Synthase Activity Levels Research Articles

In vitro nitric oxide effects on basal and gonadotropin-releasing hormone-induced gonadotropin secretion by pituitary gland of male crested newt (Triturus carnifex) during the annual reproductive cycle.

The objective of this study was to test the possible nitric oxide (NO) involvement in pituitary gonadotropin secretion in the male crested newt, Triturus carnifex. Pituitaries were incubated in vitro with medium alone, GnRH, NO donor (NOd, sodium nitroprusside), NO synthase inhibitor (NOSi, Nomega-nitro-L-arginine methyl ester), cGMP analogue (cGMPa, 8-bromo-cGMP), soluble guanylate cyclase inhibitor (sGCi, cystamine), GnRH plus NOSi, GnRH plus sGCi, and NOd plus sGCi during the annual reproductive cycle: pre-reproduction, reproduction (noncourtship and courtship), and the refractory, recovery, and estivation periods. To determine pituitary gonadotropin secretion indirectly, newt testes were superfused in vitro with preincubated pituitaries, and androgen release was determined. NO synthase (NOS) activity and cGMP levels were assessed in the preincubated pituitaries. Medium alone- and GnRH-preincubated pituitary increased androgen secretion during pre-reproduction, noncourtship, courtship, and recovery; the GnRH-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOd and cGMPa increased androgens in all reproductive phases considered except courtship; the NOd- and cGMP-induced increase was higher than the medium alone-induced increase during pre-reproduction, noncourtship, and recovery. NOS activity was highest during courtship and lowest during the refractory and estivation periods. GnRH increased NOS activity during pre-reproduction, noncourtship, and recovery. Cyclic GMP levels were highest during courtship and lowest during the refractory period and estivation. GnRH increased cGMP levels during pre-reproduction, noncourtship, and recovery, while NOd did so during all reproductive phases considered. These results suggest that basal and GnRH-induced gonadotropin secretion are up-regulated by NO in the pituitary gland of the male Triturus carnifex.

Long-term vascular effects of Nomega-nitro-L-arginine methyl ester are not soley mediated by inhibition of endothelial nitric oxide synthesis in the rat mesenteric artery.

Nomega-nitro-L-arginine methyl ester (L-NAME), one of the synthetic L-arginine analogues with inhibitory effects of nitric oxide (NO) synthesis, is now widely used to examine the role of NO in various organs. We and others demonstrated that long-term treatment with L-NAME causes hypertension and cardiovascular lesions (perivascular fibrosis and medial thickening), especially at microvascular levels. However, convincing evidence is still lacking that these long-term cardiovascular effects of L-NAME are solely mediated by the inhibition of the synthesis of endothelium-derived NO (EDNO). This study was thus designed to better understand the effects of long-term treatment with L-NAME with special reference to EDNO synthesis. Male Wister-Kyoto rats were orally administered L-NAME for 8 weeks. Blood pressure significantly increased at 3 days and 1 and 8 weeks of the treatment. Endothelium-dependent relaxations to acetylcholine (ACh) of the aorta were reduced 3 days after the treatment, recovered at 1 week, and again reduced at 8 weeks, whereas the relaxations of the small mesenteric artery were unaltered throughout the experimental periods. At 8 weeks, indomethacin-sensitive, endothelium-dependent contractions to ACh were noted. The relative contributions of NO and endothelium-derived hyperpolarizing factor also were unchanged. Citrulline assay demonstrated that substantial levels of constitutive NO synthase activity remained in the aorta during the experiments. The long-term treatment with L-NAME caused perivascular fibrosis and medial thickening, not only in the aorta but also in the mesenteric artery. These results suggest that mechanism(s) other than simple inhibition of EDNO synthesis is involved in the long-term cardiovascular effects of L-NAME in the rat mesenteric artery.

