Survey of the prevalence of Salmonella species on laying hen farms in Kosovo.

A survey on the prevalence of Salmonella (S) species was carried out on 39 layer farms in Kosovo between April and September 2012. In total 367 samples, comprising feces, dust, eggs, and internal organs from dead birds, were investigated using bacteriological culture methods. Additionally, data on the location of the farm, the total number of birds on the farm, age of birds, and laying performance were collected. Salmonella were isolated from 38 samples obtained from 19 (49%) farms. The most common serovar identified was Salmonella enteritidis, found on 18 farms. The most common S. enteritidis phage type was PT29 followed by PT6, PT7, PT21, PT13a, PT8, PT14b, and PT4. One S. enteritidis isolate was not typable. Six farms had more than one phage type. Furthermore, serovar S. Bovismorbificans also was found in samples from 3 farms. Flock size or production stage was not associated with the probability of isolating Salmonella. The only flock factor found to be significantly associated was percent hen/day production: It was 2.8 times more likely to isolate Salmonella from flocks with production above 80% hen/day production compared to flocks producing at a lower level. Analysis of antimicrobial resistance patterns of 30 isolates revealed that all isolates were sensitive to gentamicin, ampicillin, sulphamethoxazole trimethoprim, and oxytetracycline, and 29 (97%) were sensitive to ciprofloxacin. All isolates showed intermediate resistance or were resistant to minocycline and cloxacillin. Twenty-six isolates (86%) had intermediate resistance to amoxicillin and 27 isolates (90%) were fully resistant to streptomycin. The present survey revealed a high prevalence of Salmonella enteritidis in layer flocks in Kosovo, indicating that table eggs have to be suspected as an important source of human salmone-llosis.

A web-based pedometer programme in women with a recent history of gestational diabetes.

AIMS: Women with remote histories of gestational diabetes mellitus can reduce their diabetes risk through lifestyle changes, but the effectiveness of interventions in women with more recent histories of gestational diabetes has not been reported. Therefore, we conducted a pilot study of a low-intensity web-based pedometer programme targeting glucose intolerance among women with recent gestational diabetes. METHODS: Women with a gestational diabetes delivery within the past 3 years were randomized to a 13-week intervention consisting of a structured web-based pedometer programme which gave personalized steps-per-week goals, pedometers and education regarding lifestyle modification, or to a letter about diabetes risk reduction and screening after delivery for gestational diabetes (control condition). The main outcome measures were change in fasting plasma glucose and 2-h glucose levels on a 75-g oral glucose tolerance test between baseline and 13-week follow-up. Weight was a secondary outcome and behavioural constructs (self-efficacy, social support, risk perception) were also assessed. RESULTS: Forty-nine women were enrolled. At 13-week follow-up, women randomized to the intervention did not have significant changes in behavioural constructs, physical activity or anthropometrics compared with women in the control group. Changes in fasting plasma glucose (-0.046 mmol/l vs. 0.038 mmol/l, P = 0.65), 2-h glucose values (-0.48 mmol/l vs. -0.42 mmol/l, P = 0.91) and weight (-0.14 kg vs. -1.5 kg, P = 0.13) were similar between the control and intervention groups, respectively. CONCLUSIONS: Structured web-based education utilizing pedometers is feasible although uptake may be low. Such programmes may need to be supplemented with additional measures in order to be effective for reduction of diabetes risk.

Utility of long-term surveillance endomyocardial biopsy: a multi-institutional analysis.

BACKGROUND: The utility of long-term endomyocardial biopsy surveillance in heart transplant recipients has been questioned. This study was undertaken to identify risk factors for late rejection and to examine the impact of different biopsy surveillance protocols on outcomes using the registry of the Cardiac Transplant Research Database. METHODS: The study group consisted of all adult patients who underwent heart transplantation at the 33 centers participating in this investigation between January 1, 1993 and January 1, 2002, survived past the second post-transplant year, and were followed-up by a defined surveillance biopsy protocol. RESULTS: During a follow-up that consisted of 24,137 patient-years, 1,626 late rejections occurred. Shorter time since transplant, history of rejection, younger age and African-American ethnicity of the recipient were strong risk factors for late rejection. The practice of surveillance biopsy varied among institutions. Continued surveillance increased the rate of diagnosis of late rejection (RR = 1.3, p = 0.002). There was no reduction in the incidence of hemodynamically compromising rejection and no increase in survival in patients with long-term vs intermediate-term surveillance. Short-term surveillance was associated with an increased incidence of hemodynamically compromising rejection, particularly among high-risk patients, and increased mortality in African-American patients. CONCLUSIONS: There are no apparent benefits from surveillance biopsy beyond 5 years post-transplant. Surveillance biopsy between 2 and 5 years post-transplant was found to reduce mortality in African-American recipients. Non-African-American recipients at high risk for late rejection will likely benefit from surveillance up to 5 years post-transplant.

