Microbial population dynamics in a thermophilic methane digester fed with garbage.

The diversity of microbial communities in three full-scale thermophilic anaerobic digesters which treated garbage, sewage sludge and livestock wastes (hereafter called TGD, TSD and TLD, respectively) was investigated using 16S rDNA clone libraries in triplicate. The population dynamics of TGD were also studied. The purposes were to show the microbial diversity in each reactor and to suggest which key microbes in a thermophilic methane digester fed with garbage, including a check of reproducibility and the suggestion of an error range in this molecular biology method. 736 clones were identified, and the maximum error was estimated to be around +/-10% for the same OTU (operational taxonomic unit) and for most detected OTUs. The most frequently detected OTU shows a close relationship to Uncultured bacterium clone MBA08, Unidentified bacterium clone TUG22 and Uncultured archaeal symbiont PA204 in TGD, TSD and TLD, respectively. The microbial population dynamics in TGD were studied over a period of 90 days, and the occupying ratios of Bacillus infernus and Methanothermobacter wolfeii were shown to change with the change in VFA concentration. From the dynamic change and characteristics of the microbes, it is concluded that Bacillus infernus and Methanothermobacter wolfeii played an important role and were recommended as key microbes in TGD.

Plasma homovanillic acid, plasma anti-D1 and -D2 dopamine-receptor activity, and negative symptoms in chronically mediated schizophrenia.

We have investigated the relationship between the concentration of homovanillic acid in human plasma (pHVA) and plasma anti-D1 and anti-D2 dopamine receptor activity in chronic schizophrenic patients whose neuroleptic dosage was changed. The change in pHVA level correlated with that in anti-D1, not anti-D2 activity, thus suggesting that the neuroleptic-induced changes in pHVA concentration may be associated with the blocking of D1- as well as D2- receptors. The change of scores on the Scale for the Assessment of Negative Symptoms did not significantly correlate with changes in anti-D1 or anti-D2 activity, but did so correlated with the change in pHVA level.

Comprehensive site-directed mutagenesis of L-2-halo acid dehalogenase to probe catalytic amino acid residues.

L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration. Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis. The roles of these residues are discussed.

Stimulatory effect of dibutyryl cyclic GMP on acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells.

We previously reported that endogenous nitric oxide (NO) is involved in the peripheral control of gastric acid secretion induced by some secretagogues, and that endogenous NO is involved in the acid secretion process via histamine release from histamine-containing cells. However, the stimulus-secretion coupling in the cells remains to be clarified. In the present study, we investigated the effect of dibutyryl cyclic GMP on gastric acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells, in comparison with those of dibutyryl cyclic AMP. Dibutyryl cyclic GMP (300 microM) produced a slight but significant increase of gastric acid secretion, which was completely inhibited by the histamine-H2 receptor antagonist famotidine. In contrast, dibutyryl cyclic GMP (1 mM) markedly inhibited histamine-induced acid secretion. Dibutyryl cyclic AMP (100 microM) produced a sustained increase of gastric acid secretion. The pretreatment with famotidine partially inhibited dibutyryl cyclic AMP-induced gastric acid secretion. Dibutyryl cyclic GMP and dibutyryl cyclic AMP significantly increased the histamine release from gastric mucosal cells. These results suggest that both intracellular cyclic GMP and cyclic AMP act as second messengers for histamine release in the histamine-containing cells, probably ECL cells. On the other hand, in gastric parietal cells, cyclic AMP has a stimulatory effect on gastric acid secretion, whereas cyclic GMP has an inhibitory effect.

Beta3-adrenoceptors mediate relaxation of guinea pig taenia caecum by BRL37344A and BRL35135A.

Beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum was investigated by studying the effects of the beta3-adrenoceptor agonists, BRL37344A [(R*,R*)-(+/-)-4-[2'-[2-hydroxy-2-(3-chlorophenyl) ethylamino] propyl] phenoxyacetic acid sodium salt sesquihydrate] and BRL35135A [(R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl) ethylamine] propyl] phenoxyacetate hydrobromide]. BRL37344A and BRL35135A caused dose-dependent relaxation of the guinea pig taenia caecum. The concentration-response curves for BRL37344A and BRL35135A were unaffected by propranolol, ICI118551 [erythro-1-(7-methylindan-4-yloxy)-3-(isopropylamine)-but an-2-ol], atenolol, butoxamine, prazosin, yohimbine and phentolamine. Bupranolol produced shifts of the concentration-response curves for BRL37344A and BRL35135A. Schild regression analyses carried out for bupranolol against BRL37344A and BRL35135A gave pA2 values of 5.79 and 5.84, respectively. These results suggest that the relaxant response to BRL37344A and BRL35135A of the guinea pig taenia caecum is mediated by beta3-adrenoceptors.

The first open study of zonisamide, a novel anticonvulsant, shows efficacy in mania.

1. Zonisamide, an anticonvulsant developed in Japan, is structurally similar to serotonin. Zonisamide has been proven to have a pharmacological profile that is very similar to that of carbamazepine. Thus, the effect of zonisamide was examined in 24 psychiatric patients: 15 with bipolar manic state, 6 with schizoaffective manic state, and 3 schizophrenic excitement. 2. Approximately 25% of all the patients and 33% of the bipolar manic patients showed remarkable global improvement with the addition of zonisamide. Approximately 71% of all the patients and 80% of the bipolar group had more than moderate global improvement. 3. No serious adverse reactions were found and no patients required zonisamide withdrawal. One patient developed both leukocytosis and mildly abnormal liver function test. One developed leukocytosis and another reported mild sleepiness. These reactions disappeared when zonisamide was discontinued.

Severe hypertriglyceridemia with plasma inhibitory factor(s) on lipoprotein lipase activity in a patient with a common Ser(447)-Ter LPL mutation.

Severe hypertriglyceridemia is a major risk for acute pancreatitis. So far, several mutations on the lipoprotein lipase (LPL) gene causing type I hyperlipidemia have been identified. However, the common mutation Ser(447)-Ter has been recently proposed to have a lowering effect on serum triglyceride concentrations in the general population. In this study, we analyzed blood from a patient suffering from severe hypertriglyceridemia and pancreatitis with the mutation on the lipoprotein lipase gene, Ser(447)-Ter. The patient's plasma showed inhibitory effects on the LPL activities from normal subjects. The bottom fraction separated by ultracentrifugation revealed enhanced effects as an inhibitory factor. The inhibitory effect observed in the bottom fraction was dose-dependent, stable at treatment of 65 degrees C for 30 min, and decreased significantly after being dialyzed using membranes with a cut-off molecular weight of 3500 or 6000 Da. The inhibitory effect was significantly higher when the post-heparin plasma was used from the patient or a subject with the same LPL mutation as an LPL source, compared to that from normal subjects. These results suggest that the patient has inhibitory factors in his plasma. Such inhibitory factors might cause severe hypertriglyceridemia in a case with the common mutation, which has been proposed to show the lowing effect on serum triglyceride concentrations in the general population.

Determination of bioconcentration potential of tetrachloroethylene in marine algae by 13C.

The use of stable isotope of organic-carbon, organic-13C, as a tracer for the determination of the concentration of tetrachloroethylene (PCE), CA, in Heterosigma akashiwo and Skeletonema costatum was examined. CA determined by the 13C and GC methods showed good agreement with each other. This suggests that it is reasonable and reliable to determine the bioconcentration potential of PCE in marine algae. Fitting values of bioconcentration potential parameters, including uptake rate constant k1, elimination rate constant k2 and bioconcentration factor on the basis of dry weight BCFD, were done not only to the time course for PCE uptake by the algae with the bioconcentration model, but also to experimental data for "percent inhibition(%) approximately exposure concentration of PCE approximately time" with the combined bioconcentration and probability model. The values obtained from the bioconcentration model were consistent with those from the combined bioconcentration and probability model. With the parameters (such as k1, k2, growth rate constant kG, critical concentration of HOCs in the organism resulting in growth inhibition CA* and spread factor S) the variability in toxicity (such as EC10, EC50, EC70) can be estimated from the combined bioconcentration and probability model, which fits well with the experimental observations.

