Effect of ranolazine on glycaemic control in patients with type 2 diabetes treated with either glimepiride or metformin.

AIM: To report the results of two phase III trials assessing the efficacy of ranolazine for glycaemic control in patients with type 2 diabetes on metformin or glimepiride background therapy. METHODS: In two double-blind trials we randomized 431 and 442 patients with type 2 diabetes to ranolazine 1000 mg twice daily versus placebo added to either glimepiride (glimepiride add-on study) or metformin background therapy (metformin add-on study). Patients receiving ranolazine added to metformin had their metformin dose halved (with the addition of a metformin-matched placebo) relative to the placebo group to correct for a metformin-ranolazine pharmacokinetic interaction. The primary endpoint of the trials was the change from baseline in glycated haemoglobin (HbA1c) at week 24. RESULTS: When added to glimepiride, ranolazine caused a 0.51% least squares mean [95% confidence interval (CI) 0.71, 0.32] decrease from baseline in HbA1c at 24 weeks relative to placebo and roughly doubled the proportion of patients achieving an HbA1c of <7% (27.1 vs 14.1%; p = 0.001). When added to metformin background therapy, there was no significant difference in the 24-week HbA1c change from baseline [placebo-corrected LS mean difference -0.11% (95% CI -0.31, 0.1)]. CONCLUSIONS: Compared with placebo, addition of ranolazine in patients with type 2 diabetes treated with glimepiride, but not metformin, significantly reduced HbA1c over 24 weeks. The decreased dose of metformin used in the metformin add-on study complicates the interpretation of this trial. Whether an effective regimen of ranolazine added to metformin for glycaemic control can be identified remains unclear.

Comparison of the antilipolytic effects of an A1 adenosine receptor partial agonist in normal and diabetic rats.

INTRODUCTION AND AIMS: Elevated plasma free fatty acid (FFA) concentrations play a role in the pathogenesis of type 2 diabetes (2DM). Antilipolytic agents that reduce FFA concentrations may be potentially useful in the treatment of 2DM. Our previous observation that CVT-3619 lowered plasma FFA and triglyceride concentrations in rats and enhanced insulin sensitivity in rodents with dietary-induced forms of insulin resistance suggested that it might be of use in the treatment of patients with 2DM. The present study was undertaken to compare the antilipolytic effects of CVT-3619 in normal (Sprague Dawley, SD) and Zucker diabetic fatty (ZDF) rats. RESULTS: ZDF rats had significantly higher fat pad weight, glucose, insulin and FFA concentrations than those of SD rats. EC(50) values for forskolin-stimulated FFA release from isolated adipocytes from SD and ZDF rats were 750 and 53 nM, respectively (p < 0.05). Maximal forskolin stimulation of FFA release was significantly (p < 0.01) less in ZDF rats (133 +/- 60 microM) compared with SD rats (332 +/- 38 microM). EC(50) values for isoproterenol to increase lipolysis in adipocytes from SD and ZDF rats were 2 and 7 nM respectively. Maximal isoproterenol-stimulated lipolysis was significantly (p < 0.01) lower in adipocytes from ZDF rats (179 +/- 23 microM) compared with SD rats (343 +/- 27 microM). Insulin inhibited lipolysis in adipocytes from SD rats with an IC(50) value of 30 pM, whereas adipocytes from ZDF rats were resistant to the antilipolytic actions of insulin. In contrast, IC(50) values for CVT-3619 to inhibit the release of FFA from SD and ZDF adipocytes were essentially the same (63 and 123 nM respectively). CVT-3619 inhibited lipolysis more than insulin in both SD (86 vs. 46%, p < 0.001) and ZDF (80 vs. 13%, p < 0.001) adipocytes. In in vivo experiments, CVT-3619 (5 mg/kg, PO) lowered FFA to a similar extent in both groups. Plasma concentrations of CVT-3619 were not different in SD and ZDF rats. There was no significant difference in the messenger RNA expression of the A(1) receptors relative to beta-actin expression in adipocytes from SD (0.98 +/- 0.2) and ZDF rats (0.99 +/- 0.3). CONCLUSION: The antilipolytic effects of CVT-3619 appear to be independent of insulin resistance and animal model.

