on nitroglycerin as the sole carbon, nitrogen, and energy source by a mixed bacterial culture

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1, 2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 +/- 0.005 h-1 (mean +/- standard deviation) was estimated for the mixed culture at 25 degreesC. Evidence of GTN toxicity was observed at GTN concentrations above 0. 3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.     

Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii

The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level. The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed. Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level. The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase. The DAS1 gene was disrupted in one step in the host genome (das1Delta strain), and the growth of the das1Delta strain in various carbon and nitrogen sources was compared with that of the wild-type strain. The das1Delta strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y. Sakai et al., J. Bacteriol. 179:4480-4485, 1997). These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Delta strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.     

Regulation of peroxisomal proteins and organelle proliferation by multiple carbon sources in the methylotrophic yeast, Candida boidinii

A methylotrophic yeast, Candida boidinii, was grown on various combinations of peroxisome-inducing carbon source(s) (PIC(s)), i.e. methanol, oleate and D-alanine, and the regulation of peroxisomal proteins (both matrix and membrane ones) and organelle proliferation were studied. This regulation was followed (1) at the protein or enzyme level by means of the peroxisomal enzyme activity and Western analysis; (2) at the mRNA level by Northern analysis; and (3) at the organelle level by direct observation of peroxisomes under a fluorescent microscope. Peroxisomal proliferation was followed in vivo by using a C. boidinii strain producing a green fluorescent protein having peroxisomal targeting signal 1. When multiple PICs were used for cell growth, C. boidinii induced specific peroxisomal proteins characteristic of all PIC(s) present in the medium, responding to all PIC(s) simultaneously. Thus, these PICs were considered to induce peroxisomal proliferation independently and not to repress peroxisomes induced by other PICs. Next, the sensitivity of the peroxisomal induction to glucose repression was studied. While the peroxisomal induction by methanol or oleate was completely repressed by glucose, the D-alanine-induced activities of D-amino acid oxidase and catalase, Pmp47, and the organelle proliferation were not. These results indicate that peroxisomal proliferation in yeasts is not necessarily sensitive to glucose repression. Lastly, this regulation was shown to occur at the mRNA level.     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

AIMS AND BACKGROUND: Altered oncogenic activity is a feature associated with many malignant and premalignant conditions. Among the many oncogenes, ras and myc are commonly altered in many tumors. This study aims to evaluate the expression of ras and c-myc oncoproteins in a total of 204 cervical tissue samples, including premalignant and malignant lesions as well as apparently normal cervical tissue. METHODS AND STUDY DESIGN: Mouse monoclonal antibodies against the three mammalian ras gene products (c-H-ras, c-K-ras, c-N-ras) and the c-myc protein were used to evaluate oncoprotein expression by immunocytochemistry. RESULTS: None of the samples analyzed displayed immunoreactivity for H-ras and K-ras. Normal cervical epithelium showed minimal immunoreactivity for N-ras with about 33% of the samples expressing the protein. More conspicuous expression in normal tissue was displayed by c-myc, with about 90% of the samples expressing the protein (mean value of cells positive=34%). The immunoreactivity for N-ras increased with increasing histological abnormality from low-grade squamous intraepithelial lesions (SIL) to invasive carcinoma. Increased immunoreactivity for N-ras was evident in the basaloid cells of malignant lesions, with the maximum value of 66% found in poorly differentiated squamous cell carcinoma (PDSCC). The percentage of nuclei positive for c-myc also showed a gradual increase from low-grade SIL onwards, the highest positivity being found in PDSCC, where the mean value was 85%. Statistical analysis revealed a good correlation between the expression of N-ras (r=0.8922, P=0.001) and c-myc (r=0.8856, P=0.001) and various histological stages of tumor progression in the cervical epithelium. CONCLUSIONS: These results therefore suggest that c-myc and N-ras oncoproteins are important during tumor progression in the uterine cervix.     

Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme

Two brush border peptidases have been isolated from the particulate fraction of the rat intestinal mucosa and purified to homogeneity as judged by polyacrylamide gel electrophoresis, starch gel electrophoresis, isoelectric focusing, and double immunodiffusion. For convenience, the peptidases have been designated peptidase F (fast) and S (slow) on the basis of their anodic mobilities. The isoelectric point of peptidase F was 4.76 and of peptidase S, 5.10. Both enzymes are glycoproteins. The amino acid compositions of the two peptidases are similar. The same carbohydrates are found in both enzymes, but there are differences in the molar concentrations of individual sugars. Peptidase S has greater concentrations of mannose and galactose and of hexosamines than peptidase F, while sialic acid is slightly greater in peptidase F. Carbohydrate accounted for approximately 19% and 23% of the weight of peptidases F and S, respectively. Estimates of the molecular weights of both enzymes by gel filtration gave values of 280,000. Electrophoresis of the enzymes under denaturing conditions on sodium dodecyl sulfate polyacrylamide gels indicated that each enzyme is a dimer consisting of two subunits of equal molecular weight, 140,000.     

Studies on the glycosidases of semen. Further purification and characterization of two hexosaminidases from bull seminal plasma

Two isozymes of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxy glucohydrolase, EC 3.2.1.30) (A and B) from bull seminal plasma were purified to homogeneity by isoelectric focusing having pI values of 5.31 and 6.78. The two proteins were glycoproteins with very similar amino acid composition but isozyme A contained more sialic acid than isozyme B. The molecular weights of isozyme A and B were estimated at 200 000 and 190 000 by gel filtration. Two identical subunits corresponding to molecular weights of 53 000 and 13 400 were obtained from hexosaminidase A and B when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Similar results were obtained when dissociation of the isozymes was effected with mercaptoethanol, guanidine hydrochloride and urea in presence of sodium dodecyl sulphate and the subunits separated by acrylamide gel electrophoresis. The two isozymes were more stable in frozen conditions than at the refrigerated temperature. Of the divalent ion tested, glucosaminidase and galactosaminidase activities of isozymes A and B were strongly inhibited by Hg2+ and Ag+ thus suggesting the presence of thiol groups in the two proteins. The two isozymes were active on natural substrates; isozyme B being more active than isozyme A.     

Production of the carotenoids lycopene, beta-carotene, and astaxanthin in the food yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, beta-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promotes and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, beta-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Studies on purification and some properties of nisin from Lactococcus lactis subsp. lactis Al2

Protoporphyria is a genetic disorder in which patients may develop severe protoporphyrin-induced liver damage and require transplantation. Because unique problems occur in the perioperative period and because excess production of protoporphyrin by the bone marrow continues after liver transplantation, the efficacy of this procedure for protoporphyric liver disease is uncertain. We present follow-up of nine patients who underwent liver transplantation. Two patients died within 2 months of transplantation, one from complications of abdominal bleeding and the other from sepsis after bowel perforations. The remaining seven patients had follow-up at 14 months to 8 years after transplantation (mean, 3.8 years). Two of the seven had suffered skin burns from exposure to operating room lights, which healed without scarring. Three had axonal neuropathies in the postoperative period requiring prolonged mechanical ventilation, and motor defects persisted in two. Five patients had normal liver chemistries at follow-up (mean, 3.5 years), with liver biopsy results normal or showing mild portal triad abnormalities, but erythrocyte protoporphyrin levels remained significantly elevated (1,765 +/- 365 mcg/dL; normal, < 65). The other two patients, both of whom had rejection, cytomegalovirus infection, and biliary tract obstruction requiring endoscopic therapy, had a recurrence of protoporphyric liver disease as indicated by liver biopsy features. One died 5 years after transplantation from complications of the liver disease. The other was stable 3.3 years after transplantation and was being monitored for possible retransplantation. Thus, liver transplantation can be performed successfully in patients with protoporphyric liver disease, with intermediate survival rates comparable to the general transplant population. However, disease may recur in the graft, particularly if there are complications that cause cholestasis.     

Molecular cloning, purification and characterization of two endo-1,4-beta-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-beta-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 degrees C.     

