Constructed wetlands for water quality improvements: Benefit transfer analysis from Ohio.

Water resources provide many benefits that generate value for residents and recreation users alike but run-off from agricultural and impervious surfaces can impair water quality, reducing any generated value. A possible solution to this problem is the construction of treatment wetlands to remove excessive nutrients from water bodies. This study uses environmental and economic data to approximate the costs of constructing and operating free surface water wetlands to remove phosphorus and estimates the amenity and recreational benefits of the resulting improvements in water quality for 24 lakes in Ohio. A ten percent improvement in water quality from a decrease in phosphorus loadings generates positive net benefits for all lakes in the sample with a lifetime cost benefit ratio of 2.92. The study also examines the potential use of constructed wetlands as the sole strategy to achieve a reduction goal for phosphorus loadings and find that the costs of doing so are prohibitive. Constructed wetlands can be a cost-effective component of a comprehensive strategy for small-scale nutrient reduction and water quality improvements for surface water bodies, but other treatment methods would be required to achieve any proposed targeted improvements.

Decreased plasma folate concentration in young and elderly healthy subjects after a short-term supplementation with isotretinoin.

BACKGROUND: In the last two decades, there has been an increasing use of isotretinoin (13-cis-retinoic acid or 13-CRA) for treatment of severe, and recently mild and moderate, acne in Westernized populations. Recent human and animal studies emphasized alterations caused by 13-CRA administration on folate-dependent, one-carbon metabolism. Folate deficiency and subsequent hyperhomocysteinemia increase the risk of degenerative diseases. OBJECTIVES: We determine whether a short-term supplementation with 13-CRA alters folate status and homocysteinemia in young and elderly healthy human subjects. METHODS: Twenty young and 20 elderly (age mean, 26.1 and 65.4 years, respectively) healthy male volunteers were supplemented with approximately 0.5 mg/kg/day of 13-CRA for 28 days. Fasting plasma concentrations of 13-CRA, 5-methyltetrahydrofolate (5-mTHF) as the main circulating form of folate, and homocysteine (Hcy), as well as haematologic parameters and biochemical markers of liver and renal function, were measured at baseline and at the end of supplementation. Statistical analyses were carried out using two-way anova and standard tests. RESULTS: In both groups, isotretinoin supplementation caused a dramatic increase in the circulating concentration of 13-CRA and its derivatives. It also led to significant increases in serum triglyceride (P < 0.0001) and creatinine (P = 0.002) concentrations and gamma-glutamyltranspeptidase activity (P = 0.0001) and decrease in serum level of urea (P = 0.027). However, the latter four parameters remained within normal ranges. These changes were accompanied by a 17.7% and 13.5% decrease in the plasma level of 5-mTHF (P = 0.001) in the young and elderly volunteers, respectively. Supplementation with 13-CRA did not cause significant variations in their plasma Hcy concentration. However, the latter parameter seemed to respond differently in each group of age (P = 0.046). CONCLUSIONS: Our data indicate that a 28-day supplementation with isotretinoin alters the plasma folate in young and old healthy individuals. This stresses the necessity of studying the long-term effects of retinoid therapy on folate status and homocysteinemia in acne patients, given that alteration in the latter parameters is known to increase the risk of degenerative diseases.

Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine.

The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.

Conversion of pipecolic acid into lysine in Penicillium chrysogenum requires pipecolate oxidase and saccharopine reductase: characterization of the lys7 gene encoding saccharopine reductase.

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.

Effect of exogenous lysine on the expression of early cephamycin C biosynthetic genes and antibiotic production in Nocardia lactamdurans MA4213.

In beta-lactam producing microorganisms, the first step in the biosynthesis of the beta-lactam ring is the condensation of three amino acid precursors: alpha-aminoadipate, L-cysteine and D-valine. In Nocardia lactamdurans and other cephamycin-producing actinomycetes, alpha-aminoadipate is generated from L-lysine by two sequential enzymatic steps. The first step involves a lysine-6-aminotransferase activity (LAT), considered to be one of the rate-limiting steps for antibiotic biosynthesis. Here, we report the effect of exogenous lysine on antibiotic production by N. lactamdurans MA4213. Lysine-supplemented cultures showed higher titers of cephamycin C, an effect that was more significant at early fermentation times. The increase in cephamycin C production was not quantitatively correlated with specific LAT activity in lysine-supplemented cultures. Observation of a positive effect of lysine on cephamycin C production by N. lactamdurans was dependent on carbon source availability in the culture media. Supplementation of the culture media with exogenous lysine did not affect the mRNA levels of the early biosynthetic genes controlled by the bidirectional promoter. These results indicate that L-lysine is required not only for antibiotic biosynthesis, but particularly as carbon or nitrogen source.

