Mild therapeutic hypothermia for postischemic vasoconstriction in the perfused rat liver.
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BACKGROUND: Mild hypothermia, a promising therapy being evaluated for various clinical situations, may suppress the formation of reactive oxygen species during reperfusion and may ameliorate microcirculatory perfusion failure (the "no-reflow phenomenon"). METHODS: Isolated rat livers underwent 30 min of perfusion, 2.5 h of ischemia, and 3 h of reperfusion. The temperature was maintained at 34 degrees C (mild hypothermia, n = 5) or 38 degrees C (normothermia, n = 6) for all three periods by perfusion of a modified Krebs Henseleit solution, air surface cooling, or both. A third group of livers was normothermic before and during ischemia and mildly hypothermic during reperfusion (reperfusion hypothermia, n = 6). Control livers had 3 h of perfusion at normothermia. Chemiluminescence (a measure of the generation of reactive oxygen species) and hepatic vascular resistance were monitored simultaneously to evaluate the effect of temperature on the formation of reactive oxygen species and the development of no reflow. Also measured were thiobarbituric acid reactive species and lactate dehydrogenase, as indicators of oxidative stress and cell injury. RESULTS: Mild hypothermia decreased formation of reactive oxygen species and postischemic increases in vascular resistance. Reperfusion hypothermia also decreased postischemic increases in vascular resistance, but not as effectively as did mild hypothermia. Levels of thiobarbituric acid reactive species were lower for reperfusion hypothermia than for mild hypothermia at only 0 and 30 min of reperfusion. Lactate dehydrogenase was significant only at 0 min of reperfusion for the normothermic group. Oxygen consumption did not change. CONCLUSION: The prevention of hepatic vascular injury by suppression of oxidative stress may be an important protective mechanism of mild hypothermia. (+info)
Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.
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Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs. (+info)
Structure and transcriptional regulation of the gene encoding pyruvate formate-lyase of a ruminal bacterium, Streptococcus bovis.
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The gene (pfl) encoding pyruvate formate-lyase (Pfl) from Streptococcus bovis was sequenced. The deduced amino acid sequence of Pfl was similar to Streptococcus mutans Pfl, and included the conserved regions necessary for free-radical formation and a catalytic site. The Pfl of S. bovis appeared to be a free-radical-containing enzyme because of its dioxygen sensitivity and its amino acid sequence similarity with the Escherichia coli enzyme. The pfl mRNA of S. bovis was approximately 2.3 kb and was transcribed in a monocistronic fashion. When cells were grown in batch culture at pH 6.9, the level of pfl transcript increased as the growth phase changed from exponential growth to stationary phase. This result was in constrast to the previous observation that the level of lactate dehydrogenase (Ldh) mRNA decreased during the later stages of growth. Continuous culture experiments conducted at pH 6.9 under glucose-limited and ammonia-limited conditions revealed that pfl mRNA was decreased by an excess supply of glucose, as well as by a high growth rate. On the contrary, ldh mRNA increased when excess glucose was supplied and the growth rate was high. The amount of pfl mRNA in cells was lower at pH 4.5 than pH 6.9, whereas the level of ldh mRNA was higher at pH 4.5. This result was consistent with the amounts of Pfl and Ldh in cells and the proportion of formate and lactate produced. These results support the hypothesis that S. bovis regulates Pfl and Ldh synthesis at the transcriptional level in response to growth conditions. (+info)
A randomized comparison of intra-aortic balloon pumping after primary coronary angioplasty in high risk patients with acute myocardial infarction.
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AIMS: Intra-aortic balloon pumping reduces afterload and may be effective in improving reperfusion in high risk infarct patients treated with primary angioplasty. METHODS: High risk infarct patients referred from other centres for primary PTCA were randomized to treatment with or without an intra-aortic balloon pump. The primary end-point consisted of the combination of death, non-fatal reinfarction, stroke or an ejection fraction <30% at the 6 month follow-up. A weighted unsatisfactory outcome score (as previously described by Braunwald), enzymatic infarct size and left ventricular ejection fraction were secondary end-points. RESULTS: During a 3.5 year period, 238 patients were randomized, 118 to intra-aortic balloon pump therapy and 120 to no intra-aortic balloon pump therapy. Cross-over (25% in the intra-aortic balloon pump group and 31% in the no-intra-aortic balloon pump group) occurred in both treatment arms. The primary end-point was reached in 31 (26%) patients assigned to an intra-aortic balloon pump and in 31 (26%) assigned to no intra-aortic balloon pump (P=0.94). Enzymatic infarct size (LDHQ72) was calculated in 163 (68%) patients and was not significantly different between either group (intra-aortic balloon pump: 1616+/-1148, no intra-aortic balloon pump: 1608+/-1163). The left ventricular ejection fraction was measured at the 6 month follow-up in 168 patients (80% of patients alive). No difference in ejection fraction was found in either group (intra-aortic balloon pump: 42+/-13%, no intra-aortic balloon pump: 40+/-14%, P=0.51). Major complications occurred in 8% of patients treated with an intra-aortic balloon pump. CONCLUSIONS: Systematic use of intra-aortic balloon pumping after primary angioplasty does not lead to myocardial salvage or to a better clinical outcome in high-risk infarct patients. Use of intra-aortic balloon pumping after primary PTCA for acute myocardial infarction should be reserved for patients with severe haemodynamic compromise. (+info)
Size of lipid microdroplets effects results of hepatic arterial chemotherapy with an anticancer agent in water-in-oil-in-water emulsion to hepatocellular carcinoma.
