Synthase Activity Levels Research Articles

Glucosylceramide synthase activity and ceramide levels are modulated during cerebral ischemia after ischemic preconditioning.

After 24-hour middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats, brain ceramide level increased from baseline reached 595% (ischemic core) and 460% (perifocal/penumbral areas); brain glucosylceramide synthase (GCS) activities in these areas simultaneously decreased by 70% and 50%, respectively. Ten-minute MCAO preconditioning significantly attenuated 24-hour MCAO-induced ceramide accumulation by 40% to 60% in ischemic core and perifocal areas, and GCS activities improved by 60% to 70% in both areas. Thus, potentially toxic levels of brain ceramide induced by MCAO were attenuated to intermediate levels in preconditioned animals; brain GCS activity was relatively preserved. In ischemic tolerance, GCS appears to modulate otherwise high levels of brain ceramide.

Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet.

A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the.NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular.NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, l-arginine, and l-citrulline were also evaluated in homogenates. Superoxide anion (O(2)(-).) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%);.NO production increased by 90%, cGMP levels increased by 100%, and l-arginine decreased by 30%. No change in aortic O(2)(-). production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.

The role of renal natriuretic and depressor systems in insulin-resistant hypertensive rats.

Insulin resistance and impairment of the renal depressor system have been thought to be involved in the development of essential hypertension. However, the relationship between insulin resistance and this system is still unclear. To clarify this relationship, we investigated the role of the renal depressor system in a rat model of insulin-resistant hypertension. Sprague-Dawley rats were fed a standard diet (control) or a fructose-rich diet (FFR), and their blood pressures were measured every week. Urinary dopamine (uDA), urinary kallikrein (uKAL) activity and urinary nitric oxide (uNOx) levels were also measured each week, and the renal mRNA expression levels of endothelial nitric oxide synthase (eNOS), aromatic-L-amino-acid decarboxylase (AADC), and kallikrein (KAL) activity were compared at the end of the study. The blood pressure of FFR was elevated significantly from 2 weeks after the start of fructose loading. The uDA level was lower in FFR than in control rats throughout the study period (p<0.01), and the expression level of AADC mRNA was enhanced in FFR (p<0.05). There was a tendency of negative correlation between uDA level and systolic blood pressure (SBP) (r=-0.49, p=0.056). uNOx level was lower in FFR throughout the study period (p<0.05), and the eNOS mRNA expression level in the kidney was lower in FFR than in control rats (p<0.05). There was a negative correlation between uNOx level and SBP (r=-0.68, p <0.01). On the other hand, there was no significant difference in the kallikrein-kinin system between FFR and control rats. In conclusion, impairment in functions of the renal dopamine and NO systems occur in FFR, and this impairment may be caused by insulin resistance and may contribute to the development of hypertension.

Loss of virulence in Ustilago maydis by Umchs6 gene disruption

A gene encoding a sixth chitin synthase ( Umchs6, sequence GenBank accession No. AF030554) from the plant pathogenic hemibasidiomycete Ustilago maydis (DC.) Cda. was isolated and characterized. The predicted protein is 1103 amino acids in length with a calculated molecular mass of 123.5 kDa. a2b2 null mutants were obtained by substitution of a central fragment of the Umchs6 gene with the hygromycin resistance cassette, and a1b1 null mutants were obtained by genetic recombination in plants of an a2b2 Δ ch6 and a wild-type a1b1 strain. The mutation had no effect on the dimorphic transition in vitro or on mating, and growth rate of the mutants was only slightly reduced. On the other hand, they displayed important alterations in cell morphology, particularly at the mycelial stage, and in the staining pattern with calcofluor white. Levels of chitin synthase activity in vitro and chitin content were reduced. The most noticeable characteristic of the mutants was their almost complete loss of virulence to maize ( Zea mays L.). This was a recessive character. Microscopic observations during the infectious process suggest that chitin synthase 6 activity is very important for growth of the fungus into the plant. Transformation of a2b2 Δ ch6 mutants with an autonomous replicating plasmid carrying the full Umchs6 gene restored their normal morphological phenotype and virulence. These results are evidence that the mutation in the Umchs6 gene was solely responsible for the phenotypic alterations observed.