[Experimental study on cell damage of ischemic liver in obstructive jaundiced rats].

The hepatic cellular damage induced by liver ischemia was investigated in rats with obstructive jaundice. Hepatic tissue blood flow in obstructive jaundice was decreased in the relation to the duration of jaundice. The value of lipid peroxide and cathepsin D activity of the hepatic tissue increased in the obstructive jaundice. Therefore, it was suggested that cell membrane and lysosomal membrane injury were induced in obstructive jaundice. The value of lipid peroxide of hepatic tissue in obstructive jaundice was more increased after partial liver ischemia. The survival rate following hepatic ischemia in jaundiced rats was remarkably lower than that of normal rats, and also it related to the duration of jaundice. In addition, histological changes of the liver after partial ischemia are severe in obstructive jaundiced liver. These data suggest that more remarkable hepatic cellular damage than in normal liver may be induced by liver ischemia in obstructive jaundice.

[Hepatic functional evaluation using the galactose tolerance test in patients with obstructive jaundice].

Hepatic functional mass was evaluated in patients with obstructive jaundice using the galactose tolerance test (GaTT), which reflected cytosolic function of hepatocyte. The T-1/2 values as an index on the GaTT were significantly prolonged in patients with obstructive jaundice in comparison with control subjects whether before or after percutaneous transhepatic biliary drainage (PTBD). But in each cases, some showed nearly normal GaTT-T/2 value and others showed severely prolonged value. Patients with obstructive jaundice could be divided into two groups according to the GaTT-T/2 value before PTBD. The decreasing rate of serum bilirubin level "b" after PTBD was significantly fair in the group A patients (good GaTT-T/2 value before PTBD) than the group B (poor GaTT-T/2 value before PTBD) (P less than 0.05). It was that GaTT-T/2 before PTBD which represented hepatic cytosolic functional mass could predict the effect of PTBD in patients with obstructive jaundice.

[Effect of prostaglandin E1 derivative on labilization of liver lysosomal membrane in partial liver ischemia].

The change of liver lysosomal enzymes in tissue and serum during a reperfusion period was studied in partial liver ischemic model in rats and effect of Prostaglandin E1 (PGE1) derivative on partial liver ischemia was investigated. Partial liver ischemia was induced by clamping the branches of the vessels to the right and caudate lobes of rat liver. The clamp was released after 30 minutes of ischemia. Ischemic and nonischemic lobes of the liver were separately removed and the serum was also collected immediately and two hours after the release of the clamp. Lysosomal enzyme activities from free and bound lysosomal fraction were measured separately and the fragility index (F.I.) was calculated. PGE1 derivative was administered intraperitoneally 24, 6, 0.5 hours prior to the induction of ischemia at each dose of 0.05 microgram/kg. Pretreatment with PGE1 derivative prevented lysosomal labilization in ischemic lobe, since there was a significant decrease in F.I. of cathepsin D in the PGE1-pretreated group (preischemia; 28.3 +/- 2.4%, immediately after reperfusion; 30.3 +/- 2.5%, two hours after reperfusion; 30.3 +/- 2.5%) compared to the placebo group (immediately after reperfusion; 40.9 +/- 3.4%, two hours after reperfusion; 41.7 +/- 3.4%, p less than 0.05, p less than 0.05, p less than 0.01, respectively). Pretreatment with PGE1 derivative also significantly suppressed the increase of serum lysosomal enzyme activity. These results showed that PGE1 derivative improved liver lysosomal labilization in partial liver ischemia.

