Purification and characterization of bifunctional alginate lyase from Alteromonas sp. strain no. 272 and its action on saturated oligomeric substrates.

A marine bacterium (strain No. 272) isolated from sea mud in Omura Bay produced an alginate lyase and was classified as an Alteromonas species. The enzyme was purified from the culture medium of the bacterium by DEAE-Cellulofine, Sephadex G-100 gel chromatography to an electrophoretically homogeneous state in the presence and absence of SDS. The molecular mass of the enzyme was 23 and 33.9 kDa on Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis, respectively, with an isoelectric point of 3.8. The predominant secondary structure of the enzyme was found to be most likely beta-structure by circular dichroism. The enzyme was most active at pH 7.5-8.0 and stable around pH 5-11. The enzyme was more labile in Tris-HCI buffer (pH 7.0) to heat treatment, than in phosphate buffer (pH 7.0). No of metal ions significantly affected the enzyme activity. The enzyme acted on sodium alginate in an endo-type manner and on two components of alginate, poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate, as judged by routine ultraviolet assay (235 nm) and circular dichroic spectral changes of the substrates. However, the coexisting poly-alpha1,4-L-guluronate and poly-beta1,4-D-mannuronate apparently interacted with the enzyme in a competitive manner. Although the enzyme depolymerized alginate in an endo-type, it did not act on trimeric guluronate and mannuronate, but on the tetramers or more. The kinetic analyses showed that kcat/Km for each oligomer was larger for the guluronate oligomers than for the mannuronate ones, and that the subsite structure of the enzyme most likely consisted of six binding sites from the intrinsic reaction rate constant (kint) and intrinsic substrate binding constant (Kint).

Factors predicting perioperative cytokine response in patients undergoing liver transplantation.

OBJECTIVES: An exaggerated production of proinflammatory cytokines during liver transplantation stimulates the inflammatory process within the graft, and eventually promotes liver failure. This study was conducted to evaluate factors predicting perioperative response of proinflammatory cytokines during liver transplantation. DESIGN: Prospective, consecutive entry study of liver transplant candidates. SETTING: University hospital. PATIENTS: Thirty liver transplant recipients. INTERVENTIONS: Arterial blood samples were obtained perioperatively. MEASUREMENTS: Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha were measured by ELISA. Endotoxin was determined by a chromogenic endotoxin-specific method. MAIN RESULTS: The peak concentrations of IL-1beta and IL-6 in the patients with complications were significantly higher than those in the patients without complications. The peak concentration of IL-1beta was significantly correlated with the level of bilirubin at admission and the intraoperative blood product requirement. The peak concentration of IL-6 was significantly correlated with the admission bilirubin and the intraoperative blood product requirement. A multivariate regression model revealed that the serum bilirubin and the intraoperative blood product requirement were the independent factors that influenced the peak concentration of IL-1beta or IL-6. The severely jaundiced patients had a significantly higher plasma concentration of endotoxin at the end of the anhepatic phase. In addition, there was a tendency for these patients to have a higher postoperative peak concentration of endotoxin. CONCLUSIONS: Serum level of bilirubin may be a potent preoperative factor influencing perioperative cytokine response in patients undergoing liver transplantation. An enhanced perioperative response of endotoxin seen in severely jaundiced patients suggests the clinical implication of endotoxin removal during the anhepatic phase in liver transplant surgery.

Serum hepatocyte growth factor as an index of extensive catabolism of patients awaiting liver transplantation.