YY-1 and c-Jun transcription factors participate in the repression of the human involucrin promoter.

The human involucrin gene, which encodes a precursor of the keratinocyte cornified layer, is strongly expressed in response to differentiation stimuli. Earlier studies suggested that YY1 and components of the AP-1 family might participate in the silencing of involucrin in proliferating keratinocytes. This study shows that overexpression of either YY1 or c-Jun represses transcription of the human involucrin gene in multiplying keratinocytes. Transient overexpression and site-directed mutagenesis experiments of the involucrin 5'-non-coding region (5'-NCR) confirmed that YY1 and c-Jun repress involucrin transcription. This repression involves the distal zinc fingers of YY1 protein and the DNA binding and leucine zipper domains of c-Jun. The results with protein pull-down experiments are consistent with the hypothesis that interaction of YY1 with c-Jun is an important mechanism for involucrin repression. Cotransfection of YY1 modified the stimulatory function of mutant c-Jun proteins independently of their DNA binding capacity suggesting that interactions may be more complex in vivo. Additionally, c-Jun protein levels are affected by differentiation stimuli indicating the importance of c-Jun in the YY1 repression pathway. Thus YY1 and c-Jun have an important role in epidermal differentiation by negatively regulating the human involucrin gene.

Antisense activity detection by inhibition of fluorescence resonance energy transfer.

Use of antisense nucleic acids to modulate expression of particular genes is a promising approach to the therapy of human papillomavirus type 16 (HPV-16)-associated cervical cancer. Understandably, evaluation of the in vivo performance of synthetic antisense oligodeoxynucleotides (AS-ODNs) or ribozymes is of ultimate importance to development of effective antisense tools. Here we report the use of a bacterial reporter system based on the inhibition of fluorescence resonance energy transfer (FRET) to measure the interaction of AS-ODNs with HPV-16 target nt 410-445, using variants of the green fluorescent protein (GFP). An optimal FRET-producing pair was selected with GFP as the donor and yellow fluorescent protein (YFP) as the acceptor molecule. Hybridization of AS-ODNs with a chimaeric mRNA containing the antisense target site flanked by GFP variants resulted in the inhibition of the FRET effect. Use of different linkers suggested that the amino acid content of the linker has no significant effect on FRET effect. Antisense accessibility, tested by RNaseH assays with phosphorothioated target-specific and mutant AS-ODNs, suggested a specific effect on the chimaeric mRNA. FRET inhibition measurements correlated with the presence of truncated proteins confirming true antisense activity over the target. Therefore, FRET inhibition may be used for the direct measurement of AS-ODNs activity in vivo.

Gene transfer of heat-shock protein 70 reduces infarct size in vivo after ischemia/reperfusion in the rabbit heart.

BACKGROUND: Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. METHODS AND RESULTS: Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5x10(9) pfu, 50 microL/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5+/-2.8 in Ad.HSP70-injected hearts compared with 41.9+/-2.8 and 42.7+/-2.5 in the vehicle- and Ad.LacZ-treated hearts (P<0.01). The infarct size was not different between the vehicle- and Ad.LacZ-treated hearts (P>0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1+/-5.2, 47.7+/-3.5, and 53.3+/-2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (P>0.05). CONCLUSIONS: Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart.

Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning.

We investigated the role of tyrosine kinase (TK) signaling in the opening of the ATP-sensitive K(+) (K(ATP)) channel and 72-kDa heat shock protein (HSP72) expression during late preconditioning. Rabbits were subjected to surgical operation (sham) or were preconditioned (PC) with four cycles of 5 min of ischemia and 10 min of reperfusion. Twenty-four hours later, animals were subjected to 30 min of ischemia and 180 min of reperfusion. Genistein (1 mg/kg ip) was used to block the receptor TK. Six groups were studied: control, sham, genistein-sham, PC, genistein-PC, and vehicle-PC group (1% dimethyl sulfoxide). Genistein or vehicle was given 30 min before the surgical procedure. Genistein pretreatment decreased the expression of HSP72 in PC hearts and suppressed action potential duration shortening during ischemia in sham and PC groups. Infarct size (%risk area) was reduced in the PC (11.6 +/- 1.0%) and vehicle-PC (19.3 +/- 2.0%) compared with the control (40.0 +/- 3.8%) or sham (46.0 +/- 2.0%) groups (P < 0.05). Genistein pretreatment increased infarct size to 46.4 +/- 4.1% in the PC hearts. We conclude that TK signaling is involved in K(ATP) channel opening and HSP72 expression during late PC.

