A nonconserved carboxy-terminal segment of GroEL contributes to reaction temperature.

The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 degrees C, but also at 10 degrees C, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 degrees C and 12 degrees C respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

Induction of specific stress response increases resistance of rat liver allografts to cold ischemia and reperfusion injury.

Heme oxygenase-1 (HO-1) has been shown to increase cellular resistance against oxidative injury, but the functional significance of this is currently obscure. We investigated the protective role of HO-1, induced by tin-protoporphyrin IX (SnPP), in attenuating liver transplantation injury. Lewis rats were intraperitoneally treated with saline as control, 50 micro mol/kg of SnPP, or 2 mg/kg of cycloheximide (CHX) before SnPP injection. Gene expression of HO-1 was induced after either treatment with SnPP- or CHX + SnPP instead of saline, whereas HO-1 protein synthesis was enhanced in Kupffer-like dendritic cells of the SnPP-treated group. Following reperfusion of liver grafts preserved for 30 h, there were fewer intercellular adhesion molecule-1-positive cells in SnPP-treated livers, significantly reduced numbers of dead cells, and enhanced graft viability. The present data suggest that increased synthesis of HO-1 protein by SnPP pre-conditioning is linked to the improved liver graft viability through inhibition of inflammatory adhesion molecules.

Pyrrolidine dithiocarbamate provides protection against hypothermic preservation and transplantation injury in the rat liver: the role of heme oxygenase-1.

BACKGROUND: Pyrrolidine dithiocarbamate (PDTC) represents a class of antioxidants and is a potent inducer of the heme oxygenase-1 (HO-1) gene and an inhibitor of nuclear factor-kappa B (NF-kappa B). We examined the impact of PDTC preconditioning against cold ischemia and reperfusion injury in the rat liver. METHODS: Lewis rats were treated subcutaneously with saline or PDTC solution 24 hours before harvesting. Some animals pretreated with PDTC were also given zinc protoporphyrin IX intravenously immediately after reperfusion. HO-1 expression and enzyme activity in liver tissues were analyzed at different time points after each treatment. After transplantation of 24-hour preserved livers, serum levels of transaminases and gene expression of tumor necrosis factor-alpha, interleukin-1 beta, and NF-kappa B were measured. Animal survival and cellular viability were monitored. RESULTS: HO-1 gene expression and protein synthesis were enhanced in PDTC-treated livers, leading to increased enzyme activity (P <.05). The PDTC treatment group showed lower transaminase levels (P <.05), lower cytokine and NF-kappa B messenger RNA expression (P <.05), and fewer nonviable cells (P <.05) than did the control group, whereas these PDTC effects were abolished with zinc protoporphyrin injection after reperfusion (P <.05). The best animal survival rate was observed in the PDTC group (P <.05). CONCLUSION: PDTC preconditioning reduces inflammatory responses during reperfusion. PDTC appears to exert this protective effect by induction of an antioxidative stress protein and inhibition of proinflammatory cytokines.

Preconditioning with tin-protoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney.

BACKGROUND: Heme oxygenase (HO)-1 is induced as a unique stress response and leads to a transient resistance against oxidative damage, including ischemia and reperfusion (I/R) injury. In the present study, we examined whether HO-1 induction may confer a protection against I/R injury in the rat kidney. METHODS: Lewis rats were divided into four groups as follows: (1) vehicle group; (2) group treated with ferri-protoporphyrin IX (hemin), an inducer of HO; (3) group treated with low-dose tin-protoporphyrin IX (SnPP), an inhibitor of HO; and (4) group treated with high-dose SnPP. Renal warm ischemia for 60 minutes was performed 24 hours after each treatment. RESULTS: At 24 hours after treatment, hemin induced a significant increase in renal HO activity, but failed to induce HO-1 protein synthesis. Although both low- and high-dose SnPP reduced HO activity, a marked HO-1 expression was observed only in the high-dose SnPP-treated kidney. Hemin exacerbated the renal function after reperfusion, while high-dose SnPP significantly suppressed the intercellular adhesion molecule (ICAM)-1 expression, the infiltration of ED-1-positive macrophages and the expression of activated caspase-3, which resulted in attenuation of apoptotic cell death and ameliorated I/R injury. CONCLUSION: These results suggest that prior induction of HO-1 protein by high-dose SnPP may lead to anti-inflammatory and antiapoptotic effects on warm renal I/R injury independently of its enzyme activity, and that HO enzyme activation may not always act as an antioxidant, especially under I/R-induced oxidative stress.

Donor-specific blood transfusion prolongs cardiac allograft survival in rats by low nitric oxide production and elevated serum levels of prostaglandin E(2).

Donor-specific blood transfusion (DST) improves allograft survival, although the exact mechanism(s) is not completely understood. The purpose of this study was to determine the role of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in DST-protective effects, using a rat cardiac transplantation model. Lewis rats (RT-1(1)) were transfused with fully allogeneic ACI rats (RT-l(a)) blood (DST group) or Lewis syngeneic blood (ST) through the portal vein 7 days prior to allogeneic cardiac transplantation. Posttransplantation, aminoguanidine (AG)-treated rat received continuous intravenous infusion of AG, an inhibitor of inducible nitric oxide synthase (iNOS). The survival times of grafted hearts were 5.7+/-0.5, 6.8+/-0.8 and 9.2+/-1.2 days in ST, ST/AG, and DST groups (n=6 for each group), respectively. Inhibition of iNOS in ST/AG group prolonged allograft survival, but it was shorter than that in DST rats. Serum nitrite/nitrate levels on postgrafting day 5 were significantly higher in ST than in ST/AG and DST groups, but were similar in ST/AG and DST groups. These results were confirmed by immunohistochemical staining with anti-iNOS antibody. Serum PGE(2) concentrations in DST rats were significantly higher than in ST and ST/AG groups. Our results suggest that DST prolongs graft survival by enhanced production of PGE(2) with a resultant suppression of immune activation. In addition, DST-induced prolongation of graft survival may be partially mediated by NO suppression.