BACKGROUND: Whole body catabolism as the result of intrahepatic metabolic derangement is common in liver transplant candidates. However, individual nutritional assessment parameters lack sensitivity and specificity in determining energy status of these patients. Recently, serum hepatocyte growth factor (HGF) has been shown to reflect the recovery of hepatic energy metabolism after liver transplantation. AIMS: The relation between preoperative levels of serum HGF and metabolic variables was investigated to clarify the clinical value of measuring HGF in evaluations of the catabolism. PATIENTS/METHODS: Blood samples were obtained from 30 liver transplant recipients, and biopsy specimens were taken from each recipient's rectus muscle and the explanted liver. Preoperative serum concentration of HGF was determined. Whole body energy metabolism was assessed by measuring glycogen contents of biopsy specimens and plasma or serum levels of glucose, insulin, total ketone bodies, total carnitine, and amino acids. RESULTS: Serum HGF concentration was elevated in 22 of 30 patients and correlated with the Child-Pugh score. It showed a negative association with muscle glycogen content, and a positive correlation with serum levels of glucose, total carnitine, and total ketone bodies. Patients with elevated serum HGF concentrations had higher preoperative plasma levels of aromatic amino acids and branched chain amino acids, associated with lower branched chain to aromatic amino acid ratios. CONCLUSIONS: The elevated serum concentration of HGF in liver transplant candidates reflected inhibition of peripheral glucose storage, enhanced lipid oxidation, and increased peripheral release of branched chain amino acids, and thus extensive energy catabolism.

Energy storage and cytokine response in patients undergoing liver transplantation.

Overproduction of pro-inflammatory cytokines during surgery has been known to exert tissue-damaging and lethal effects on the host. Hypermetabolism-associated malnutrition, a common feature of patients with end-stage liver diseases, is related to the presence of a systemic inflammatory response, as reflected by enhanced levels of proinflammatory cytokines. The present study was designed to evaluate energy status of 29 liver transplant recipients, and to assess the relation of energy storage to post-operative cytokine response. The glycogen contents of the graft, and the recipient's abdominal muscle and old liver were measured. The plasma concentrations of tumour necrosis factor alpha, interleukin 1beta, interleukin 6, lactate, pyruvate and total ketone bodies were determined during and after surgery. In undernourished patients, ketone bodies seemed to be the major fuel available to muscle. The concentration of ketone bodies decreased rapidly after the incision, and remained at a low level after reperfusion. These patients had higher plasma levels of lactate/pyruvate ratio and aromatic amino acids during the anhepatic phase, followed by an exaggerated response of cytokines. Depletion of energy storage of the recipients may be involved in the deterioration of peri-operative energy metabolism and the exaggerated post-operative cytokine response.

Inhibitory effects of prior low dose X-ray irradiation on Fe(3+)-NTA-induced hepatopathy in rats.

Blood activities of hepatocellular enzymes such as lactate dehydrogenase (LDH), glutamic pyruvic transaminase (GPT) and glutamic oxalacetic transaminase (GOT) peaked at 12 hours after a single intraabdominal injection of ferric nitrilotriacetate (Fe(3+)-NTA) in rats. Enzymes such as alkaline phosphatase (ALP) and leucin amino peptidase (LAP) originating in the capillary bile ducts or bile secretory liver cells were also released into the blood between 6-24 hours after intraabdominal injection of Fe(3+)-NTA in rats. Furthermore, hyperoxidation of lipids occurred in rat hepatic cell membranes, reaching a peak 6 hours after intraabdominal injection of Fe(3+)-NTA. It was found that a single prior 0.5 Gy whole body X-ray irradiation significantly increased superoxide dismutase (SOD) activities and suppressed above-mentioned symptoms of transient hepatopathy in rats.

Change of glutathione peroxidase synthesis along with that of superoxide dismutase synthesis in mice spleens after low-dose X-ray irradiation.