Delta-1-piperideine-6-carboxylate dehydrogenase, a new enzyme that forms alpha-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes.

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

Inducible nitric oxide synthase gene transcription and protein activity in the rat heart during endotoxaemia.

Septicaemia leads to an impairment of myocardial contractility in animals and humans. Cytokines released during endotoxaemia are capable of increasing inducible nitric oxide synthase (iNOS) expression in vitro in myocytes, endothelial cells and macrophages. The aim of this study was to assess whether iNOS gene transcription occurs in the myocyte in vivo. Rats were injected with intraperitoneal endotoxin. Myocardial sections obtained 4, 6 and 8 hours after infection were hybridised with oligonucleotides complementary to iNOS cDNA. Myocardial homogenates were used to measure NOS enzyme activity and to detect iNOS mRNA. Uninfected control animals did not demonstrate myocardial iNOS expression. Myocardium from endotoxaemic animals contained iNOS mRNA and high calcium-independent NOS enzyme activity. In situ hybridisation did not localise iNOS to myocytes but to cells located between myocytes. Endotoxaemia leads to iNOS gene transcription and calcium-independent NOS enzyme activity in the rat myocardium. In situ hybridisation demonstrates that iNOS is not transcribed by the myocyte in vivo.

Nitric oxide generation. A predictive parameter of acute allograft rejection.

The L-arginine:nitric oxide (NO) biosynthetic pathway has been proposed as an important mediator in host defense mechanisms and may therefore play a role in the acute allograft response. We have studied NO generation in liver allograft rejection and determined its value in immunological monitoring. Stable end products of this pathway have been determined serially in 50 primary liver recipients and compared with 2 known mediators and markers of acute allograft rejection (IL-2R positive lymphocytes and circulating TNF alpha). Plasma concentrations of acid-labile nitrosocompounds (NOx), which increased during acute allograft rejection (P < 0.0001), correlated with rejection severity and were reduced after administration of supplemental high dose glucocorticoids. Concentrations were significantly lower in nonrejection graft complications but were elevated during episodes of sepsis. Correlations between plasma NOx levels and circulating TNF-alpha (r = 0.451, P < 0.001) and IL-2R-positive lymphocytes in peripheral blood (r = 0.781, P < 0.001) were demonstrated. In a logistic analysis of these variables, plasma NOx was the most predictive parameter of an episode of acute cellular rejection. Nitric oxide generation in FK506-treated patients was lower compared with patients receiving a CsA-based immunosuppression regimen and was associated with a reduced frequency of acute rejection in the FK506 group. These data are consistent with a role for NO in the cellular alloantigen immune response and indicate that monitoring of plasma levels of NOx may be useful in the detection of acute allograft rejection.

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.

Biosynthesis and phosphate control of candicidin by Streptomyces acrimycini JI2236: effect of amplification of the pabAB gene.

Biosynthesis of candicidin by Streptomyces acrimycini JI2236 was strongly inhibited by phosphate. p-Aminobenzoic acid (PABA) synthase activity, required for the synthesis of PABA, a candicidin precursor, was reduced by 72% in cells grown in medium supplemented with 7.5 mM phosphate. Hybridization studies showed that the DNA region of S. acrimycini carrying the pabAB gene (encoding PABA synthase) is very similar to the homologous region of S. griseus 3570. S. acrimycini was easily transformed with plasmids containing the pabAB gene of S. griseus. Four transformants were studied in detail; three of the transformants synthesized higher levels of PABA synthase and two transformants produced more candicidin than control cultures transformed with pIJ699. The fourth transformant was unable to synthesize the antibiotic. Formation of PABA synthase and candicidin production was equally sensitive to phosphate regulation in transformants with the pabAB than in the untransformed S. acrimycini strain.

Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants.

No DNA sequence homologous to the penDE gene of Penicillium chrysogenum was found in the genome of three different strains of Cephalosporium acremonium. The pcbC-penDE gene cluster of P. chrysogenum complemented the isopenicillin N synthase deficiency of C. acremonium mutant N2 and resulted in the production of penicillin, in addition to cephalosporin, in cultures supplemented with phenylacetic acid. The penicillin formed was identified as benzylpenicillin by HPLC and NMR studies. The penDE gene of P. chrysogenum is expressed in C. acremonium forming a transcript of 1.15 kb. The transcript is processed and translated in C. acremonium resulting in the formation of acyl CoA: isopenicillin N acyl transferase. When the penDE gene was introduced into a cephalosporin producing strain, the total titre of beta-lactam antibiotics comprised distinct proportions of penicillin and cephalosporin in different transformants. Analysis of the hybridization patterns of the DNA of C. acremonium transformed with the pcbC or penDE genes indicated that integration occurs by non-homologous recombination.

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift.

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.

Myocardial creatine kinase exchange rates and 31P NMR relaxation rates in intact pigs.

In this study, we used 31P NMR to investigate the relationship between cardiac workload and creatine kinase flux in intact pigs. NMR measurements were performed on anesthetized miniature swine in which a surface coil was surgically implanted on the surface of the left ventricle. Cardiac workload was varied by infusion of norepinephrine. Phosphate exchange between creatine phosphate and ATP was measured by a combined saturation transfer, saturation recovery pulse sequence. Exchange measurements showed that creatine kinase flux and concentrations of PCr and ATP were independent of workload for a 2.5-fold range of cardiac rate-pressure products. It appears that, if creatine kinase flux is coupled to work load, the pig heart operates in a regime where small changes in metabolite concentrations or creatine kinase flux are sufficient to maintain elevated workloads. Exchange and relaxation measurements, at 2.0 and 4.7 T, yielded T1 relaxation times for creatine phosphate and ATP which are longer than most reported values. Analysis of the T1 data indicates that chemical-shift anisotropy is a plausible mechanism for a portion of the spin-lattice relaxation rate at high field strengths.

Regional myocardial blood flow, function and metabolism using phosphorus-31 nuclear magnetic resonance spectroscopy during ischemia and reperfusion in dogs.

Postreperfusion regional myocardial dysfunction may be associated with depletion of high energy phosphate compounds during ischemia and with their relatively slow repletion during reperfusion. However, few studies have correlated relatively rapid changes in regional myocardial function (sonomicrometers) and blood flow (microspheres) with high energy phosphate concentrations measured using phosphorus-31 nuclear magnetic resonance spectroscopy in intact large animal models of regional myocardial ischemia. The left anterior descending coronary artery of mongrel dogs was abruptly occluded for 17.1 +/- 1.9 minutes and then completely released; measurements were made for an additional 22 minutes. Transmural blood flow decreased from 1.07 +/- 0.25 to 0.25 +/- 0.10 ml/(min X g) and holosystolic expansion was observed in all dogs (segmental systolic shortening decreased from 9.3 +/- 3.7 to -6.3 +/- 6.0%). Phosphocreatine (PCr) measured during 4.4 minute sampling intervals decreased to steady state within the first sampling period after occlusion and was 45.9 +/- 17.0% of control at the end of the occlusion, whereas beta-adenosine triphosphate (beta-ATP) reached its lowest level early after reperfusion (72.7 +/- 13.3% of control). The ratio of PCr to inorganic phosphate (Pi) decreased during the occlusion (3.34 +/- 0.75 versus 1.01 +/- 0.61) but returned to control level early during reperfusion. The ratio of PCr to beta-ATP also decreased during coronary occlusion (2.16 +/- 0.39 versus 1.29 +/- 0.39) but did not return to control level during reperfusion. Significant correlations were observed between the intensity of ischemia (reduced blood flow) and reductions in regional contractile function, PCr, beta-ATP, myocardial pH and the increase in Pi during the coronary occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.

Glucose regulation of cephamycin biosynthesis in Streptomyces lactamdurans is exerted on the formation of alpha-aminoadipyl-cysteinyl-valine and deacetoxycephalosporin C synthase.

