Synthase Enzyme Activity Research Articles

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.

Genotype-specific influence on nitric oxide synthase gene expression, protein concentrations, and enzyme activity in cultured human endothelial cells.

The results of studies on the association of ecNOS polymorphisms and vascular diseases are inconsistent. To explore the nature of this interaction in the absence of confounding factors, such as smoking, we measured ecNOS mRNA, protein, and enzyme activity in cultured human umbilical vein endothelial cells (HUVECs) with and without ecNOS polymorphisms. We identified a T(-786)-->C polymorphism in the promoter region, the intron 4 variable number of tandem repeats (VNTR), the E298A polymorphism in exon 7, and the G10-T polymorphism in intron 23 of the ecNOS gene in the DNA from 43 human umbilical cords. We measured ecNOS and GAPDH mRNA from the cultured HUVECs by reverse transcription-PCR and ecNOS protein and enzyme activity by Western blotting (as ratio to positive control band) and by determining the conversion of [(3)H]arginine to [(3)H]citrulline, respectively. The T(-786)-->C polymorphism showed the same allelic distribution as the intron 4 VNTR. Mean (SD) ecNOS protein from the cultured HUVECs was significantly lower in the 4a/4b genotype [0.84 (1.23); n = 9] of the intron 4 VNTR than in the 4b/4b genotype [2.14 (2.26); n = 34; P = 0.0300]. The enzyme activity was also significantly lower in the 4a/4b genotype [0.84 (0.21) pmol.min(-1).mg protein(-1); n = 9] than in the 4b/4b genotype [1.07 (0.31) pmol.min(-1).mg protein(-1); n = 34; P = 0.0197]. ecNOS gene expression, protein concentrations, and enzyme activity are genotype-dependent in HUVECs. The intron 4 VNTR has a consistent influence that may be mediated by the T(-786)-->C polymorphism in the promoter region.

The Importance of Cationic Amino Acid Transporter Expression in Human Skin

Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells.

Interactive effects of PTH and mechanical stress on nitric oxide and PGE2production by primary mouse osteoblastic cells

Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9 M human PTH-(1-34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 +/- 0.3 Pa at 5 Hz) stimulated both NO and PGE2 production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2 production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2 production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.

Citrate synthase expression and enzyme activity after endurance training in cardiac and skeletal muscles.

The present study was designed to examine the acute and chronic effects of endurance treadmill training on citrate synthase (CS) gene expression and enzymatic activity in rat skeletal and cardiac muscles. Adult rats were endurance trained for 8 wk on a treadmill. They were killed 1 h (T(1), n = 8) or 48 h (T(48), n = 8) after their last bout of exercise training. Eight rats were sedentary controls (C) during the training period. CS mRNA levels and enzymatic activities of the soleus and ventricle muscles were determined. Training resulted in higher CS mRNA levels in both the soleus muscles (21% increase in T(1); 18% increase in T(48), P < 0.05) and ventricle muscles (23% increase in T(1); 17% increase in T(48), P < 0.05) when compared with the C group. The CS enzyme activities were 42 (P < 0.01) and 25% (P < 0.01) greater in the soleus muscles of T(1) and T(48) groups, respectively, when compared with that of the C group. Soleus CS enzyme activity was significantly greater in the T(1) vs. T(48) groups (P < 0.05). However, no appreciable alterations in CS enzyme activities were observed in the ventricle muscles in both training groups. These findings suggest differential responses of skeletal and cardiac muscles in CS enzymatic activity but similar responses in CS gene expression at 1 and 48 h after the last session of endurance training. Moreover, our data support the existence of an acute effect of exercise on the training-induced elevation in CS activity in rat soleus but not ventricle muscles.

Effective anti-platelet and COX-1 enzyme inhibitors from pungent constituents of ginger