Characterization of an NBTI-sensitive equilibrative nucleoside transporter in vascular smooth muscle.

Adenosine plays a significant role in various physiological and regulatory processes including coronary vasodilatation. In the current study, a high-affinity adenosine transporter in freshly dissociated porcine coronary smooth muscle (PCSM) cells and cultured human coronary smooth muscle (HCSM) cells was characterized. Kinetic analysis of the transport process revealed a V(max) of 82+/-17 pm/mg protein/min and a K(m) of 4.3+/-2.1 microm for PCSM cells, whereas a K(m) of 4.8 microm and V(max) of 254 pm/mg/min was observed for cultured HCSM. Concentration-dependent inhibition of adenosine uptake by S-(4-nitrobenzyl)-6-thioinosine (NBTI) was observed in both PCSM (IC(50), 0.08 microm) and HCSM (0.1 microm) cells. Both cell types also demonstrate a high-affinity, single binding site for NBTI (PCSM, B(max) 144.8+/-23 fmol/mg protein and K(d) 1.1+/-0.35 nm; HCSM, B(max) 672+/-62 fmol/mg protein and K(d) 0.45+/-0.14 nm). Adenosine uptake in these cells was not affected by extracellular sodium concentration. RT-PCR analysis of mRNA from individually selected PCSM and HCSM cells demonstrated expression of an NBTI-sensitive equilibrative transporter. Smooth muscle cells isolated from porcine brachial and femoral arteries also transported adenosine at levels similar to that of coronaries. These data demonstrate that vascular coronary smooth muscle possess an NBTI-sensitive equilibrative transporter for adenosine which could function in regulation of vasodilation.

Selective transport of adenosine into porcine coronary smooth muscle.

Adenosine (ADO), an endogenous regulator of coronary vascular tone, enhances vasorelaxation in the presence of nucleoside transport inhibitors such as dipyridamole. We tested the hypothesis that coronary smooth muscle (CSM) contains a high-affinity transporter for ADO. ADO-mediated relaxation of isolated large and small porcine coronary artery rings was enhanced 12-fold and 3.4-fold, respectively, by the transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI). Enhanced relaxation was independent of endothelium and was selective for ADO over synthetic analogs. Uptake of [(3)H]ADO into freshly dissociated CSM cells or endothelium-denuded rings was linear and concentration dependent. Kinetic analysis yielded a maximum uptake (V(max)) of 67 +/- 7.0 pmol. mg protein(-1). min(-1) and a Michaelis constant (K(m)) of 10. 5 +/- 5.8 microM in isolated cells and a V(max) of 5.1 +/- 0.5 pmol. min(-1). mg wet wt(-1) and a K(m) of 17.6 +/- 2.6 microM in intact rings. NBTI inhibited transport into small arteries (IC(50) = 42 nM) and cells. Analyses of extracellular space and diffusion kinetics using [(3)H]sucrose indicate the V(max) and K(m) for ADO transport are sufficient to clear a significant amount of extracellular adenosine. These data indicate CSM possess a high-affinity nucleoside transporter and that the activity of this transporter is sufficient to modulate ADO sensitivity of large and small coronary arteries.

chk-YB-1b, a Y-box binding protein activates transcription from rat alpha1(I) procollagen gene promoter.

Type-I collagen, the predominant component of extracellular matrix, is a triple-helical protein consisting of two alpha1 polypeptides and one alpha2 polypeptide. Expression of alpha1 and alpha2 procollagen genes is co-ordinately regulated under both normal and various pathological conditions. However, the basis of this co-ordinate regulation is not well known. YB-1b, a Y-box protein, has been shown to bind to the polypyrimidine tract present in the alpha2 procollagen gene. Here, we show that chk-YB-1b, a YB-1 homologue, binds in a single-strand-sequence-specific manner to the highly conserved pyrimidine-rich sequences in both alpha1(I) and alpha2(I) procollagen promoters from different species, as demonstrated by electrophoretic-mobility-shift assays and by DNaseI footprinting experiments. Transiently transfected and retrovirally expressed antisense oligonucleotides directed against chk-YB-1b specifically inhibited the alpha1(I) procollagen promoter-driven transcription in cultured fibroblasts. Considering these data and the fact that the chk-YB-1b binding site is one of the few sites between alpha1(I) and alpha2(I) procollagen promoters that is conserved from chicken to human, it is proposed that chk-YB-1b may be involved in co-ordinate expression of these two collagen genes.