Myocardial ischemia/reperfusion injury in the inducible nitric oxide synthase knockout mice.

Inducible nitric oxide synthase (iNOS) plays an important role in the inflammatory process of certain major cardiac disorders including myocardial infarction and allograft rejection. However, the role of iNOS in acute myocardial ischemia has not been well defined. We determined the effects of genetically disruption of the intact iNOS system on cardiac tolerance to ischemia/reperfusion injury. Adult male wild-type (WT) and iNOS knockout (KO) B6,129 mice were subjected to 20 min global ischemia and 30 min reperfusion in a Langendorff isolated perfused heart model (37 degrees C, n = 10/each group). Ventricular contractile function, heart rate, coronary flow, and leakage of intracellular enzymes (CK and LDH) were not significantly different between the groups during pre-ischemia as well as reperfusion period (P > 0.05). Myocardial infarct size was also not significantly different between WT (20.2+/-2.0% of risk area) and KO mice (23.5+/-3.8%; Mean+/-SEM, P > 0.05). However, the post-ischemic heart rate was significantly preserved in KO as compared to WT (P < 0.05). We conclude that disruption of iNOS gene does not exacerbate ischemia/ reperfusion injury in the heart.

Essential role of inducible nitric oxide synthase in monophosphoryl lipid A-induced late cardioprotection: evidence from pharmacological inhibition and gene knockout mice.

BACKGROUND: Monophosphoryl lipid A (MLA), a nontoxic analogue of endotoxin, is a pharmacological agent that is known to have anti-ischemic effects. Mechanisms involved with the cardioprotection are still unclear. A role for inducible nitric oxide synthase (iNOS) was recently proposed. We tested this hypothesis using S-methylisothiourea (SMT), one of the specific pharmacological inhibitors of iNOS, as well as iNOS gene knockout mice. METHODS AND RESULTS: Adult male ICR or B6,129 mice were pretreated with either MLA 35 or 350 microg/kg IP (MLA35 or MLA350) or vehicle 24 hours before global ischemia/reperfusion, which was carried out in a Langendorff isolated perfused heart model (n=8 to 9 per group). Another group of MLA350 mice received SMT 3 mg/kg IP 30 minutes before heart perfusion. Ventricular contractile function and heart rate were not different between the groups during the preischemia and reperfusion periods (P>0.05). Preischemic basal coronary flow was significantly increased in all MLA350 but not MLA35 mice. Myocardial infarct size was reduced significantly, from 26.9+/-2.9% of risk area in vehicle-treated mice to 13.5+/-2.4% in the MLA350 group (mean+/-SEM, P<0.05). This reduction in infarct size was accompanied by augmented nitrite/nitrate accumulation, from 0.23+/-0. 05 nmol/mg protein in the vehicle group to 0.97+/-0.27 nmol/mg protein in MLA350 mice (P<0.01). Infarct size increased significantly, to 22.2+/-2.8% after treatment with SMT in the MLA350 group. Furthermore, MLA350 failed to reduce infarct size in iNOS knockout mice (25.5+/-3.6%). CONCLUSIONS: These results demonstrate a direct association of infarct size reduction with increased NO production with MLA350. An obligatory role for iNOS in mediating the cardioprotective effect induced by MLA was confirmed with the pharmacological inhibition and gene knockout mice.

Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of post-ischemic ventricular function.