Background: Based on recent studies, pungent constituents of ginger ( Zingiber officinale) and related substances represent a potential new class of anti-platelet agents. The ability of 20 pungent constituents of ginger and related substances to inhibit arachidonic acid (AA) induced platelet activation in human whole blood was studied. Methods: Anti-platelet activity of the compounds was measured in vitro by the Chrono Log whole blood platelet aggregometer. Molecular hydrophobicity (log P) was measured by reversed-phase high-performance liquid chromatography. COX-1 (ovine) inhibitory effect of [8]-paradol and analogues 1 and 5 was carried out using a COX-1 inhibitor assay kit. Results: [8]-Gingerol, [8]-shogaol, [8]-paradol and gingerol analogues ( 1 and 5) exhibited anti-platelet activities with IC 50 values ranging from 3 to 7 μM, whilst under similar conditions the IC 50 value for aspirin was 20±11 μM. The COX-1 inhibitory activity of [8]-paradol (IC 50=4±1 μM) was more potent than the gingerol analogues ( 1 and 5) (IC 50 approximately 20 μM). Conclusion: The above findings show that gingerol compounds and their derivatives are more potent anti-platelet agents than aspirin under the conditions described in this study. [8]-Paradol, a natural constituent of ginger, was found to be the most potent COX-1 inhibitor and anti platelet aggregation agent. The mechanism underlying AA-induced platelet aggregation inhibition may be related to attenuation of COX-1/Tx synthase enzymatic activity. Lastly, important features of phenolic compounds for inhibition of AA-induced platelet aggregation and COX-1 activity were revealed in this study.

Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity.

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Seasonal differences in citrate synthase and digestive enzyme activity in larval and postlarval antarctic krill, Euphausia superba

Antarctic krill maintain large population sizes despite dramatic seasonal fluctuations in food availability, but the mechanisms for this are still debated. The aim of this study was to compare seasonal differences in enzyme activity and respiration rates of larval and postlarval krill to provide insights into their overwintering strategies. Respiration rates, activity of the metabolic enzyme citrate synthase (CS), and those of the digestive enzymes laminarinase and total proteinase were measured in austral summer west of the Antarctic Peninsula, and in autumn in the southwestern Lazarev Sea. The 100-fold difference in chlorophyll a concentrations between the two studies is representative of the classic transition from a summer bloom to sparse winter conditions. Correspondingly, adult krill showed reduced respiration rates and CS activity in autumn. However, their digestive enzyme activity was significantly higher, suggesting more efficient assimilation of food at low food levels. Similar-sized larvae showed no summer-autumn differences in respiration rates and enzyme activity, supporting suggestions that they need to feed and grow year-round. However, trends in enzymatic activity varied between the larval stages measured, implying ontogenetic changes in body structure and function.

Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco.

Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps.

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.

Ethanol extract of propolis inhibits nitric oxide synthase gene expression and enzyme activity

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. However, the molecular basis for anti-inflammatory properties of propolis has not yet been established. Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. The present study, therefore, examined effects of ethanol extract of propolis (EEP) on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with EEP significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ). EEP also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-κB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, revealed that EEP inhibited the iNOS promoter activity induced by LPS plus IFN-γ through the NF-κB sites of the iNOS promoter. In addition, EEP directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that EEP may exert its anti-inflammatory effect by inhibiting the iNOS gene expression via action on the NF-κB sites in the iNOS promoter and by directly inhibiting the catalytic activity of iNOS.

Calculated background elimination in quantifying nitric-oxide synthase enzyme activity

Measuring nitric-oxide synthase (NOS) activity by monitoring the conversion of l-arginine to l-citrulline is currently the standard assay for NOS activity. We describe a simple method of quantifying low values of NOS activity by removing the background mathematically. When performing NOS activity studies in samples with low protein amount (<25 μg/μl), we encountered the problem of sample values that can hardly be differentiated from blank values probably originating from radioactive-labeled arginine in the final eluate. Our method determines mathematically these background values and may be an improvement of the citrulline assay.

Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity

Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, has been identified to show anti-inflammatory, anti-viral and anti-cancer activities. The present study, therefore, examined effects of CAPE on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with CAPE significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ). CAPE also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-κB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase reporter gene, revealed that CAPE inhibited the iNOS promoter activity induced by LPS plus IFN-γ through the NF-κB sites of the iNOS promoter. In addition, CAPE directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression at the transcriptional level through the suppression of NF-κB activation, and by directly inhibiting the catalytic activity of iNOS.

Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Nuclear Factor κB Is a Molecular Target for Sulforaphane-mediated Anti-inflammatory Mechanisms