Platelet nitric oxide synthase activity and plasma cyclic GMP levels suggests dysregulation in nitric oxide metabolism in schizophrenia

Platelet nitric oxide synthase activity and plasma cyclic GMP levels suggests dysregulation in nitric oxide metabolism in schizophrenia

A RANDOMIZED DOUBLE-BLIND TRIAL OF ORAL L-ARGININE FOR TREATMENT OF INTERSTITIAL CYSTITIS

Nitric oxide synthase activity is decreased in the urine of patients with interstitial cystitis compared to the urine of controls. In a preliminary trial oral L-arginine, the substrate for nitric oxide synthase, increased urinary nitric oxide synthase activity and improved interstitial cystitis symptoms. This randomized, double-blind, placebo controlled study further investigates the efficacy of L-arginine treatment for interstitial cystitis. A total of 53 interstitial cystitis patients were assigned to receive daily 1,500 mg. L-arginine or placebo orally for 3 months. Interstitial cystitis symptoms were assessed by interviews at 2 weeks, and 1, 2 and 3 months. The trial was completed by 21 of 27 patients in the L-arginine group and 25 of 26 in the placebo group. Using per protocol analysis 29% (6 of 21 patients) in the L-arginine group and 8% (2 of 25) in the placebo group were clinically improved by the end of the trial (p = 0.07). A Likert scale showed greater global improvement in the L-arginine group (48%, 10 of 21) compared to the placebo group (24%, 6 of 25) at 3 months (p = 0.05) with a decrease in pain intensity (p = 0.04), and tendency toward improvement in urgency (p = 0.06) and frequency of pain (p = 0.09). Using an intention to treat approach to analysis there were no differences between groups. Oral L-arginine (1,500 mg. daily) may decrease pain and urgency in a subset of interstitial cystitis patients.

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells.

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Temporal bone surgery causes reduced nitric oxide synthase activity in the ipsilateral guinea pig hippocampus

There is a lack of data on the neurochemical basis for the interaction between the vestibular system and the hippocampus. The aim of the present study was to determine levels of nitric oxide synthase (NOS) activity in the ipsilateral and contralateral hippocampi at 10 h following unilateral deafferentation of the peripheral vestibular nerve (UVD) in guinea pig, using a radioenzymatic technique. The levels of NOS activity were similar in the contralateral hippocampus following either a sham temporal bone operation or the UVD. However, NOS activity was significantly lower in the ipsilateral hippocampus following both the UVD and the sham temporal bone surgery (P<0.05 for both comparisons). These results suggest that even sham temporal bone surgery results in a reduction in NOS activity in the ipsilateral hippocampus.

Cloning and Functional Expression of a cDNA Encoding Geranylgeranyl Diphosphate Synthase fromTaxus canadensisand Assessment of the Role of this Prenyltransferase in Cells Induced for Taxol Production

Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-inducedTaxus canadensissuspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an inducedT. canadensiscell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90–100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative “mature proteins” from which the transit peptide was deleted, were transformed into a yeast mutant defective for the β-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction.In vitroactivity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS–PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in inducedT. canadensissuspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.

Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate.

Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, encoding malate synthase from Streptomyces clavuligerus NRRL 3585, was cloned using PCR and fully sequenced. The ORF obtained encodes 541 amino acids with a deduced Mr of 60000, consistent with the observed Mr (62000-64000) of most malate synthase enzymes reported so far. The aceB gene has a high G+C content (71.5 mol%), especially in the third codon position. A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region. The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated. Growth of S. clavuligerus on acetate as the major carbon source was delayed, compared to that on glycerol. Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium. Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol. The cloned S. clavuligerus aceB gene was expressed in Escherichia coli BL21(DE3). Transformants exhibited an approximately 71-fold increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.

Preservation with 8-bromo-cyclic GMP improves pulmonary function after prolonged ischemia