Separation, purification, and properties of multiple forms of cytochrome P-450 from the liver microsomes of phenobarbital-treated mice

Four distinct cytochrome P-450 fractions (A1, A2, C1, and C2) have been separated and purified from the liver microsomes of phenobarbital-treated hybrid mice (B6D2F1/J). Fractions A2 and C2 were highly purified with specific contents of 16.5 and 17.5 nmol of cytochrome P-450/mg of protein, respectively, based on their amino acid compositions. The major hemeprotein bands of A2 and C2 have different minimum molecular weights (50,000 and 56,000, respectively) on polyacrylamide gels in the presence of sodium dodecyl sulfate. All four fractions with respect to their spectral and catalytic properties, thereby demonstrating that mouse liver microsomes from phenobarbital-treated hybrid mice contain at least four forms of cytochrome P-450.     

Effects of silicon on the oxidation,hot-corrosion,and mechanical behavior of two cast nickel-base superalloys

Cast specimens of nickel-base superalloys 713C and Mar-M200 with nominal additions of 0, 0.5, and 1 wt pct Si were evaluated for oxidation and corrosion resistance, tensile and stress-rupture properties, microstructure, and phase relations. Results are com-pared with those of an earlier study of the effects of Si in B-1900. Si had similar effects on all three superalloys. It improves oxidation resistance but the improvement in 713C and Mar-M200 was considerably less than in B-1900. Hot-corrosion resistance is also improved somewhat. Si is, however, detrimental to mechanical properties, in particular, rupture strength and tensile ductility. Si has two obvious microstructural effects. It in-creases the amount of γ precipitated in eutectie nodules and promotes a Mo(Ni,Si)₂ Laves phase in the alloys containing Mo. These microstructural effects do not appear responsible for the degradation of mechanical properties, however.     

Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris

A cDNA coding for porcine leukocyte 12-lipoxygenase was expressed intracellularly in the methylotrophic yeast Pichia pastoris under the regulatory control of the alcohol oxidase promoter. The recombinant 12-lipoxygenase contained in the yeast cell lysate was soluble, displayed the catalytic properties of the native enzyme, and was recognized by antibodies prepared against native 12-lipoxygenase derived from porcine leukocytes. The catalytically active enzyme of the 100,000 x g supernatant obtained from the yeast lysate was readily purified by immunoaffinity chromatography to near homogeneity. Porcine leukocyte 12-lipoxygenase is the first arachidonic acid oxygenase to be expressed in yeast, an easy, inexpensive, and rapid method of expressing native and site-directed mutants of recombinant proteins.     

Effect of the physiological properties of yeast strains on their formation of higher alcohols and aldehydes and on the composition of nitrogen compounds in the fermented wort

Dynamics of formation of by-products was studied with the following yeast races: Sacch. cerevisiae, strain Odesskaya-14 (baker's yeast); Sacch. vini, strain Prikumskaya 80/9 (wine yeast). The yeast cultures were found to be very similar by the rate of biomass accumulation, ethanol production, and the fractional composition of nitrogen compounds. The concentration of accumulated higher alcohols depended on the mass of yeast cells, their growth rate, and the duration of cultivation.     

Effect of zirconium,cerium, and nitrogen on the properties of a cast chromium-base dispersion-strengthened alloy

Conclusions An analysis of the chemical inertness and wettability of various nonmetallic compounds has shown zirconium nitride to be the most suitable strengthening phase for a chromium-base alloy. Endogeneously strengthened alloys have been produced having values of high-temperature strength _t ⁹⁰⁰ of between 270 and 340 MPa and of room-temperature ductility ²⁰ of between 5 and 44%. The properties of such an alloy are determined by the size and uniformity of distribution of its strengthening particles, while these in turn depend on the zirconium-tonitrogen ratio in the alloy.Translated from Poroshkovaya Metallurgiya, No. 11(227), pp. 81–84, November, 1981.     

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.     