Pouch tissue and angiotensin peptide generation.

Myofibroblasts and their potential to generate angiotensin (Ang) II and transforming growth factor beta 1 (TGF-beta 1) at sites of infarction in the rat heart have been implicated in tissue repair. These cells likewise contribute to repair in a subcutaneous pouch model of fibrous tissue formation. Their appearance in pouch tissue coincides with high density ACE and Ang II receptor binding, suggesting a role for Ang II in tissue repair. Using pouch tissue studied at different time points of repair, the present study examined the expression of requisite mRNA for Ang peptide generation: angiotensinogen, Ao; an aspartyl protease, either cathepsin-D, Cat-D, or renin: and angiotensin converting enzyme, ACE, TGF-beta 1 and type I collagen mRNA expression was also addressed. Unlike pouch studied on day 2 and 4, at 7, 14 and 21 days, we found: (a) expression of Ao, Cat-D but not renin, ACE and TGF-beta 1 mRNA; (b) Ang I and Ang II peptides in pouch tissue and exudate; (c) the presence of Cat-D activity but no renin activity; (d) an increase in type I collagen mRNA with time; (e) upregulation of pouch tissue ACE mRNA expression by lisinopril treatment, whereas AT1 and AT2 receptor antagonists (losartan and PD 123177, respectively) downregulated the expression of mRNA for ACE, when compared to untreated controls; (f) downregulation of TGF-beta 1 mRNA expression by lisinopril and losartan compared to untreated controls; and (g) PD 123177 had no effect, whereas lisinopril and losartan treatment significantly (P < 0.05) reduced type I collagen mRNA expression. Thus, in this model of fibrous tissue formation, we found expression of component genes involved in Ang peptide (I and II) and TGF-beta 1 generation and Ang II upregulation of TGF-beta 1 expression, suggesting Ang II and/or TGF-beta 1 may upregulate type I collagen expression during tissue repair. Pharmacologic intervention studies with lisinopril or losartan indicate Ang II plays a role in the reciprocal regulation of ACE mRNA expression, which modulates Ang II levels at sites of repair.

Inhibition of tissue repair by spironolactone: role of mineralocorticoids in fibrous tissue formation.

Mineralocorticoids have been implicated in promoting fibrous tissue formation in various organs. In the present study, we sought to address the potential contribution of mineralocorticoids to fibrous tissue formation using a skin pouch model which has proved valuable for the analysis of inflammatory and wound healing responses. Skin pouches were induced in rats by administration of a phorbol ester, croton oil (0.5 ml of a 1% solution). After 2 weeks, rats were killed and intact pouch tissue collected. Pouch weights of control and aldosterone-treated (0.75 microg/h via osmotic minipump) rats were similar (3.33 +/- 0.44 g vs. 3.70 +/- 0.28 g respectively). However, pouch weights were reduced by more than 50% in spironolactone-treated (25 mg/day powdered in food) animals (1.62 +/- 0.22 g and 1.27 +/- 0.23 g respectively in aldosterone and spironolactone alone groups). To ascertain the effects of different treatments on collagen accumulation, hydroxyproline concentration was measured. Compared with controls, hydroxyproline concentration was significantly reduced following spironolactone treatment (17.1 +/- 0.08 vs. 7.5 +/- 2.0 microg/mg dry wt, respectively, p < 0.01). This response to spironolactone was negated by coadministration of aldosterone (hydroxyproline concentration was 18.6 +/- 2.1 microg/mg dry wt). Following bilateral adrenalectomy, spironolactone reduced pouch weight and hydroxyproline concentration, which was not the case for adrenalectomy alone. Two week aldosterone administration in uninephrectomized rats on high salt diet was deemed ineffective in modulating pouch development (pouch wet wts were 3.48 +/- 0.4 g vs. 3.00 +/- 0.19 g in controls and aldosterone-treated rats, respectively). Mineralocorticoid receptor expression in pouch tissue was demonstrated by RT/PCR. Furthermore, NADP+-dependent 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity was detected in pouch tissue, together with lower levels of NAD+-dependent 11beta-HSD2. Spironolactone (p < 0.05) significantly reduced 11beta-HSD1 activity compared with controls. Thus, fibrous tissue possesses requisite components of MC action, and antagonism of mineralocorticoid receptors by spironolactone attenuates its formation. Pouch formation is under the influence of circulating MC and, we would like to propose, is also mediated through corticosteroids generated de novo at the site of tissue repair.