[Changes in lysosomal enzymes and cell damage of the liver in obstructive jaundiced rats].

The changes of hepatic lysosomal enzymes and the hepatic cellular damage were investigated in rats with obstructive jaundice, phospholipase A2 (PL-A2) which is a strong labilizer of lysosomal membrane was added in the lysosomal fraction of rat's liver with various concentration. The activities of cathepsin D and beta-glucuronidase those were released by PL-A2 from lysosomal fraction were measured. The values of both lysosomal enzyme activities showed positive relation to the concentration of PL-A2, and were remarkably increased in obstructive jaundiced rats than in normal rats. We also measured the activity of cathepsin D released by Triton X-100 from lysosomal fraction of normal and jaundiced rat liver. The amount of lysosomal enzyme was more increased in obstructive jaundiced liver than in normal liver. Fragility score as the indicator for lysosomal membranous fragility was calculated as the ratio of cathepsin D released by PL-A2 to that released by Triton X-100. Fragility score was more increased in obstructive jaundiced rats than in normal rats. In conclusion, these data suggest that the fragility of lysosomal membrane could be enhanced in obstructive jaundiced liver.

[Enhancement of selective drug accumulation and retention in normal and regenerating liver by the use of lipiodol in portal venous infusion in rats].

We studied selective accumulation and retention of lipiodol (LP) and anticancer agents in normal and regenerating liver tissue following portal infusion in rats. Total concentration of Aclarubicin (ACR) and its metabolites in liver tissue was higher in ACR + LP portal infusion group than in ACR portal infusion group both in normal and regenerating liver, concentration of active metabolites of ACR was higher in ACR portal infusion group than in ACR peripheral infusion group. Much higher concentration was found in ACR + LP portal infusion group. Histologic examination revealed more toxic effect on regenerating liver in ACR portal infusion group than in ACR + LP portal infusion group. Oil red staining demonstrated the retention of lipiodol more than 7 days following intraportal infusion in regenerating liver tissue. This study confirms that the ACR + LP portal infusion induces selective accumulation and long-term retention in normal and regenerating liver tissue, and may enhance the antitumor effect of drugs.

[Relation of hepatic protein synthesis and hepatic functional mass in obstructive jaundiced rats].

In obstructive jaundiced rat, the change of hepatic functional mass assessed by [14C]-aminopyrine breath test (ABT) and galactose tolerance test (GaTT) and hepatic protein synthesis measured by [14C]-leucine incorporation into hepatic protein fraction were investigated. Bile duct ligation (BDL) for 5 and 14 days was followed by choledocho-duodenal fistula as the relief of obstruction. Hepatic functional mass measured by ABT and GaTT revealed a remarkable decrease at 5 and 14 days after BDL without differences in grades. These depressed values returned to the preoperative ones in 10 to 20 days after the relief of obstruction. On the contrary, hepatic protein synthesis was reciprocally enhanced after BDL. After the relief of obstruction the enhancement of hepatic protein synthesis was prolonged and then returned to the normal level in 20 days. These data suggested that in obstructive jaundice hepatic protein synthesis was stimulated by several stress and continued to be enhanced even after the relief of obstruction. These enhancement of hepatic protein synthesis would induce to decrease hepatic functional mass.

[Inhibition of hepatic DNA synthesis after partial hepatectomy in the rats with obstructive jaundice with special reference to the enhancement of hepatic protein synthesis].