Genetically engineered mice provide an excellent tool to study the role of a particular gene in biological systems and will be increasingly used as models to understand the signal transduction mechanisms involved in ischemic preconditioning (IP). However, the phenomenon of IP has not been well characterized in this species. We therefore attempted to examine whether IP could protect isolated mouse heart against global ischemia/reperfusion (GI/R) injury. Thirty adult mice hearts were perfused at constant pressure of 55 mmHg in Langendorff mode. Following 20 min equilibration, the hearts were randomized into three groups (n = 10/each): (1) Control Group; (2) IP2.5 Group: IP with two cycles of 2.5 min GI + 2.5 min R; (3) IP5 Group: IP with 5 min GI + 5 min R. All hearts were then subjected to 20 min of GI and 30 min R (37 degrees C). Ventricular developed force was measured by a force transducer attached to the apex. Leakage of CK and LDH was measured in coronary efflux. Infarct size was determined by tetrazolium staining. Following sustained GI/R, infarct size was significantly reduced in IP2.5 (13.8+/-2.3%), but not in IP5 (20.1+/-4.0%), when compared with non-preconditioned control (23.6+/-3.8%) hearts. CK & LDH release was also reduced in both IP2.5 and IP5 groups. No significant improvement in post-ischemic ventricular contractile function was observed in either IP groups. We conclude that IP with repetitive cycles of brief GI/R is able to reduce myocardial infarct size and intracellular enzyme leakage caused by a sustained GI/R in the isolated perfused mouse heart. This anti-necrosis cardioprotection induced by IP was not associated with the amelioration of post-ischemic ventricular dysfunction.

Whole body heat shock fails to protect mouse heart against ischemia/reperfusion injury: role of 72 kDa heat shock protein and antioxidant enzymes.

The transgenic mice overexpressing heat shock protein 72 (HSP72) or antioxidants have been reported to be more resistant to myocardial ischemia/reperfusion injury. However, it remains unknown whether whole body heat stress (HS) which may induce HSP72 or endogenous antioxidants affords similar protection in the mouse heart. Adult male mice were treated with either HS (42 degrees C for 15 min) or anesthesia only (SC) against a group of non-stressed controls (NC). At 6 or 24 h later, the hearts were excised and perfused at a constant pressure of 55 mmHg in Langendorff mode. Following 30 min equilibration, hearts were subjected to 20 min of global ischemia and 30 min reperfusion (37 degrees C). Ventricular force was measured by a force-displacement transducer attached to the apex. Leakage of intracellular enzymes (CK, LDH) was measured in coronary efflux. Infarct size was determined by tetrazolium staining. The results showed that no significant differences between HS, SC, and NC groups in ventricular contractile function, CK and LDH release, or infarct size were observed at either time window. HS enhanced the expression of HSP72 in mouse hearts by two- to three-fold, whereas antioxidant enzyme activities (catalase and MnSOD) did not change significantly. We conclude that HS does not precondition the isolated perfused mice hearts against ischemia/reperfusion injury, despite induction of HSP72.

Heat shock provides delayed protection against oxidative injury in cultured human umbilical vein endothelial cells.

During both mild and severe ischemia, vascular endothelial cells lining large and small vessels of the ischemic organ are exposed to oxygen-derived free radicals resulting in oxidative damage to the organ. Heat shock has been shown to induce thermotolerance and also protect against ischemic injury, possibly via increased synthesis of heat shock proteins (HSPs). We hypothesized that heat shock preconditioning may protect human endothelial cells against oxidative damage. Cultured human umbilical vein endothelial cells (HUVEC) were subjected to heat shock (42 degrees C, 1 h) and allowed to recover for 2 or 20 h, at which times the cells were oxidatively stressed for 1 h by exposing them to 100-200 mumol/l of hydrogen peroxide (H2O2). Cellular damage was assessed immediately and 18 h later by morphology and release of lactate dehydrogenase (LDH). No protection of HUVEC was seen using the 2-hour recovery interval, but a significant protection (P < 0.05) was observed after the 20-hour delay. Northern blot analysis at 1 and 2 h after heating showed induction of HSP-70 mRNA. Western blot analysis demonstrated a significant increase in HSP-72 protein after 2 as well as 20 h of recovery from heat shock, although the amounts of protein at the two times were not significantly different. Furthermore, no differences in the activity of the antioxidant enzyme catalase were observed between heated and unheated HUVEC at 2 and 20 h after heat preconditioning. Thus, heat shock preconditioning induces delayed protection against oxidative injury in HUVEC, and the mechanism of protection appears to involve more than the expression of HSP-72 or activity of catalase.

Delayed preconditioning of cultured adult rat cardiac myocytes: role of 70- and 90-kDa heat stress proteins.