Oxalate oxidase from barley roots: purification to homogeneity and study of some molecular, catalytic, and binding properties

Cross-sectional estimates of immigrant wage growth have painted an optimistic picture of the ability of immigrants to adapt to the U.S. labor market: Studies using cross-sectional data have generally found the wage growth of immigrants to exceed that of the native born. This optimistic picture of immigrant economic assimilation was challenged by the important finding that compared to earlier immigrant cohorts, recent immigrants started at much lower wages. As such, the high wage growth of immigrants relative to the native born measured in cross-sectional data may simply be the spurious result of declining immigrant earnings ability. In this paper, we match Current Population Survey samples so that the wages of individual immigrant and native-born men can be followed for one year. We find that the wage growth of immigrants does exceed that of the native born. The general finding of faster immigrant wage growth also holds when imposing the foreign-born geographic distribution upon natives, but not when imposing the native-born geographic distribution on the foreign born-a result consistent with some theories of immigrant assimilation. In each comparison, however, the actual wage growth of immigrants relative to natives is similar to the predictions of cross-sectional regressions. This similarity suggests that either there is no cohort quality bias in the cross-sectional estimates of immigrant wage growth, or that there has been a coincidental increase in immigrant wage growth as the entry wages of immigrants have fallen.     

Purification and properties of two fibrinolytic enzymes from Fusarium oxysporum N.R.C.1

OBJECTIVE: To find differences in effect on sperm motility of agents that increase intracellular cAMP: manganese ion, methyl-isobutyl-xanthine (MIX), 2-deoxyadenosine, glucose, and Mn-MIX and Mn-glucose. DESIGN: Nine men with asthenozoospermia vs. fertile donors. METHODS: Sperm was washed in Hepes-buffered saline, motility tested by laser-Doppler technique. RESULTS: Best activation was obtained with Mn and 2-deoxyadenosine; generally poor response to MIX or glucose. CONCLUSIONS: Usually, poor endogenous stimulation of adenylyl cyclase, and probably not limited energy supply, is the cause of impaired motility.     

Forage characteristics, steer performance, and water quality from bermudagrass pastures fertilized with two levels of nitrogen from swine lagoon effluent

Four .8-ha pastures of bermudagrass (Cynodon dactylon [L.] Pers.) were fertilized with either 456 or 873 kg/ha of nitrogen (N) from swine lagoon effluent (two replicates per treatment) and grazed by steers over two summers. Within each pasture, steers received forage only, an energy source (corn), a mixture of corn and soybean meal, or a mixture of corn and blood meal via electronic Calan feeders. All supplements were offered at a level of 1.36 kg/d, and the soybean meal and blood meal supplements provided similar among quantities of protein. Weight gains were similar among supplemented steers, but supplemented steers gained faster (P < .05) than controls. Nitrogen fertilization level had no effect on steer gains, steer grazing days per hectare, or in vitro dry matter disappearance, NDF, and ADF of clipped forage samples. Plant protein and nitrate ion concentrations were greater (P < .06) in clipped forage samples receiving the higher N application rate. Nitrate ion concentrations were greater in available forage samples from the pastures with the high N application rate. Mean total N and nitrate N concentrations were similar in water samples obtained from monitoring wells for the two N treatments over the 2 yr and there were no year x N interactions. Chloride concentrations were greater (P < .05) and pH and specific conductance were less in water samples collected from the 873 kg than from the 456 kg/ha N treatment. Long-term studies are needed to examine the possible cumulative effects of applying various levels of swine waste to the same land area.     

Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing

From the approximately 5000 open reading frames presently identified by systematic sequencing of the yeast genome, 100 Saccharomyces cerevisiae transport proteins belonging to the major facilitator superfamily (MFS), were assigned to 17 families on the basis of extensive database searches and binary comparisons. These families include multidrug resistance proteins and transport proteins for sugars, amino acids, uracil/allantoin, allantoate, phosphate, purine/cytosine, proteins, peptides, potassium, sulfate, and urea. Four new families of unknown function have been identified. For the sugar and amino acid transport proteins, alignments were made and phylogenetic trees were constructed allowing the identification of several clusters of proteins presumably exhibiting similar transport functions.