We studied the liver regeneration after partial (68%) hepatectomy in rats with obstructive jaundice followed by the relief of obstruction. Rats received bile duct ligation, then 5 or 14 days later choledocho-duodenostomy was performed. Partial hepatectomy was done at various intervals after the relief of obstruction. DNA synthesis of the regenerating liver, hepatic protein synthesis and mitochondrial swelling induced by exogenous phospholipase A2 (PLA2) were determined. Hepatic DNA synthesis was significantly inhibited in obstructive jaundiced rats compared to controls. While the inhibition disappeared 5 days after the relief of obstruction in 5-day-obstructed group, it was still detectable as late as 21 days after the drainage in 14-day-obstructed group. Hepatic protein synthesis was markedly increased by obstructive jaundice, and this increase continued until 10 days after drainage in 14-day-obstructed group. Partial hepatectomy also increased the hepatic protein synthesis significantly in normal rats, but failed to show any significant changes in obstructive jaundiced rats. Any difference could not be found in PLA2-induced hepatic mitochondrial swelling between obstructive jaundiced rats and normal rats. We concluded the preceding energy-requiring responses in obstructive jaundiced liver resulted in the reduction of hepatic DNA synthesis and in the lack of additional increase of hepatic protein synthesis as the responses to a further insult of partial hepatectomy.

[Cyclic AMP- and ATP-mediated stimulation of DNA synthesis following partial hepatectomy by prostaglandin-E1 in D-galactosamine injured liver].

D-galactosamine (D-gal) damaged rats were infused with Prostaglandin E1 (PGE1) through a peripheral vein for 40 min. before and after partial hepatectomy. DNA synthesis following 68% partial hepatectomy was severely inhibited by the pretreatment of D-galactosamine. PGE1 infusion (0.5, 1.0 microgram/kg/min) enhanced the DNA synthesis inhibited by D-gal 600 mg/kg significantly (p less than 0.01). After 20 min. of PGE1 infusion cyclic AMP levels of liver tissue was increased as compared with saline infusion in D-gal (600 mg/kg)-damaged rat (p less than 0.05). Also 20 min. and 3 hour after partial hepatectomy. ATP levels of liver tissue was enhanced in PGE1 treated group (p less than 0.05). However the doses of PGE1 infused in this investigation could not increase the hepatic tissue blood flow measured by hydrogen gas clearance method. These results suggest that PGE1 enhance DNA synthesis of injured liver after partial hepatectomy by the mechanism which PGE1 stimulate cyclic AMP production and increase ATP level in hepatic tissue.

[Enhancement of anti-tumor effect of adriamycin by verapamil in a intrahepatic arterial infusion in the rats].

In a intrahepatic arterial infusion, the enhancement of cytotoxicity of adriamycin (ADR) by verapamil (VER), a calcium antagonist was investigated in male Wistar rat with liver tumor of Walker 256 carcinosarcoma. The accumulation of ADR in tumor tissue at 2 hrs after a bolus intrahepatic arterial injection of ADR with VER (4 mg/kg) was 1.9-fold more than without VER. But VER did not enhance it in normal liver tissue and heart tissue. In the continuous intrahepatic arterial infusion therapy (cia) of ADR and VER (1.5 mg/kg/day) for 6 days, tumor weight and the accumulation of ADR in tumor tissue, normal liver tissue and heart tissue were assessed. Tumor weight of ADR-cia+VER-cia group was significantly less than ADR-cia group (p less than 0.05). And the accumulation of ADR in tumor tissue of ADR-cia+VER-cia group was significantly higher than ADR-cia group (p less than 0.05). But VER did not enhance it in normal tissue. The administration of VER in a intrahepatic arterial infusion is shown to be able to enhance the cytotoxicity of ADR in tumor tissue but not to enhance in normal tissue. The continuous intrahepatic arterial infusion of VER enhanced cytotoxicity of ADR at clinical dose and it suggests the clinical applicability of VER in cancer chemotherapy.

[Transcatheter embolization of splenic artery aneurysm; a case report].

A 76-year old woman with splenic artery aneurysm was treated by nonsurgical treatment. Surgical treatment has been recommended for the treatment of visceral aneurysms because these aneurysms may rupture and lead to fatal hemorrhage. This report describes the successful interventional coil and Gelfoam occlusion of splenic aneurysm.