We have previously demonstrated that the activity of superoxide dismutase (SOD), an antioxidant, is enhanced by low-dose X-ray irradiation in various organs of animals such as rats. Since SOD is an enzyme that mediates the dismutation of O2- to H2O2, the question as to whether the resultant H2O2 is further detoxicated into H2O and O2 or not must still be evaluated. Hence, we studied the effect of low-dose X-ray irradiation on the synthesis of glutathione peroxidase (GSHPx), which is an antioxidant that catalyzes this reaction. The results suggest that H2O2 produced by increased SOD activity can be detoxicated into H2O and O2 due to simultaneous enhancement of the GSHPx activity by X-ray irradiation at 20 cGy, in contrast to irradiation at 400 cGy. The results also show the enhancement in enzyme activities by induction of their synthesis shortly after irradiation at 20 cGy. Moreover, as this phenomenon was observed in BALB/c mice (which are more radiation-sensitive compared to other mouse strains) and radiation-resistant C57BL/6NJcl mice, it was considered to be a common phenomenon in the rat spleen.

Prognostic significance of the DNA index in a colorectal cancer.

DNA flow cytometry was performed on paraffin-embedded tissue blocks made from 230 surgically resected colorectal cancers, 109 (47.4%) of which were diploid tumors, and 121 (52.6%) aneuploid tumors. The DNA index (DI) was calculated as the ratio of the G0/G1 channel number of tumor cells to the G0/G1 channel number of stromal cells. There was no significant difference in survival between patients with diploid tumors and those with aneuploid tumors (P = 0.322), although the survival rate was significantly lower in patients with a high DI (> or = 1.5) than in those with a low DI (< 1.5) (P = 0.004). A multivariate analysis of prognostic factors using Cox's proportional hazard model showed that Dukes' staging was the strongest predictor of survival, followed by the DI of tumor cells, then histological differentiation. In conclusion, it is suggested that the DI of tumor cells is instructive for predicting the survival of patients with colorectal cancer.

Hepatic infarction as a complication of gastric cancer surgery: report of four cases.

Four cases of patients who developed hepatic infarction caused by an operative injury to the hepatic circulation during gastric cancer surgery are reported herein. In two patients, the hepatic infarction resulted from accidental injury to the proper hepatic artery, and in the other two, it was possibly due to persistent pressure on the folded liver by a retractor during surgery. In the former two patients, the proper hepatic artery had been collapsed by the spread of enlarged metastatic lymph nodes before the onset of the arterial injury. In the latter two patients, postoperative laboratory data and computed tomography scanning revealed hepatic infarction even though preservation of the proper hepatic artery was confirmed by angiography. Elevated serum levels of hepatic enzymes released from the infarcted tissue recovered to the normal range within three weeks in all four patients. In conclusion, when an operative injury to the hepatic artery is encountered, the hepatoduodenal ligament should not be manipulated any more than necessary to preserve the collaterals, and the gallbladder should be removed to prevent necrotic perforation. Although close observation is mandatory, conservative therapy seems to be sufficient when an infarcted area is restricted to the lateral segment and a small part of the medial segment of the liver.

Serum cell adhesion molecules in patients with colorectal cancer.

The serum levels of intercellular adhesion molecule-1 (sICAM-1) and endothelial leukocyte adhesion molecule-1 (sELAM-1) were determined in 40 patients with colorectal cancer. The sICAM-1 and sELAM-1 levels in the drainage venous blood adjacent to a tumor were significantly correlated with those in the peripheral venous blood in patients without evident hematogenous dissemination of tumor cells. The sICAM-1 levels in peripheral venous blood were significantly higher in patients with hepatic metastases, while the sELAM-1 levels were significantly higher in those with pulmonary metastases. An immunohistochemical study of metastatic sites in the liver revealed that ICAM-1 was expressed in cancer stroma, but not in the cancer cells. In conclusion, the sICAM-1 and sELAM-1 levels in the peripheral venous blood in colorectal cancer patients without any distant metastasis are likely to reflect the topical production of these cell adhesion molecules, and appear to be instructive in predicting hematogenous dissemination in patients with colorectal cancer.

Constant infusion rates of lipid emulsions to stabilize plasma triglyceride concentrations: medium-chain triglyceride/long-chain triglyceride emulsions (MCT/LCT) versus LCT.

