Screening and characterization of trehalose-oleate hydrolyzing lipase.

Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE. One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity. The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X. The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1). The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme. The pI of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C. Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3). High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane. The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species. Thus the enzyme was concluded to be a kind of lipase.

Opsonin-independent and -dependent Phagocytosis in the Ascidian Halocynthia roretzi: Galactose-specific Lectin and Complement C3 Function as Target-dependent Opsonins.

To clarify the molecular mechanisms of phagocytosis, we have been preparing monoclonal antibodies that inhibit phagocytosis by the hemocytes of the ascidian Halocynthia roretzi. A monoclonal antibody, RA5, inhibited the phagocytosis of non-treated sheep red blood cells (SRBCs) and yeast cells. It was demonstrated that the phagocytosis by the hemocytes was enhanced by pretreatment of target cells, SRBCs or yeast cells, with H. roretzi plasma. However, the RA5 antibody was unable to inhibit the phagocytosis of plasma-treated target cells. These results strongly suggest that the molecule recognized with the RA5 antibody is involved in the opsonin-independent phagocytosis. Western blot analysis showed that this antibody recognized a 200 kDa protein in H. roretzi hemocytes. On the other hand, flow cytometry analyses showed that a galactose-specific lectin (Gal-lectin) and complement C3 (AsC3), present in H. roretzi plasma, can bind to SRBCs and yeast cells, respectively, to enhance the phagocytosis of the respective target cells. Thus, H. roretzi hemocytes undergo opsonin-independent and -dependent phagocytosis, and Gal-lectin and AsC3 both function in the opsonin-dependent phagocytosis.

A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma.

The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.

Suppression of reperfusion arrhythmia by ischemic preconditioning in the rat: is it mediated by the adenosine receptor, prostaglandin, or bradykinin receptor?

The mechanism for the suppression of reperfusion arrhythmia by preconditioning (PC) remains unknown. This study aimed to examine the roles of the adenosine receptor, prostaglandin (PG), and bradykinin (BK) receptor in PC. Under pentobarbital anesthesia, the coronary artery of the rat was occluded for 5 min and then reperfused. In untreated controls, this protocol induced ventricular tachycardia (VT) in 100% of the rats and ventricular fibrillation (VF) in 60%. PC with 2 min ischemia/5 min reperfusion prior to the 5 min coronary occlusion significantly reduced the incidence of reperfusion VT and VF to 30% and 0%, respectively. This antiarrhythmic effect of the PC was not blocked when rats were pretreated with 8-phenyltheophylline (8-PT, 10 mg/kg), aspirin-DL-lysin (18 mg/kg), or a specific BK receptor antagonist, Hoe140 (20 nmol/kg). None of these agents alone significantly modified the incidence of reperfusion VT or VF. These results suggest that neither the adenosine receptor, endogenous PG, nor BK receptor play a major role in the mechanism of suppression of perfusion arrhythmias by PC in the rat heart.

[A case of Buerger's disease solitary involved in the left subclavian and axillary artery].

A 26-year-old male with about a ten-year history of smoking was admitted to our hospital for evaluation of ischemic symptoms including numbness, easy fatigability on exercise and lack of pulse in his left arm. His left axillary, brachial and radial pulse could not be palpated and a needle reaction was negative on physical examination. Laboratory data showed no diabetes mellitus or hyperlipidemia. C-reactive protein, Wassermann's reaction, rheumatoid reaction, anti-nuclear factor, anti-DNA antibody, hypocomplementemia and circulating immune complex were negative. Invasive arteriography using contrast medium revealed segmental occlusion with multiple collateral arteries showing a typical "corkscrew" appearance at the left subclavian artery. However, no stenotic and aneurysm-like lesions suggesting aortitis syndrome, vasculo-Beçhet disease and giant arteritis were found on the aorta or other arteries including the pulmonary artery. Although no pathological study could be carried out, the angiographic and laboratory findings strongly suggested Buerger's disease as a possible cause of solitary stenosis of the left subclavian artery in this patient.

Superoxide dismutase attenuated post-ischaemic contractile dysfunction in a myocardial xanthine oxidase deficient species.

1. We assessed the effect of polyethylene glycol conjugated superoxide dismutase (PEG-SOD) on myocardial stunning in the rabbit heart in which xanthine oxidase level is extremely low. 2. In open-chest anaesthetized rabbits, the left marginal branch of the coronary artery was occluded for 10 min and then reperfused for 30 min. A group of rabbits (PEG-SOD group) received 1000 units/kg of PED-SOD and another group (control group) was given saline 15 min before the coronary occlusion. 3. Regional systolic thickening fraction (TF) was similarly reduced to approximately -25% of baseline value during ischaemia in both groups. However recovery of TF after reperfusion was significantly better in the PEG-SOD group (n = 9) and TF at 30 min after reperfusion was 70.1 +/- 3.9% of baseline value compared with 44.9 +/- 3.4% in the control group (n = 9; P less than 0.05). Rate-pressure products, left ventricular pressure, and LV dP/dt max were not significantly different between the PEG-SOD treated and untreated control rabbits at any time during the experiment. PEG-SOD did not modify the regional myocardial blood flow (coloured microsphere method) during ischaemia/reperfusion, which was assessed by using separate groups of rabbits. 4. These findings indicate that oxygen free radicals are important in the pathogenesis of myocardial stunning in xanthine oxidase deficient hearts.

Effect of prednisolone on myocardial infarct healing: characteristics and comparison with indomethacin.