Changes in sodium, potassium-ATPase induced by repeated fencamfamine: the roles of cyclic AMP-dependent protein kinase and the nitric oxide–cyclic GMP pathway

Fencamfamine (FCF) is an indirect dopamine agent with effects similar to amphetamine and cocaine. In the present study, we investigate changes in Na,K-ATPase, cyclic AMP-dependent protein kinase (PKA) and nitric oxide synthase (NOS) activity and cyclic GMP levels in the nucleus accumbens (NAc) and striatum (ST) of animals acutely or repeatedly treated with FCF (3.5 mg/kg). Na,K-ATPase had a similar activity in control and repeatedly treated animals, but was reduced in the NAc of the acute group. This enzyme was reduced in the ST in acute and repeatedly treated animals, compared to the control group. Expression of the α 1,2,3-Na,K-ATPase isoforms in the NAc and the ST was not altered in all groups studied. Acute FCF induced a significant increase in PKA activity in both the ST and the NAc. Repeatedly treated animals showed a higher increase in PKA activity in the NAc, but not in the ST, when compared to the acute group. There was also an increase in both NOS activity and cyclic GMP levels only in the NAc of FCF repeatedly treated animals compared to the acute and control groups. We suggest that chronic FCF treatment is linked to a modification in Na,K-ATPase activity through the PKA and NO–cyclic GMP pathway.

Nitric oxide and cyclic nucleotides are regulators of neuronal migration in an insect embryo.

The dynamic regulation of nitric oxide synthase (NOS) activity and cGMP levels suggests a functional role in the development of nervous systems. We report evidence for a key role of the NO/cGMP signalling cascade on migration of postmitotic neurons in the enteric nervous system of the embryonic grasshopper. During embryonic development, a population of enteric neurons migrates several hundred micrometers on the surface of the midgut. These midgut neurons (MG neurons) exhibit nitric oxide-induced cGMP-immunoreactivity coinciding with the migratory phase. Using a histochemical marker for NOS, we identified potential sources of NO in subsets of the midgut cells below the migrating MG neurons. Pharmacological inhibition of endogenous NOS, soluble guanylyl cyclase (sGC) and protein kinase G (PKG) activity in whole embryo culture significantly blocks MG neuron migration. This pharmacological inhibition can be rescued by supplementing with protoporphyrin IX free acid, an activator of sGC, and membrane-permeant cGMP, indicating that NO/cGMP signalling is essential for MG neuron migration. Conversely, the stimulation of the cAMP/protein kinase A signalling cascade results in an inhibition of cell migration. Activation of either the cGMP or the cAMP cascade influences the cellular distribution of F-actin in neuronal somata in a complementary fashion. The cytochemical stainings and experimental manipulations of cyclic nucleotide levels provide clear evidence that NO/cGMP/PKG signalling is permissive for MG neuron migration, whereas the cAMP/PKA cascade may be a negative regulator. These findings reveal an accessible invertebrate model in which the role of the NO and cyclic nucleotide signalling in neuronal migration can be analyzed in a natural setting.