Identification of negative and positive regulatory elements in the rat alpha 1(I) collagen gene promoter.

Type I collagen is the main constituent of extracellular matrix found in various organs including the heart. Under some pathological conditions accumulation of excess type I collagen in the interstitium leads to organ dysfunction. In order to identify the regulatory elements in the rat alpha 1(I) collagen gene promoter, deletions were made in the promoter region. Various plasmid constructs were transfected into different fibroblasts using LipofectAMINE. The results indicated a negative cis-element between nucleotides -310 to -440 in the rat alpha 1(I) collagen gene promoter. Presence of this sequence significantly diminished the reporter gene activity. In addition we have observed that the sequence between -220 to -330 contained a positively acting cis-element, which is highly active in rat fibroblasts. Analysis of the nuclear factors binding to the negative element by electrophoretic mobility shift assays indicated that similar or identical factors are present in different fibroblasts as well as human HeLa cells and that these factors appear to bind to a composite sequence within -325 to -400. Competition with different oligonucleotides suggested that two distinct but contiguous sequence motifs may constitute the negative regulatory element. Our results with the rat alpha 1(I) collagen promoter confirm the presence of a negative cis-element previously described for the mouse promoter and provided additional information on the bipartite nature of this element.

Anti-free radical mechanisms in captopril protection against reperfusion injury in isolated rat hearts.

OBJECTIVE: Captopril, an angiotensin-converting enzyme (ACE) inhibitor, is known to modulate ischemia-reperfusion injury in the isolated hearts. This study was designed to examine the involvement of anti-free radical mechanisms in this protection. METHODS: Isolated perfused rat hearts were subjected to 60 mins of global ischemia and 30 mins of reperfusion with or without captopril (100 mumol/L). Myocardial resting tension and contractile force were recorded. At the end of reperfusion, hearts were analyzed for the activities of antioxidant enzymes, superoxide dismutase, glutathione peroxidase and catalase, as well as for the extent of lipid peroxidation. Another potent ACE inhibitor, enalapril (100 mumol/L) was used for comparison. RESULTS: Captopril significantly improved the recovery of contractile function as well as attenuated the rise in resting tension in the ischemic-reperfused hearts as compared to the control. Captopril-exposed ischemic-reperfused hearts showed an increase in the activity of superoxide dismutase with no change in glutathione peroxidase and catalase enzyme activities. Lipid peroxidation at the end of reperfusion was significantly attenuated in the captopril-exposed hearts compared to the control. Enalapril had no protective effect against ischemia-reperfusion induced contractile failure or rise in resting force. CONCLUSIONS: These results suggest that cardioprotection by captopril, against ischemia-reperfusion injury, may involve an anti-free radical mechanism independent of its ACE inhibition property.

Complete stunning in hypertrophied guinea pig heart. Defects in sarcolemmal calcium influx.