We investigated the protective effect of heat stress and metabolic preconditioning in cultured adult rat cardiac myocytes and correlated this effect with induction of heat shock proteins (HSP). Myocytes were preconditioned with sublethal heat shock or metabolic preconditioning for 30 min. Twenty hours later, preconditioned myocytes were subjected to lethal heat shock (46 degrees C for 2 h) or ischemia by incubation in ischemic buffer for 2 h. Cellular injury index was reduced from 69 +/- 4.0% in lethally heat-shocked cells to 27.0 +/- 1.6% with heat shock preconditioning (mean +/- SE; P < 0.01) and 19.0 +/- 3.0% with metabolic preconditioning (P < 0.01). Cellular injury index was 81.0 +/- 1.0% in ischemic myocytes and was reduced to 25.9 +/- 2.7 and 21.4 +/- 2.6% in heat shock- and metabolic-preconditioned myocytes, respectively (P < 0.01). A significant cross-tolerance of myocytes against lethal injury was observed with the two preconditioning methods. Western blot analysis revealed 3.3- and 2.5-fold increases in HSP 90 and 500- and 15-fold increases in HSP 70 with heat shock and metabolic preconditioning, respectively. HSP 27 expression remained unaltered relative to control cells. We conclude that heat shock and metabolic preconditioning induce delayed tolerance against lethal injuries in adult cardiac myocytes with elevated levels of HSP 70 and HSP 90.

Milrinone: basic and clinical pharmacology and acute and chronic management.

Milrinone (Inocor-Sanofi-Winthrop) represents a second generation phosphodiesterase inhibitor currently approved for intravenous administration in the treatment of decompensated congestive heart failure. By inhibiting Type III phosphodiesterase, milrinone increases intracellular cyclic adenosine monophosphate. This results in a positive inotropic effect on the heart and vasodilatation in the periphery. The hemodynamic consequences of this action produce left ventricular afterload reduction, with an increase in cardiac output and a reduction in total peripheral resistance. Unlike the sympathomimetic amines, milrinone produces no tolerance and possesses the distinct advantage of directly decreasing pulmonary vascular resistance. Short-term intermittent infusion by peripheral administration, continuous infusion, long-term therapy, and intermittent outpatient therapy was demonstrated to be safe, efficacious, and cost effective. It is hypothesized that intravenous milrinone, by producing biventricular afterload reduction, offers an efficacious, cost-effective tool for the treatment of decompensated heart failure.

Dehydroepiandrosterone inhibits human platelet aggregation in vitro and in vivo.

The hypothesis has been advanced that the adrenal steroids dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) exert antiatherogenic and cardioprotective actions. Platelet activation has also been implicated in atherogenesis. To determine if DHEA and DHEAS affect platelet activation, the effects of these steroids on platelet aggregation were assessed both in vitro and in vivo. When DHEAS was added to pooled platelet-rich plasma before the addition of the agonist arachidonate, either the rate of platelet aggregation was slowed or aggregation was completely inhibited. Inhibition of platelet aggregation by DHEA was both dose- and time-dependent. Inhibition of platelet aggregation by DHEA was accompanied by reduced platelet thromboxane B2 (TxB2) production. Inhibition of platelet aggregation by DHEA was also demonstrated in vivo. In a randomized, double-blind trial, 10 normal men received either DHEA 300 mg (n = 5) or placebo capsule (n = 5) orally three times daily for 14 days. In one man in the DHEA group arachidonate-stimulated platelet aggregation was inhibited completely during DHEA administration, whereas in three other men in the DHEA group the rate of platelet aggregation was prolonged, and the sensitivity and responsiveness to agonist were reduced. None of the men in the placebo group manifested any change in platelet activity. These findings suggest that DHEA retards platelet aggregation in humans. Inhibition of platelet activity by DHEA may contribute to the putative antiatherogenic and cardioprotective effects of DHEA.

Inotropic therapy for the failing myocardium.

Congestive heart failure (CHF) is a common clinical entity with diverse causes. Patients may present with acute decompensation or have a more indolent course, with diminishing exercise tolerance and increasing dyspnea. The management of this clinical entity traditionally has focused on restricting fluid intake, decreasing dietary sodium, decreasing afterload with vasodilatory agents, and supporting the failing myocardium with agents which produce a positive inotropic response. In the acutely decompensated patient, short-term therapy with positive inotropics is clearly beneficial. The role of long-term inotropic therapy for chronic CHF remains less clear. A number of clinical trials have recently evaluated the effects of long-term therapy on morbidity and mortality, with disappointing results. For a number of the newer, nonglycocide oral positive inotropics, at doses of drug which produce measurable hemodynamic improvement, increased mortality in treatment groups has been unacceptably high. Ironically, patients with worse left ventricular dysfunction show the most clinical improvement, but have the highest increased mortality. However, as with digoxin, there is some evidence that employing lower doses of drug that do not produce measurable improvement in hemodynamic parameters may result in both improved clinical state and decreased mortality. This review discusses the role of both oral and intravenous inotropic agents, discusses the difficulty in translating short-term hemodynamic improvement into long-term clinical benefit, and presents a rationale for the use of lower-dose inotropic therapy to improve long-term clinical outcome.