Sulforaphane (SFN), an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate anti-inflammatory mechanisms of SFN, we here report a potent decrease in lipopolysaccharide (LPS)-induced secretion of pro-inflammatory and pro-carcinogenic signaling factors in cultured Raw 264.7 macrophages after SFN treatment, i.e. NO, prostaglandin E(2), and tumor necrosis factor alpha. SFN did not directly interact with NO, nor did it inhibit inducible nitric-oxide synthase enzymatic activity. Western blot analyses revealed time- and dose-dependent reduction of LPS-induced inducible nitric-oxide synthase as well as Cox-2 protein expression, which was suppressed at the transcriptional level. To reveal the target of SFN beyond its anti-inflammatory action, we performed electrophoretic mobility shift assay analyses of transcription factor-DNA binding. Consequently, nuclear factor kappa B (NF-kappa B), a pivotal transcription factor in LPS-stimulated pro-inflammatory response, was identified as the key mediator. SFN selectively reduced DNA binding of NF-kappa B without interfering with LPS-induced degradation of the inhibitor of NF-kappa B nor with nuclear translocation of NF-kappa B. Because SFN can interact with thiol groups by dithiocarbamate formation, it may impair the redox-sensitive DNA binding and transactivation of NF-kappa B. Sulforaphane could either directly inactivate NF-kappa B subunits by binding to essential Cys residues or interact with glutathione or other redox regulators like thioredoxin and Ref-1 relevant for NF-kappa B function. Our data provide novel evidence that anti-inflammatory mechanisms contribute to sulforaphane-mediated cancer chemoprevention.

Mechanism of endothelial dysfunction in apolipoprotein E-deficient mice.

Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10(-)(9) to 10(-)(5) mol/L) and Ca(2+) ionophore (10(-)(9) to 10(-)(6) mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-)(10) to 10(-)(5) mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O(2)(-)) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn-superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10(-)(5) mol/L), reduced O(2)(-) production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [(14)C]L-Citrulline assay showed a decrease of Ca(2+)-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O(2)(-) and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O(2)(-) and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.

Circadian Expression of Clock Genes in Human Oral Mucosa and Skin: Association with Specific Cell-Cycle Phases

We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.

Effect of Vitamin C on the Availability of Tetrahydrobiopterin in Human Endothelial Cells

Vitamin C has long been known for its beneficial vascular effects, but its mechanism of action remains unclear. Recent reports suggest that vitamin C may prevent endothelial dysfunction by scavenging free radicals and increasing the bioavailability of nitric oxide. To investigate this area further, we studied the effect of vitamin C (10(-4) M) and Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP; 10(-5) M), a scavenger of superoxide, hydrogen peroxide, and peroxynitrite, on endothelial nitric oxide synthase (eNOS) enzymatic activity in cultured human umbilical vein endothelial cells. L-Citrulline formation (a measure of eNOS enzymatic activity) was significantly increased in cells treated for 24 h with vitamin C. No effect was observed after MnTBAP treatment. Chronic administration of vitamin C also had no effect on eNOS protein expression. Treatment with vitamin C for 24 h significantly increased levels of the eNOS co-factor tetrahydrobiopterin (BH4), whereas MnTBAP did not affect its levels. Sepiapterin (10(-4) M), a precursor of BH4, significantly increased eNOS activity, whereas addition of vitamin C to cells treated with sepiapterin did not cause any further increase in eNOS activity. Our results suggest that the beneficial effect of vitamin C on endothelial function is best explained by increased intracellular BH4 content and subsequent enhancement of eNOS activity. This effect appears to be independent of the ability of vitamin C to scavenge superoxide anions.

Molecular Cloning of a Divinyl Ether Synthase: IDENTIFICATION AS A CYP74 CYTOCHROME P-450

Lipoxygenase-derived fatty acid hydroperoxides are metabolized by CYP74 cytochrome P-450s to various oxylipins that play important roles in plant growth and development. Here, we report the characterization of a Lycopersicon esculentum (tomato) cDNA whose predicted amino acid sequence defines a previously unidentified P-450 subfamily (CYP74D). The recombinant protein, expressed in Escherichia coli, displayed spectral properties of a P-450. The enzyme efficiently metabolized 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid but was poorly active against the corresponding 13-hydroperoxides. Incubation of recombinant CYP74D with 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid yielded divinyl ether fatty acids (colneleic acid and colnelenic acid, respectively), which have been implicated as plant anti-fungal toxins. This represents the first identification of a cDNA encoding a divinyl ether synthase and establishment of the enzyme as a CYP74 P-450. Genomic DNA blot analysis revealed the existence of a single divinyl ether synthase gene located on chromosome one of tomato. In tomato seedlings, root tissue was the major site of both divinyl ether synthase mRNA accumulation and enzyme activity. These results indicate that developmental expression of the divinyl ether synthase gene is an important determinant of the tissue specific synthesis of divinyl ether oxylipins.

Paradoxical increase in nitric oxide synthase activity in hypercholesterolaemic rats with impaired renal function and decreased activity of nitric oxide.

We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO. Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry. Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet. Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.