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We have initially prepared a new drug delivery system for hepatocellular carcinoma (HCC). Using sonication and a detergent, iodinated poppy seed oil (IPSO) can be mixed with an aqueous solution of epirubicin to make a water-in-oil emulsion. The water-in-oil emulsion is further passed through a microporous glass membrane and split into saline to make a long-term inseparable water-in-oil-in-water emulsion (W/O/W) that consists of IPSO microdroplets. To investigate the effect of the size of IPSO microdroplets on the efficacy of injection chemotherapy with W/O/W in patients with HCC, 32 HCC patients were randomly assigned and treated with W/O/W of small IPSO microdroplets (30 micrometers in diameter) containing 60 mg of epirubicin (n = 16, group A) or W/O/W of large IPSO microdroplets (70 micrometers) containing the same amounts of epirubicin (n = 16, group B). Effects were assessed by measuring the percentage of decline of the alpha-fetoprotein (AFP) level in a week from the AFP level immediately before the treatment. The decline was significantly larger in group B (50.5 +/- 19.8, mean +/- S.D.) compared with group A (18.9 +/- 33.1; p <.005). The size of IPSO microdroplets injected into the hepatic artery determines the decrease of serum AFP levels of the patients with HCC. (+info)
ANIT-induced disruption of biliary function in rat hepatocyte couplets.
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alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect. (+info)
Genetic vulnerability of cortical neurons isolated from stroke-prone spontaneously hypertensive rats in hypoxia and oxygen reperfusion.
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Severe hypertension and cerebrovascular diseases develop in stroke-prone spontaneously hypertensive rats (SHRSP). Cortical neurons from SHRSP are more vulnerable than those from Wistar Kyoto rats (WKY) to the effects of nitric oxide (NO)- and N-methyl-D-aspartate (NMDA)-mediated neurotoxic agents. Growth factors, idebenone, and nilvadipine (a Ca2+ channel blocker) can reduce neuronal damage caused by hypoxia or neurotoxic agents. This study was designed to determine 1) whether cortical neurons from SHRSP are more vulnerable than those from WKY and 2) whether neuronal damage is minimized by the so-called neuroprotective agents in cells exposed to hypoxia and oxygen reperfusion. We demonstrated that 6 to 24 h of hypoxia did not increase cell death in either WKY or SHRSP, whereas 36 h of hypoxia significantly increased cell death in SHRSP (p < 0.01). Furthermore, 6 to 36 h of hypoxia and 1.5 to 5 h of reperfusion heavily damaged cells from both strains of rats, and most cells became apoptotic or necrotic. We also verified that the ability to protect neurons in hypoxia and oxygen reperfusion was as follows: idebenone > insulin-like growth factor-1 (IGF-1) > nilvadipine. These data indicate that oxygen radical generation occurs and the free radicals heavily damage neurons in hypoxia and oxygen reperfusion. SHRSP neurons are weaker than WKY neurons in these conditions. Furthermore, we surmise that idebenone, an antioxidant, decreases free radicals, and IGF-I attenuates p53-mediated apoptosis and thereby prevents cell death. We conclude that antioxidants are more potent than IGF-1 in protecting cortical neurons from damage caused by hypoxia and oxygen reperfusion, although both are very useful in minimizing damage to cortical neurons. (+info)
Establishment of an activated macrophage cell line, A-THP-1, and its properties.
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A new macrophage cell line with activated character and unique morphology was isolated by selecting adherent cells from the human monocytic cell line THP-1. The original THP-1 cells had been cultured for more than 9 years using 25 cm2 flasks, when cells with a different morphology appeared, adhering to the bottoms of the culture flasks. These were selected by discarding floating nonadherent cells at every subculture. Enrichment of adherent THP-1 cells with long processes proceeded during the cultivation. These adherent THP-1 showed remarkable phenotypic changes, not only morphologically, but also functionally. Namely, increased phagocytic activity, HLA-DR expression and MLR stimulator activity were remarkable. This adherent cell line was designated as activated-THP-1 (A-THP-1), since it demonstrated characteristics of activated macrophages continuously without exogenous stimulation. A cloned A-THP-1 cell line (A-THP-1 C1) also showed the same features and contained about 10% multinucleated giant cells probably caused by cell fusion. This A-THP-1 cell line, the first activated macrophage cell line to be established, provides a good model for understanding of activation mechanisms of macrophages and multinucleation. In this paper, morphological, immunological, and biological characters of this cell line are described. (+info)