Background. Cyclic guanosine monophosphate (cGMP) is a potent second messenger for the nitric oxide pathway in the pulmonary vasculature. Increased cytosolic cGMP levels elicit pulmonary vasodilatation resulting in decreased pulmonary vascular resistance and maximized pulmonary function after ischemia-reperfusion injury. We hypothesized that the addition of a membrane-permeable cGMP analogue (8-bromo-cGMP) to a Euro-Collins (EC) preservation solution would ameliorate pulmonary reperfusion injury better than prostaglandin E 1 injection alone after prolonged hypothermic ischemia. Methods. All lungs from New Zealand White rabbits (weight, 3 to 3.5 kg) were harvested en bloc, flushed with EC solution, and reperfused with whole blood for 30 minutes. Group 1 lungs (immediate control) were immediately reperfused. Group 2 lungs (control) were stored inflated at 4°C for 18 hours before reperfusion. Groups 3 and 4 lungs were flushed with EC solution containing 200 μmol/L 8-bromo-cGMP and stored at 4°C for 18 and 30 hours, respectively. Fresh, nonrecirculated venous blood was used to determine single-pass pulmonary venous–arterial oxygen gradients at 10-minute intervals. Assays for cGMP, cyclic adenosine monophosphate, nitric oxide synthase activity, and myeloperoxidase were performed on all lung tissue samples. Wet to dry weight ratios were determined after 2 weeks of passive desiccation. Results. Oxygenation (venous-arterial oxygen gradient), pulmonary artery pressure, pulmonary vascular resistance, and edema formation were significantly improved in groups 3 and 4 (addition of 8-bromo-cGMP to EC plus 18 or 30 hours of hypothermic ischemia). Hypothermic storage (groups 2, 3, and 4) decreased both nitric oxide synthase activity and myeloperoxidase levels compared with immediate reperfusion (group 1). Conclusions. These results suggest that the addition of a membrane-permeable cGMP analogue to an EC pulmonary flush solution improves pulmonary function after prolonged storage compared with EC and prostaglandin (E 1) preservation alone. The finding of myeloperoxidase reduced levels after hypothermic storage and subsequent reperfusion may suggest a more important role for pulmonary hemodynamic control in mitigating pulmonary reperfusion injury.

Response to cadmium in carrot in vitro plants and cell suspension cultures

In vitro grown plants and cell suspension cultures of carrot ( Daucus carota L.) were treated with various Cd concentrations. Stress ethylene production in carrot plants was highly stimulated by 1 mM Cd. A pre-treatment with buthionine sulfoximine (BSO) did not further increase ethylene production. After being treated with Cd, both plants and cell suspensions produced phytochelatins, and no lipid peroxidation was detected. In cell cultures, the in vitro activity of phytochelatin synthase was assayed in the presence of Cd and glutathione: the first product (PC 2) was detected in less than 30 min. Absence of ethylene (after treatment with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene-biosynthesis, or use of ethylene traps) caused both a decrease in the phytochelatin synthase activity of cell suspensions and a strong lowering in the Cd-induced SH groups in plants. However 1-aminocyclopropane-1-carboxylic acid (ACC) supply did not increase either phytochelatin synthase activity or total SH level.

Effect of Desmethyl Tirilazad, Dizocilpine Maleate and Nimodipine on Brain Nitric Oxide Synthase Activity and Cyclic Guanosine Monophosphate during Cerebral Ischemia in Rats

The present study was designed to evaluate the effects of pretreatment with a combination of desmethyl tirilazad (21-aminosteroid) plus dizocilpine maleate (N-methyl-D-aspartate receptor antagonist) and nimodipine (calcium channel antagonist) on constitutive nitric oxide synthase (cNOS) activity and cyclic guanosine monophosphate (cGMP) levels in brain homogenates of rats subjected to global cerebral transient ischemia induced by bilateral clamping of the carotids for 30 min and reduction of arterial pressure (to 50–60 mm Hg) by intravenous infusion of trimethaphan (30 mg/kg). Our results show that cerebral ischemia produced an increase in cNOS activity and cGMP levels in brain homogenates. Pretreatment with desmethyl tirilazad or dizocilpine maleate or nimodipine individually significantly suppressed (p < 0.01) the increase in cNOS activity and cGMP levels induced by cerebral ischemia, which may be related to their neuroprotective effect. Similar results were obtained with pretreatment by a combination of desmethyl tirilazad plus dizocilpine maleate plus nimodipine.

Molecular cloning and characterization of Ca2+-dependent inducible nitric oxide synthase from guinea-pig lung.