Enzymatic control of the accumulation of verbascose in pea seeds

ABSTRACTVerbascose, the pentasaccharide of the raffinose family of oligosaccharides, consists of galactose units joined to sucrose. In pea (Pisum sativum) seeds, the content of verbascose is highly variable. In a previous study on a high‐verbascose pea cultivar, the present authors have demonstrated that verbascose is synthesized by a multifunctional stachyose synthase (EC 2.4.1.67), which utilizes raffinose as well as stachyose as a galactosyl acceptor. Herein the results of a study of the cloning and functional expression of stachyose synthase from the low‐verbascose genotype SD1 are reported and it is demonstrated that this line contains a protein with a reduced ability to synthesize verbascose. Analysis of seeds from seven pea lines revealed a positive correlation between verbascose synthase activity and verbascose content. Among these genotypes, only the SD1 line showed low verbascose synthase activity when the data were normalized to stachyose synthase activity. These results suggest that differences in the level of verbascose synthase activity could be caused by mutations in the stachyose synthase gene as well as by variation in the amount of the protein. The lines were also analysed for activity of α‐galactosidase, a catabolic enzyme that could limit the extent of verbascose accumulation. No relationship was found between α‐galactosidase activity and the amount of raffinose family oligosaccharides.

Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts.

We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts. Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts. A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group. We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Fat feeding impairs glycogen synthase activity in mice without effects on its gene expression

To examine whether the effects of high-fat feeding on glycogen synthase (GS) activity and mRNA levels differ between diabetes-prone (C57BL/6J) and diabetes-resistant mice (NMRI), we measured GS activity and mRNA levels in muscle from C57BL/6J and NMRI mice fed a high-fat or normal chow diet for 3, 6, and 15months. As compared with chow feeding, fat feeding increased plasma insulin levels in C57BL/6J mice at 15 months (464 ± 29 v 267 ± 47 pmol/L, P =.005), which was associated with elevated plasma glucose levels at 15 months (5.3 ± 0.3 v 3.8 ± 0.2 mmol/L, P =.001). Fat feeding increased plasma insulin levels also in NMRI mice at 15 months (705 ± 145 v 275 ± 64 pmol/L, P =.01) without, however, a rise of plasma glucose levels. In parallel with increased insulin levels, decreased muscle GS fractional velocity (FV) was observed at 6 (49.0% ± 2.6% v 69.1% ± 7.3%, P =.04) and 15 (45.8% ± 1.8% v 53.4% ± 1.6 %, P <.01) months but not at 3 months in the fat-fed C57BL/6J mice. Similarly, there was a significant decrease in GS fractional activity at 3 (57.9% ± 4.3% v 70.4% ± 2.6 %, P <.03) and 15 (47.3% ± 2.4% v 56.4% ± 2.1%, P =.02) but not at 6 months in the fat-fed NMRI mice. The decrease in GS activity was not associated with changes in mRNA levels at any time points. We conclude that (1) fat feeding results in similar elevation of plasma insulin levels and impairs GS activity in C57BL/6J and NMRI mice, and (2) the changes in GS activity do not involve effects on gene expression. © 2003 Elsevier Inc. All rights reserved.

The N-acetylglutamate synthase/N-acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesis.

In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869-42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.

Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.

Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R.

Melanin-concentrating hormone, hippocampal nitric oxide levels and memory retention

The present study attempts to determine, if the effect of melanin-concentrating hormone (MCH) upon memory retention is correlated with changes in nitric oxide synthase (NOS) activity and tissue levels of nitric oxide (NO) and cGMP. We used a behavioral experiment using a step-down inhibitory avoidance test, the biochemical determinations of NO and cGMP, and electrophysiological model. Results of behavioral studies (step-down test) showed that MCH administration reverts the amnesic effects induced by N G-nitro- l-arginine ( l-NOArg). Moreover, electrophysiological studies demonstrated that l-NOArg did not block the potentiation induced by the peptide. Hippocampal NO and cGMP levels increased after MCH injection.

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck.