Free radicals, calcium homeostasis, heat shock proteins, and myocardial stunning.

Repeated brief ischemic episodes result in prolonged depression of contractile function despite the absence of irreversible damage, a phenomenon called myocardial stunning. Considerable evidence exists to suggest that oxygen radicals, particularly the hydroxyl radical formed as a result of Fenton reaction or nitric oxide-peroxynitrite pathway, may contribute to the pathogenesis of myocardial stunning. The generation of free radicals may cause sarcoplasmic reticulum dysfunction, and both of these mechanisms may lead to calcium overload, which in turn could exacerbate the damage initiated by oxygen radicals. Antioxidant therapy has been shown to effectively attenuate or even prevent the development of prolonged depression of contractility in many studies. In addition, preconditioning with brief ischemic insults is able to trigger protection, which appears to attenuate stunning 24 to 48 hours later. The mechanism of this protection is not known, although one or more members of the heat shock protein family may have a role in protection against stunning.

Sustained inhibition of nitric oxide by NG-nitro-L-arginine improves myocardial function following ischemia/reperfusion in isolated perfused rat heart.

It has been postulated that nitric oxide (NO) can react with superoxide anion (.O2-) to generate hydroxyl (.OH) radical. If this is correct, inhibition of NO synthesis could attenuate .OH radical mediated ischemia/reperfusion injury. Therefore we studied the effects of NG-nitro-L-arginine (L-NNA), a competitive inhibitor of the NO synthase enzyme on ischemia/reperfusion injury injury in isolated perfused rat hearts. Three groups of rats (n = 12-15) were studied. Group I: Untreated ischemia/reperfusion control (37.5 min of global ischemia followed by 20 min reperfusion); Group II: ischemia/reperfusion with 25 microM NG-nitro-L-arginine; and Group III: ischemia/reperfusion in the presence of L-NNA and 2 mM L-arginine, the substrate for NO synthase. Coronary flow (in ml/min) and ventricular developed pressure, +dP/dt and -dP/dt were measured 5 min prior to ischemia and at the end of reperfusion. Baseline preischemic developed pressure was significantly lower in L-NNA perfused hearts than controls (76.8 +/- 5.9 v 97.6 +/- 2.9 mmHg, P < 0.05). However, the developed pressure following reperfusion was significantly greater in L-NNA perfused hearts (57.4 +/- 7.4 v 20.8 +/- 6.4 mmHg in control). This protective effect was reversed by the addition of L-arginine. Preischemic coronary flow was decreased significantly in the L-NNA group (6.4 +/- 0.5 ml/min) compared to controls (11.6 +/- 0.7 ml/min). The duration of sinus rhythm was significantly improved from 3.8 +/- 1.2 min in controls to 15.1 +/- 0.8 min in L-NNA perfused hearts. A corresponding significantly lower incidence of arrhythmias was observed (10.2 +/- 1.5 in ischemia/reperfusion group v 1.7 +/- 0.8 min with L-NNA).(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidant stress increases heat shock protein 70 mRNA in isolated perfused rat heart.

Ischemia/reperfusion (I/R) and preconditioning of the heart by coronary artery occlusions increase expression of heat shock protein 70 (HSP 70). Because free radicals are generated during I/R, we hypothesized that the oxidant stress might contribute to an increased expression of HSP 70. Isolated rat hearts were perfused with free radical-generating systems such as xanthine/xanthine oxidase (X/XO), irradiated rose bengal (RB) generating singlet oxygen, and H2O2 for 15 min followed by 30 min of recovery period. Significant decrease in developed pressure and coronary flow occurred after perfusion with X/XO, H2O2, and RB. During I/R, the developed pressure and coronary flow were 60 +/- 8 and 80 +/- 5%, respectively, of control, which improved significantly with superoxide dismutase. The expression of HSP 70 mRNA increased over 13-fold in hearts perfused with X/XO, 6- to 7-fold with RB, and over 5-fold with H2O2. With I/R, an over 10-fold increase in HSP 70 mRNA was observed, which decreased significantly in the presence of superoxide dismutase. These results demonstrate that oxidant stress directly increases HSP 70 mRNA in the rat heart. It is concluded that one of the potential mechanisms of expression of HSP 70 by I/R may be oxygen radicals.