Isolated sham control as well as hypertrophied guinea pig hearts were subjected to global ischemia and reperfusion. Developed force declined to zero during 5 min of ischemia without any significant change in resting tension in both sham control and hypertrophied hearts. Upon reperfusion, control hearts showed nearly complete recovery of developed force within 20 min, whereas hypertrophied hearts during this time showed no contractile function, i.e., "a complete stunning" was observed. A continued reperfusion of the stunned hypertrophied hearts ultimately resulted in complete recovery of force within 40-60 min. Data on myocardial cation content showed a relative calcium deficiency in the stunned hearts (3.4 mumol/gm dry wt) as compared to sham control hearts (5.3 mumol/gm dry wt). Stunning could be reversed sooner by isoproterenol (100 microns), and low Na+ (35 and 60 mM) perfusion. Recovery of contractile function by low Na+ was blocked by amiloride (0.17-1.2 mM) in a dose-dependent manner. Perfusion with Bay K8644 (0.1-10 microM) as well as low (0.62 mM) and high (2.5 mM) extracellular calcium concentrations failed to reverse stunning. The pharmacological interventions that were able to reverse the stunning condition also increased the myocardial calcium content. Although the possibilities of a sarcoplasmic reticular dysfunction and/or reduced sensitivity of myofilaments are not excluded, data suggest that a defect in calcium influx across the sarcolemma is an important factor in "complete stunning." It is suggested that this "potential sarcolemmal defect" in the hypertrophied heart, which is unmasked by the ischemic stress, may also represent an early abnormality in the pathogenesis of heart failure.

Role of oxidative stress in transition of hypertrophy to heart failure.

OBJECTIVES: In an attempt to define the role of increased oxidative stress in the transition from compensatory hypertrophy to heart failure, this study examined the effects of long-term vitamin E therapy on the occurrence of heart failure subsequent to chronic pressure overload in guinea pigs. BACKGROUND: Hyperfunctional heart hypertrophy has been shown to be accompanied by an increase in the endogenous antioxidant reserve, whereas congestive heart failure is accompanied by a decrease in this reserve. The effects of vitamin E, a naturally occurring antioxidant, on the development of heart failure from a hypertrophic stage were examined. METHODS: The ascending aorta in guinea pigs was coarcted. For vitamin treatment, slow-release pellets were implanted at the time of the operation. The animals were assessed at 10 and 20 weeks for hemodynamic function, myocardial structure, antioxidant agents and oxidative stress. RESULTS: Banding of the ascending aorta in guinea pigs resulted in hyperfunctional hypertrophy at 10 weeks, which was followed by congestive heart failure at 20 weeks. Hypertrophied hearts showed decreased oxidative stress, as evidenced by a higher oxidation-reduction (redox) state and less lipid peroxidation, whereas the failure stage was characterized by increased oxidative stress. Supplementation of animals with timed-release vitamin E tablets resulted in an increased myocardial content of the vitamin, and the banded animals did not develop any signs of heart failure at 20 weeks. Hemodynamic function at 20 weeks in these vitamin E-treated animals was also better maintained. The myocardial reduced glutathione/oxidized glutathione ratio of vitamin E-treated animals at 20 weeks was higher and lipid peroxidation was less compared with the untreated animals. Ultrastructural abnormalities were significantly less in the vitamin E-treated hearts compared with the untreated failing hearts at 20 weeks. CONCLUSIONS: An improved myocardial redox state with vitamin E therapy, coupled with the modulation of the development of heart failure, may indicate a pathophysiologic role for increased oxidative stress in the pathogenesis of heart failure. This study suggests the potential therapeutic value of long-term antioxidant treatment in modulating or preventing the pathogenesis of heart failure.

Crystallization and preliminary crystallographic analysis of E. coli uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase in two new crystal forms.

Uridine 5'-diphospho-N-acetylenolpyruvylglucosamine reductase (MurB), the second enzyme in the peptidoglycan synthetic pathway of Escherichia coli, has been crystallized in two previously unreported forms, one orthorhombic and the other monoclinic. MurB (molecular mass 38 kDa) crystallizes in a range of conditions that utilize polyethylene glycol fractions as precipitants, and crystals can be grown with or without the enzyme's substrate, uridine 5'-diphospho-N-acetylenolpyruvylglucosamine. X-ray diffraction from crystals of the orthorhombic form extends to 2 A resolution and shows the symmetry and systematic absences of space group P2(1)2(1)2(1). These crystals show significant variations in cell dimensions at room temperature and at 100 K. A crystal used to collect a 2.0 A resolution data set at a synchrotron source showed cell dimensions at ca 100 K of a = 51.0, b = 79.3 and c = 87.1 A, indicating one molecule peroasymmetric unit. The monoclinic crystals scatter X-rays to 3.0 A resolution consistent with space group P2(1), unit-cell dimensions (ca 100 K) a = 50.7, b = 92.4, c = 85.5 A, and beta = 104 degrees, and two molecules per asymmetric unit. Mercury derivatives have been prepared with both orthorhombic and monoclinic forms, and efforts are underway to exploit these derivatives to determine the structure of this protein.