Numerous reports have documented a direct involvement of matrix metalloproteinase (MMP) overexpression in the development and progression of head and neck squamous cell carcinoma (HNSCC). In this study, the authors examined whether the expression of MMPs in HNSCC is correlated with other steps involved in tumor growth and metastasis, like angiogenesis, activation the nitric oxide (NO) pathway, and alteration of the p53 tumor suppressor gene. MMP-1, MMP-2, and MMP-9 expression levels were examined immunohistochemically in samples from 43 patients with HNSCC. Microvessel density (MVD) was determined by immunostaining of endothelial cells with anti-CD31 monoclonal antibody. Inducible nitric oxide synthase (iNOS) activity and cyclic guanosine monophosphatate (cGMP) levels were assessed in fresh tumor samples, whereas exons 5-9 of the p53 gene were analyzed by reverse transcriptase-polymerase chain reaction, single-strand conformation polymorphism analysis and were sequenced. MMP-1 overexpression (>10% of tumor cells) was identified in 32 tumors (74.5%), whereas elevated levels of MMP-2 and MMP-9 were detected in 17 tumors (39.5%) each. Tumors with MMP-9 overexpression were characterized by significantly higher MVD (P = 0.05) and significantly higher iNOS activity and cGMP levels (P = 0.005 and P = 0.02, respectively). Moreover, p53 mutation was associated strongly with MMP-9 overexpression (P = 0.004). Conversely, no correlation was found between MMP-1 and MMP-2 expression, angiogenesis, iNOS activity, cGMP levels, and p53 mutation in this series. This study documents the existence of a correlation between MMP-9 expression, activity of the iNOS pathway, p53 status, and angiogenesis in patients with HNSCC. This raises the possibility that p53 mutation, which frequently is present in HNSCC, may result in increased angiogenesis and invasiveness related to increased nitric oxide and MMP production by tumor cells, ultimately contributing to tumor progression.

Selective endothelin receptor blockade reverses mitochondrial dysfunction in canine heart failure

Objective: Mitochondrial enzymatic activity reductions in both myocardial and skeletal muscle tissues have been reported in a canine model of pacing-induced congestive heart failure (CHF). Endothelin-1 (ET-1), a vasoconstrictor peptide with diverse biological properties, has been implicated in CHF pathogenesis, and ET-1 receptor blockade has been shown to attenuate CHF progression. We hypothesized that the beneficial effect of ET-1 receptor blockade may be mediated in part by improved mitochondrial function. Methods: Myocardium and skeletal muscle tissues were evaluated for respiratory complex I-V and citrate synthase activity levels in paced animals treated with and without LU 135252, a specific type A ET-1 receptor (ETA) antagonist. Results: Specific activity levels of complex V and III, which were 65% to 85% lower in both cardiac and skeletal muscle in paced compared to unpaced animals, were significantly increased in ETA antagonist–treated animals (50%-300% compared to untreated paced animals). Levels of other mitochondrial respiratory complex activities including complex I, II, and IV as well as citrate synthase were not significantly changed. Conclusions: These findings suggest that endothelin activation may be involved in the myocardial dysfunction and mitochondrial enzyme deficiencies observed in pacing-induced CHF. Improvement of mitochondrial function may be a novel mechanism mediating the beneficial effect of ETA receptor blockade in CHF.

Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3 protein synthesis in transgenic potato plants.

14-3-3 proteins are abundant eukaryotic proteins that interact with many other proteins, thereby modulating their function and thus cell metabolism. The data from mRNA analysis confirm the developmental regulation of 14-3-3 isoform expression in potato plants. In order to test whether or not 14-3-3 protein expression affects plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pepo 14-3-3 or underexpressing endogenous 14-3-3 isoforms were analysed. An increase in tuber number and a decrease in tuber size in the overexpressed transformant was observed; the transgenic plants contain more chlorophyll than the control and they lose it more slowly than the control when transferred to the dark. The 14-3-3-repressed transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of the transgenic tubers was also detected. The increased catecholamine level was accompanied by an increased ratio of soluble sugars to starch in overexpressed transformant. The opposite effect was detected in 14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation of NR occurs in vivo, and is not isoform-dependent. The increase in NR activity resulted in a significant decrease in nitrate level. The level of sucrose phosphate synthase activity was also significantly increased in all 14-3-3-underexpressed transgenes, and remarkably the increase in enzyme activity was accompanied by respective changes in sucrose levels in the tubers. The most intriguing finding was the significant (2-3-fold) increase in ethylene content in all the 14-3-3-repressed transgenic lines, which probably resulted from a methionine level increase. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analysed transgenic plants.