We have isolated a full-length cDNA for an inducible nitric oxide synthase (iNOS) from guinea-pig lung. The cDNA has a 3447 bp open reading frame encoding 1149 amino acid residues. The deduced amino acid sequence is approx. 80% identical with iNOS of human epithelial cells and murine macrophages. Consensus recognition sites for cofactors are highly conserved. COS cell lysate transfected with the guinea-pig iNOS shows significant levels of nitric oxide synthase (NOS) activity, and this is inhibited by 79% by chelation of Ca2+ ions. The NOS activity is restored in a concentration-dependent manner by increasing the free Ca2+ level. The NOS activity is also inhibited by trifluoperazine, a calmodulin antagonist, which suggests that the Ca2+ dependence is due to Ca2+-dependent calmodulin binding to the enzyme. Northern blot analysis reveals that the cloned iNOS mRNA is expressed in the lung and the colon in normal guinea pigs. Stimulation in vivo by lipopolysaccharide induces the expression of iNOS in the kidney, the spleen and the colon, but in the lung the same stimulation decreases its expression. These results suggest that the cloned guinea-pig iNOS is distinct in characteristics and expression from previously described iNOS forms.

Factors contributing to differences in the regulation of cGMP in isolated porcine pulmonary vessels

Guanosine 3′,5′-cyclic monophosphate (cGMP) is an important second messenger in many biological systems including vascular smooth muscle where it mediates relaxation. Cellular levels of cGMP are regulated primarily by three enzymes; nitric oxide (NO) synthase, soluble guanylate cyclase, and cGMP-phosphodiesterase. Basal cGMP levels of isolated endothelium intact porcine pulmonary vein are five fold higher than in pulmonary artery. The objective of this study was to investigate possible reasons for this difference. Therefore, we compared NO synthase activity of pulmonary vein with artery and used pharmacologic approaches to compare soluble guanylate cyclase and phosphodiesterase activities in these vessels. NO synthase activities of pulmonary vein and artery were measured by monitoring the conversion of exogenous l-[ 14 C ]arginine to l-[ 14 C ]citrulline and by quantifying NO formation from endogenous l-arginine. Rates (pM/min per mg protein) of basal l-citrulline and NO formation from endothelium intact pulmonary vein (29.0±4.8 and 44±7.1, respectively) were significantly higher than from artery (8.3±2.2 and 17.1±3.3). Western blot analysis indicated higher constitutive NO synthase protein in the vein than in artery. N-nitro- l-arginine (0–100 μM), a potent inhibitor of NO synthase, induced contractions of the pulmonary vein which were significantly higher than those of the artery. N-nitro- l-arginine (5 and 20 μM) in the presence of phosphodiesterase inhibitors, decreased basal cGMP levels of endothelium intact blood vessels. In endothelium denuded pulmonary vein and artery, basal cGMP levels were not different from each other, but increased significantly following stimulation of soluble guanylate cyclase with exogenous NO. In the presence of both non-specific and specific cGMP phosphodiesterase inhibitors, exogenous NO-induced cGMP levels of endothelium denuded tissues were not significantly different from each other. However, in the absence of the phosphodiesterase inhibitors, exogenous NO-induced cGMP was significantly less in the artery than in the vein. These results suggest that (I) the intact porcine pulmonary vein contains higher levels of NO synthase activity than pulmonary artery, and that (II) the soluble guanylate cyclase activities in pulmonary vein and artery are equally responsive to NO, and finally (III) pulmonary artery expresses greater phosphodiesterase activity than vein. Higher NO synthase and lower phosphodiesterase activity may explain the greater accumulation of cGMP in the pulmonary vein compared to the artery.

Characterization of the Difference of Starch Branching Enzyme Activities in Normal and Low-Amylopectin Maize during Kernel Development

In order to determine the reasons for the differences in structure between starch from a normal and a low-amylopectin maize variety the activities of all the enzymes in the committed pathway of starch synthesis were studied throughout kernel development. Levels of ADP glucose pyrophosphorylase and starch synthase activity were found to be broadly similar between the two varieties but the low-amylopectin starch (LAPS) maize variety showed dramatically reduced starch branching enzyme activity, with an almost total absence of the branching enzyme II isoform. Scanning electron microscopy showed a significant alteration in the morphology of the starch granules of the low-amylopectin maize. The results suggest that the increased amylose and the reduction of high molecular weight amylopectin in the LAPS starch results from the absence of the branching enzyme II isoform. This evidence supports the theory that the different branching enzyme isoforms contribute separately to the synthesis and final structure of amylopectin.

Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer.

Angiogenesis (formation of new blood vessels) is associated with tumor growth and metastasis in patients with solid tumors, including those of the head and neck. Nitric oxide (NO) production may contribute to these processes. We assessed the role of the NO pathway in angiogenesis and tumor progression in patients with head and neck cancer. Biochemical assays were used to measure NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) levels in specimens of tumor and normal mucosa obtained from 27 patients. Microvessels in tumor specimens were identified by CD-31-specific immunohistochemical staining. Associations between microvessel densities, levels of NOS, and cGMP were examined by use of two-sided statistical tests. Tumor specimens and human squamous carcinoma A-431 cells were grown as explants on the corneas of rabbits, and the effect of the NOS inhibitor N(omega)-nitro-L-arginine-methyl ester (L-NAME) was tested. Levels of total NOS, inducible NOS, and cGMP were higher in tumor specimens than in specimens of normal mucosa (all P<.0001). Tumor specimens from patients with lymph node metastases presented a higher total NOS activity (P = .005) and were markedly more vascularized than tumor specimens from patients with no lymph node involvement (P = .0002). Microvessel density at the tumor edge was an independent predictor of metastasis for this series of patients (odds ratio = 1.19; 95% confidence interval = 1.07-2.89; P = .04). A-431 cells and tumor specimens exhibiting high levels of NOS activity induced angiogenesis in the rabbit cornea assay; when NO production was blocked, tumor angiogenesis and growth were repressed. The NO pathway appears to play a key role in tumor angiogenesis and spread in patients with head and neck cancer.

Individual variation in constitutive and induced monoterpene biosynthesis in grand fir

Variations in the level and distribution of monoterpene synthase activities in stem samples of 46 grand fir saplings maintained under controlled conditions were investigated using sensitive, non-destructive bioanalytical techniques that permitted the assessment of constitutive, wound-induced and ethylene modulated biosynthetic capability of each individual. Radiochemically-based in vitro assays, with [1- 3H]geranyl diphosphate as substrate, revealed considerable quantitative variation in constitutive and induced response levels of total monoterpene synthase activity, and the analysis of monoterpene distribution generated in the cell-free extracts allowed the definition of seven distinct biosynthetic chemotypes. No correlation was observed between total monoterpene biosynthetic capacity and chemotype, indicating that both broad quantitative and qualitative variation in oleoresin monoterpene production exists in this native population of grand fir. The implications of these findings for oleoresin-based defense against bark beetle predation are described.

Effect of calcium and calmodulin antagonists on ethylene biosynthesis in tomato fruits

The aim of this work was to elucidate the mode of action of calcium in calcium-inhibited ethylene biosynthesis. In one experiment, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-1-naphthalene-sulphonamide (W5). 1.5% (w/v) CaCl 2 + 60 μM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7), 1.5% (w/v) CaCl 2 alone or distilled water (control). The control and the W7 treatment had much higher ethylene production than either the CaCl 2 treatment alone or the CaCl 2 + W5 treatment. In another experiment, similar tomatoes were treated with 1.5% (w/v) CaCl 2 + 15 μM trifluoperazine (TFP), 1.5% (w/v) CaCl 2 only, or distilled water (control). The treatment with 1.5% CaCl 2 + 15 μM TFP gave higher ethylene production than 1.5% (w/v) CaCl 2 without TFP, and nearly the same level as the control towards the end of the experimental period. For enzyme and intermediate metabolite assays, mature green tomatoes were treated with 1.5% (w/v) CaCl 2 plus either W5 or W7. W5 treatment had a lower ethylene forming enzyme (EFE) activity, higher 1-aminocyclopropane-l-carboxylic acid (ACC) concentration, similar levels of ACC synthase activity and 1-(malonylamino)-cyclopropane-1-carboxylic acid concentration, compared to the control and W7 treatment. As W7 is a more potent calmodulin antagonist than W5, these results suggest a calcium-calmodulin inactivation of EFE activity.