Oxidative stress and heart failure.

Various abnormalities have been implicated in the transition of hypertrophy to heart failure but the exact mechanism is still unknown. Thus heart failure subsequent to hypertrophy remains a major clinical problem. Recently, oxidative stress has been suggested to play a critical role in the pathogenesis of heart failure. Here we describe antioxidant changes as well as their significance during hypertrophy and heart failure stages. Heart hypertrophy in rats and guinea pigs, in response to pressure overload, is associated with an increase in 'antioxidant reserve' and a decrease in oxidative stress. Hypertrophied rat hearts show increased tolerance for different oxidative stress conditions such as those imposed by free radicals, hypoxia-reoxygenation and ischemia-reperfusion. On the other hand, heart failure under acute as well as chronic conditions is associated with reduced antioxidant reserve and increased oxidative stress. The latter may have a causal role as suggested by the protection seen with antioxidant treatment in acute as well as in chronic heart failure. It is becoming increasingly apparent that, anytime the available antioxidant reserve in the cell becomes inadequate, myocardial dysfunction is imminent.

Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.

Antioxidant changes in hypertrophied and failing guinea pig hearts.

Hypertrophy and heart failure were induced by placing a mildly constrictive band around the ascending aorta in young guinea pigs. Based on heart weight, left ventricular wall thickness, hemodynamic data, and other clinical signs, these animals were found to have physiological hypertrophy at 10 wk and congestive heart failure (CHF) at 20 wk. Hearts from these two groups of animals were examined for superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase activities as well as lipid peroxidation and glutathione [reduced glutathione (GSH)/oxidized glutathione (GSSG)] levels. There was an age-dependent increase in SOD activity and GSH content in sham controls. SOD activity was 28% higher in the 10-wk-hypertrophy group and 46% lower in the CHF group than in respective sham controls. GSHPx activity increased significantly in the hypertrophied hearts, whereas in the failing hearts, the activity was not different from the 20-wk controls but was significantly lower than in the hypertrophied hearts. Catalase activity did not change at either stage. GSH content in the hypertrophied hearts was significantly higher compared with sham controls. In the CHF group, GSH content was significantly lower and GSSG content was significantly higher than in sham controls. Lipid peroxidation, as indicated by malondialdehyde content, was significantly decreased in the hypertrophy group but increased toward control levels in the failure group. It is proposed that a relative deficit in myocardial antioxidant capacity as well as in the redox state may play a role in the pathogenesis of cardiac failure.

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions.

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during various in vivo as well as in vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.

A study of Mycobacterium tuberculosis antigen and antibody in cerebrospinal fluid and blood in tuberculous meningitis.

Radioimmunoassay (RIA) techniques have been evaluated to detect specific tubercular antigen (TB Ag) and antitubercular antibody (TB Ab) in CSF and serum of patients with tuberculous meningitis (TBM). A solid-phase RIA using H37RV sonicate antigen of Mycobacterium tuberculosis, anti-BCG antibody, and staphylococcal protein A was standardized. TB Ag and TB Ab levels were noted to be significantly elevated in cerebrospinal fluid (CSF) as well in circulating immune complexes (CIC) isolated from serum samples of TBM patients as compared to control group (P less than 0.01). Detectability of disease by demonstrating elevated TB Ag and/or TB Ab levels in either CSF or CIC or both was 95%. There was no correlation between individual levels of TB Ag and TB Ab in CSF and in circulation. A follow-up study in patient over a period of 4-12 weeks revealed that TB antigen and/or TB Ab persisted in the majority of the cases for several weeks despite chemotherapy.