Glucose exerted a concentration-dependent negative regulation on the biosynthesis of cephamycin C by Streptomyces lactamdurans. Formation of the cephamycin precursor delta(alpha-aminoadipyl)-cysteinyl-valine was greatly decreased by excess glucose. The ring-expanding enzyme deacetoxycephalosporin C synthase was strongly repressed by glucose in vivo. Isopenicillin N synthase (cyclase) and isopenicillin N epimerase were not repressed by glucose. However, the activity of isopenicillin N synthase was inhibited in vitro by glucose 6-phosphate, and the activity of deacetoxycephalosporin C synthase was inhibited by inorganic phosphate, glucose 6-phosphate, fructose 2,6-diphosphate and fructose 1,6-diphosphate. The intracellular cAMP content decreased as growth proceeded and remained lower in glucose-supplemented cells than in control cultures. cAMP did not seem to be involved in glucose control of cephamycin biosynthesis.

Isolation of mutants deregulated in phosphate control of candicidin biosynthesis.

Mutants have been isolated in which phosphate does not inhibit the biosynthesis of candicidin. At high phosphate concentrations, candicidin production by phosphate-deregulated mutants is still inhibited, but to a lesser extent than in the wild type. Some of these mutants are higher candicidin producers than the wild type, not only in phosphate-supplemented medium but also in non-supplemented production medium. The high candicidin production by these mutants is due to (1) a high specific rate of candicidin biosynthesis and (2) an extended production phase. None of the phosphate-deregulated mutants in which uptake of [32P]phosphate was measured was a phosphate-permeability mutant.

Minimal tissue concentrations of glutamate required to produce necrosis of hypothalamic neurons in newborn mice.

Groups of 4-day-old Cox Swiss albino mice were injected once subcutaneously with monosodium glutamate at several doses between 0.2 and 0.5 mg/g body weight. Glutamate, at a dose of 0.35 mg/g, produced neuronal necrosis of a very limited nature in only 60% of the animals and was defined as the minimal effective neurotoxic dose in the 4-day-old mouse. Neuronal loss was not detected in any animals treated with less than 0.35% mg/g of the amino acid whereas lesions became more extensive as the dose was increased to 0.5 mg/g. Glutamate was measured in the arcuate nucleus and plasma 0, 15, 30, 60 and 90 min after injection. These data indicated that duration of glutamate accumulation in the arcuate nucleus may be as important a variable in producing neuronal degeneration in the hypothalamus as concentration of the amino acid in that nucleus.

The use of calcium chelating agents and prostaglandin E to eliminate platelet and white blood cell losses resulting from hemoperfusion through uncoated charcoal, albumin-agarose gel, and neutral and cation exchange resins.

Hemoperfusion through adsorbents such as charcoal, cation exchange (e.g., AG 50W-X8) and uncharged (e.g., XAD-2) resins, and albumin-agarose gel (AAG) has been proposed for use in patients with hepatic failure. However, the loss of white blood cells and, particularly, platelets caused by each of these adsorbents remains a major deterrent to their clinical use. In vitro studies demonstrate that addition of citrate, ethylenediamine tetraacetate (EDTA), or oxalate to heparinized human blood eliminated this loss of formed blood elements during hemoperfusion. The improvement in postperfusion platelet counts (per cent of preperfusion values) produced by citrate were XAD-2, 13 leads to 95 per cent; AG 50W-X8, 10 leads to 94 per cent; AAG, 17 leads to 94 per cent; and charcoal, 44 leads to 96 per cent. Prostaglandin E1 in high doses (5 mug/ml.) markedly reduced platelet losses. Lower doses were less uniformly effective. Three young rhesus monkeys were hemoperfused for 160 minutes with columns containing AAG, XAD-2, and charcoal. During the first 80 minutes, citrate and calcium were infused into the column inflow and outflow lines respectively. For all three adsorbents, average platelet counts in the monkeys (115 per cent) and column effluent (95 per cent) were unchanged from preperfusion control values during the first 80 minutes but fell promptly to 13 and 7 per cent, respectively, after the citrate infusion was stopped. Each of the monkeys tolerated the procedure without ill effects. Use of a system analogous to that described here may facilitate clinical application of the technique of hemoperfusion through a variety of adsorbents.