Safety assessment of genetically engineered yeast: elimination of mutagenicity of the yeast Saccharomyces cerevisiae by decreasing the activity of methylglyoxal synthase

Summary Methylglyoxal synthase catalyses the transformation of dihydroxyacetonephosphate to methylglyoxal, a toxic 2‐oxoaldehyde which is found to be present in the yeast Saccharomyces cerevisiae DKD‐5D‐H. Yeast cells in which genes for phosphoglucose isomerase, phosphofructokinase and triosephosphate isomerase had been extrachromosomally amplified by using a multi‐copy plasmid showed an increased ability to induce mutagenesis in standard tests when compared to wild type yeast. This response is mainly due to the increased amount of methylglyoxal in the engineered cells. To decrease the mutagenic activity and make it possible to use genetically engineered yeasts for practical fermentation processes, a mutant having a decreased level of methylglyoxal synthase activity was isolated. When transformed with genes for the glycolytic enzymes, the mutant cells showed extremely low levels of methylglyoxal content and mutagenic activity, both levels being comparable with those of non‐transformed DKD‐5D‐H cells.

Glycolipid Sialyltransferases Are Enhanced during Neural Differentiation of Mouse Embryonic Carcinoma Cells, P19

We have studied ganglioside alterations and their enzymatic basis during the course of neural differentiation of mouse embryonic carcinoma cell line P19. This cell line can differentiate into neurons and astrocytes on cell aggregation after treatment with retinoic acid (RA) or into muscle cells on dimethyl sulfoxide (DMSO) treatment. GD3, detected on immunostaining after thin-layer chromatography (TLC) with monoclonal antibody (MAb) R24, was markedly present in aggregates treated with RA. GM3 synthase (α2,3-sialyltransferase, SAT-I) in neurons was found to exhibit the highest activity. GD3 synthase (α2,8-sialyltransferase, SAT-II) and GD3 synthase mRNA, as analyzed by Northern blotting, were also markedly present in aggregates and neurons induced by RA. However, on treatment with DMSO, which induces muscle cells, there was no change in the level of GD3 synthase activity, and its transcript was hardly detected during the course of muscle differentiation. GT1b synthase (α2,3-sialyltransferase, SAT-IV) was present at similar levels in undifferentiated cells and aggregates treated with RA, but a higher level was observed in neurons. On the other hand, the level of GQ1b synthase (α2,8-sialyltransferase, SAT-V) in RA-induced aggregates was significantly higher than that in neurons. These results show that RA but not DMSO induces the expression of GM3, GD3, GT1b and GQ1b synthases, and particularly GD3 synthase mRNA, in the ganglioside biosynthetic pathway during the neural differentiation of embryonic carcinoma P19 cells.

EFFECT OF LONG-TERM ORAL L-ARGININE ON THE NITRIC OXIDE SYNTHASE PATHWAY IN THE URINE FROM PATIENTS WITH INTERSTITIAL CYSTITIS

Purpose We attempted to determine whether oral L-arginine, the substrate for nitric oxide synthase, increases nitric oxide synthase activity and cyclic guanosine monophosphate (cGMP) levels in the urine from interstitial cystitis patients. Nitric oxide and cGMP are decreased in urine from interstitial cystitis patients and both induce smooth muscle relaxation and immunological responses. Increasing urinary nitric oxide and cGMP may ameliorate interstitial cystitis symptoms. Materials and Methods Eight patients with interstitial cystitis were given L-arginine (1,500 mg. a day) orally for 6 months. Before and during treatment nitric oxide synthase activity and inducible nitric oxide synthase protein, cGMP, nitrate plus nitrite and interleukin 8 (IL-8) levels were measured in urine. Results After 2 weeks to 1 month of oral L-arginine treatment, urinary levels of nitric oxide synthase related enzymes and products increased significantly, while levels of the cytokine IL-8 were not changed significantly. IL-8 was significantly elevated in interstitial cystitis patients with leukocyte esterase positive urine. Conclusions Long-term oral administration of L-arginine increases nitric oxide related enzymes and metabolites in the urine of patients with interstitial cystitis, which is associated with a decrease in interstitial cystitis related symptoms.