Molecular and biochemical characterization of the Neurospora crassa glycogen synthase encoded by the gsn cDNA.

Glycogen synthases catalyze the transfer of a glucosyl moiety from a nucleotide phosphosugar to a nascent glycogen chain via an alpha1-->4 linkage. Although many genes coding for glycogen synthases have been described, the enzymes from rabbit and yeast are the best characterized. The fungus Neurospora crassa accumulates glycogen during exponential growth, and mobilizes it at the onset of stationary phase, or when placed at high temperature or starved for carbon. Through a PCR methodology, the gsn cDNA coding for the N. crassa glycogen synthase was isolated, and the amino acid sequence of the protein was deduced. The product of the cDNA seems to be the only glycogen synthase present in N. crassa. Characterization of the gsn cDNA revealed that it codes for a 706-amino acids protein, which is very similar to mammalian and yeast glycogen synthases. Gene expression increased during exponential growth, reaching its maximal level at the end of the exponential growth phase, which is consistent with the pattern of glycogen synthase activity and glycogen level. Expression of the gsn is highly regulated at the transcriptional level. Under culture conditions that induce heat shock, conidiation, and carbon starvation, expression of the gsn gene was decreased, and glycogen synthase activity and glycogen content behaved similarly.

Slow component of [V]O(2) kinetics: the effect of training status, fibre type, UCP3 mRNA and citrate synthase activity.

In healthy individuals performing constant-load exercise at intensities above the lactate threshold a secondary rise in pulmonary oxygen uptake ([V]O(2)) occurs. [V]O(2) reaches a maximum and exhaustion rapidly prevails for a range of work rates lower than the maximal work rate achieved during a conventional rapid-incremental test. This phenomenon is called the slow component (SC) of [V]O(2) kinetics and represents an increase in [V]O(2) without an increase in work rate. Although still under debate, the magnitude of the SC is believed to be associated with the percentage of type II muscle fibres and their recruitment. In this study we investigated the relationship between the magnitude of the relative SC, citrate synthase activity, UCP2 and UCP3 mRNA levels and muscle fibre composition in both endurance-trained and recreationally active subjects. The magnitude of the relative SC was measured in 12 endurance-trained (Tr) and 15 recreationally active (RA) male subjects. The magnitude of the relative SC was determined as the difference between the end-exercise [V]O(2) and 3 min [V]O(2) divided by the difference between end-exercise [V]O(2) and baseline [V]O(2). UCP2 and UCP3 mRNA expression in the vastus lateralis was measured by RT-PCR with beta-actin mRNA used as an internal control. These values were also normalized against cytochrome-b mRNA to control for training induced changes in mitochondria when comparing the Tr and RA groups. Type I, IIa and IIx skeletal muscle fibre composition was determined using a routine myosin ATPase histochemical staining technique. Citrate synthase (CS) activity was measured using spectrophotometric detection. The magnitude of the relative SC of the Tr group had the highest correlation with citrate synthase activity (r=-0.90, P<0.001) and that of the RA group with [V]O(2) peak (r=-0.68, P<0.01). For the Tr group other correlations with the magnitude of the relative SC included UCP3 mRNA levels (r=0.69, P<0.05) and the percentage of type I fibres (r=-0.58, P<0.05), while for the RA group they included UCP3 mRNA (r=0.58, P<0.05) and the percentage of type IIa muscle fibres (r=0.59, P<0.05). The Tr subjects had a lower relative SC (P=0.04) and a lower expression of UCP2 (P=0.04), and UCP3 mRNA (P=0.01) than the RA subjects. When the groups were combined the magnitude of the relative SC correlated with UCP3 mRNA (r=0.70, P<0.01), percentage of type IIa muscle fibres (r=0.60, P<0.01) and [V]O(2) peak (r=-0.73, P<0.01). Additionally UCP3 mRNA correlated with the percentage of type IIa muscle fibres (r=0.63, P<0.001). Citrate synthase activity and [V]O(2) peak are indicators of aerobic fitness. The high negative correlations between the magnitude of the relative SC and citrate synthase activity and [V]O(2) peak, of the Tr and RA subjects, respectively, suggests that the magnitude of the relative SC is inversely correlated with aerobic fitness. Additionally the correlations between UCP3 mRNA and the magnitude of the relative SC for both groups individually and combined suggest that the uncoupling activity of the UCP3 protein may also influence the magnitude of the relative SC.