OBJECTIVE: To characterize retardation of myocardial infarct healing by corticosteroid administration, and to examine the role of suppression of prostaglandin production in its effect. DESIGN: The left circumflex coronary artery of the rabbit was occluded for 30 mins and reperfused for 72 h. Rabbits were divided into four groups: a control group, a low dose prednisolone group (L-PSL) that was treated with 5 mg/kg/24 h prednisolone, a high dose prednisolone group (H-PSL) that was treated with 10 mg/kg/24 h prednisolone, and an indomethacin group that received a 5 mg/kg intravenous bolus of indomethacin followed by 10 mg/kg/24 h. The status of infarct healing and infarcted wall thinning was assessed 72 h after ischemia by the percentage of infarct mass organized (%O/I) and the ratio of infarcted wall thickness to noninfarcted wall thickness (thinning ratio). MAIN RESULTS: The %O/I was 61.4 +/- 4.2% (mean +/- SEM) in the control group. The L-PSL and H-PSL groups had %O/Is of 48.3 +/- 3.7% and 29.1 +/- 2.1%, respectively, which were significantly lower than the control value. The difference in %O/I between the H-PSL and L-PSL groups was also significant. However, the %O/I of the indomethacin group (55.1 +/- 3.3%) was not significantly different from control. When the myocardial infarcts were retrospectively subgrouped into small infarcts (infarct volume less than 0.31 cm3) and large infarcts (greater than or equal to 0.31 cm3), infarct healing delay in large infarcts was evident only for H-PSL and not for L-PSL, while both L-PSL and H-PSL treatment retarded healing of small infarcts. No significant difference was observed in the thinning ratio for any group. CONCLUSION: Infarct healing delay by prednisolone is dosage dependent, and smaller infarcts may be more sensitive to its effect. Retardation of infarct healing by prednisolone is unlikely to be mediated by suppression of prostaglandins from the cyclo-oxygenase pathway.

Adenosine infusion during early reperfusion failed to limit myocardial infarct size in a collateral deficient species.

STUDY OBJECTIVE: Intracoronary or intravenous adenosine during reperfusion in combination with lignocaine may attenuate "reperfusion injury" and limit myocardial infarct size in the canine heart. The aim of this study was to test whether intravenous adenosine also protects myocardium in the rabbit heart, which lacks xanthine oxidase and significant coronary collaterals in contrast to the canine heart. DESIGN: Five groups of rabbits underwent a 30 min occlusion of the circumflex coronary artery, followed by reperfusion. In adenosine treated groups, either a high dose of adenosine (0.37 mg.kg-1.min-1) with lignocaine treatment (5 mg intravenously 1 min before coronary occlusion and before reperfusion) or a low dose (0.15 mg.kg-1.min-1) of adenosine with or without lignocaine was infused for 60 min starting 5 min before the onset of reperfusion. Group 1 was untreated, while group 2 received a high dose of adenosine with lignocaine. These groups were reperfused for 3 h. Group 3 was untreated, group 4 received a low dose of adenosine, and group 5 a low dose of adenosine and lignocaine. These groups were reperfused for 72 h. EXPERIMENTAL MATERIAL: 60 anaesthetised open chest rabbits were used. Groups 1 and 2 were killed after 3 h coronary reperfusion. Groups 3, 4, and 5 recovered from surgery for 72 h and were then killed for further study. MEASUREMENTS AND MAIN RESULTS: The high dose of adenosine reduced mean blood pressure to 44% of baseline value and diminished reactive hyperaemia in the area at risk by "coronary steal". The low dose of adenosine did not significantly alter systemic blood pressure or heart rate. Infarct size did not differ between groups 1 and 2, at 39.7(SD 20.1)% of area at risk v 33.2(15.9)% (by tetrazolium staining), nor between groups 3, 4, and 5: 50.3(12.6)% v 52.7(15.6)% v 47.8(9.3)% (by histology). CONCLUSION: Neither a high dose nor a low dose of adenosine limited myocardial infarct size in the rabbit heart even when adenosine was combined with lignocaine treatment.

Effects of early and later reperfusion on healing speed of experimental myocardial infarct.

The effects of reperfusion on myocardial infarct healing in the rabbit were analyzed using two morphometric indexes: the ratio of the volume of the organized portion of infarct to the volume of the whole infarct (%O/I), and the ratio of the minimal thickness of the infarct zone to the normal zone thickness (thinning ratio). In the nonreperfused infarcts, %O/I increased from 43.8 +/- 3.1% (mean +/- SEM) at 48 h to 85.7 +/- 2.5% at seven days, which supported the validity of this index. The thinning ratio was 0.50 +/- 0.03 at 48 h and did not change during the following five days. In other groups of rabbits, the coronary artery was temporarily occluded for 30 mins (early reperfusion) or 60 mins (late reperfusion), and the hearts were analyzed at 72 h or seven days. Early reperfusion limited infarct size as a percentage of the area at risk (%I/AAR) by approximately 50%, but late reperfusion did not. In both nonreperfused and reperfused infarcts, %O/I correlated significantly with absolute infarct size. Furthermore, the regression lines of the relationship of infarct size to %O/I of early and late reperfused infarcts shifted towards higher %O/I values compared with that of nonreperfused infarcts at seven days, which suggested accelerated organization. However, such a regression line shift by reperfusion was not detected at 72 h after infarction. The thinning ratio was higher in early reperfused infarcts compared to nonreperfused or late perfused infarcts, and there was a weak inverse correlation between thinning ratio and %I/AAR when the data of reperfused and nonreperfused infarcts were pooled.(ABSTRACT TRUNCATED AT 250 WORDS)