High Levels of Nitric Oxide Synthase Activity are Associated with Nasal Polyp Tissue from Aspirin-sensitive Asthmatics

The pathogenesis of aspirin intolerance remains unclear. Inducible nitric oxide synthase (iNOS) expression is upregulated in nasal polyp epithelium, implying a role for nitric oxide (NO) in its formation. We decided to compare iNOS activity in polyp tissue from patients with and without aspirin intolerance. Nasal polyp tissue was collected from 15 patients undergoing routine nasal polypectomy. These patients were classified into three groups: Group A comprised patients with nasal polyps without asthma; Group B contained patients with nasal polyps and asthma; and Group C comprised patients with nasal polyps, asthma and aspirin sensitivity. All subjects in Group C had a history of aspirin-induced reaction and a confirmatory intranasal challenge with lysine-aspirin. NOS activity was measured by the ability of tissue homogenates to convert 3,4-L-arginine to L-citrulline in an L-N G -nitro-L-arginine-inhibitable fashion. The iNOS activity (picomoles) in polyp tissue from the 3 groups was: A, 248.72 - 220.79; B, 23.71 - 41.06; and C, 549.71 - 132.11. Thus, nasal polyps from patients with Samter's triad had a significantly higher iNOS activity ( p = 0.004; one-way ANOVA). This finding does not correlate simply with disease severity or with the occurrence of asthma and could indicate another important facet of aspirin-induced airways disease.

Expression of monolysocardiolipin acyltransferase activity is regulated in concert with the level of cardiolipin and cardiolipin biosynthesis in the mammalian heart

BackgroundMonolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation.ResultsIn rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls.ConclusionWe propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart.

Analysis of the Raffinose Family Oligosaccharide Pathway in Pea Seeds with Contrasting Carbohydrate Composition

Raffinose family oligosaccharides (RFOs) are synthesized by a set of galactosyltransferases, which sequentially add galactose units from galactinol to sucrose. The accumulation of RFOs was studied in maturing seeds of two pea (Pisum sativum) lines with contrasting RFO composition. Seeds of the line SD1 accumulated stachyose as the predominant RFO, whereas verbascose, the next higher homolog of stachyose, was almost absent. In seeds of the line RRRbRb, a high level of verbascose was accumulated alongside with stachyose. The increase in verbascose in developing RRRbRb seeds was associated with galactinol-dependent verbascose synthase activity. In addition, a galactinol-independent enzyme activity was detected, which catalyzed transfer of a galactose residue from one stachyose molecule to another. The two enzyme activities synthesizing verbascose showed an optimum at pH 7.0. Both activities were almost undetectable in SD1. Maximum activity of stachyose synthase was about 4-fold higher in RRRbRb compared with SD1, whereas the activities of galactinol synthase and raffinose synthase were only about 1.5-fold higher in RRRbRb. The levels of galactinol synthase and stachyose synthase activity were reflected by steady-state levels of corresponding mRNAs. We suggest that the accumulation of verbascose in RRRbRb was controlled by a coordinated up-regulation of the last steps of verbascose biosynthesis.