As medium-chain triglyceride emulsions (MCT) are more rapidly hydrolyzed than long-chain triglyceride emulsions (LCT), MCT/LCT tends to be infused faster than LCT. The purpose of the present study was to determine the most appropriate infusion rate for MCT/LCT to stabilize plasma concentrations of triglyceride (TG), being equivalent to the optimal infusion rate of the emulsion. A TG clamp was set up by raising the mean +/- SD concentrations of TG in plasma, being 1.08 +/- 0.18 delta mmol l(-1) for LCT, and 1.65 +/- 0.31 delta mmol l(-1) for MCT/LCT after a 50-min priming infusion of each emulsion. Thereafter, the infusion rate of lipid was controlled every 10 min to maintain a steady concentration of TG for a period of 150 min. A constant infusion of glucose at 0.32 g/kg body weight (BW) per h was administered for the test period. The weight-based rate of the infusion to maintain a steady state of plasma TG concentrations did not differ between MCT/LCT and LCT, being 0.125 +/- 0.013 vs 0.117 +/- 0.021 g/kg BW per h, while the molar-based infusion rate was 0.203 +/- 0.021 mmol/kg BW per h for MCT/LCT and 0.132 +/- 0.023 mmol/kg BW per h for LCT (P < 0.05). These results suggest that although 54% more molar MCT/LCT-TG can be hydrolyzed during a constant infusion, MCT/LCT should not be infused at a rate faster than 0.1 g/kg BW per h under a steady state.

Nonadditive effects of double mutations at the flexible loops, glycine-67 and glycine-121, of Escherichia coli dihydrofolate reductase on its stability and function.

The structure, stability, and enzymatic function of dihydrofolate reductase (DHFR) from Escherichia coli are influenced by point mutations at sites 67 and 121 in two flexible loops [Gekko et al. (1994) J. Biochem. 116, 34-41; Ohmae et al. (1996) J. Biochem. 119, 703-710]. In the present study, eight double mutants at sites 67 and 121 (G67V/G121S, G67V/G121A, G67V/G121C, G67V/G121D, G67V/G121V, G67V/G121H, G67V/G121L, and G67V/G121Y) were constructed in order to identify interactions between the two sites of DHFR. The far-ultraviolet circular dichroism spectra of double mutants were clearly different from those of the respective single mutants, with significant changes being observed for three mutants, G67V/G121A, G67V/G121L, and G67V/G121S. The Gibbs free energy change of urea unfolding of double mutants could not be expressed by the sum of those of the respective single mutants except for G67V/G121H. The steady-state kinetic experiments showed that the effect of double mutations manifests itself not in Km but in k(cat), and the transition-state stabilization energy for G67V/G121A, G67V/G121C, and G67V/G121L is not equal to the sum of those for the single mutants. These results indicate that the additivity rule essentially does not hold for these double mutants, and that long-range interactions occur between sites 67 and 121, even though they are separated by 27.7 A. This is evidence that the flexible loops play important roles in the stability and function of this enzyme through structural perturbations, in which a small alteration in local atomic packing due to amino acid substitution is cooperatively magnified over almost the whole molecule.

Postperfusion energy metabolism of steatotic graft and its relation to early graft viability following liver transplantation.

The present study was designed to assess energy metabolism of steatotic grafts and to determine its relation to early graft viability. Graft biopsies were taken, and the triglyceride content was determined in 29 grafts for the assessment of steatosis. The peak aspartate aminotransferase level and the concentrations of lactate and pyruvate were strongly correlated with the triglyceride content, suggesting that steatotic grafts are more vulnerable to preservation or reperfusion injury and that glucose oxidation is inhibited postoperatively in the steatotic grafts. Ketogenesis, an alternative pathway to produce energy substrates, was not accelerated even when the steatotic grafts produced more free carnitine to enhance the beta-oxidation of fatty acids. The deterioration of energy metabolism was associated with the increase in prothrombin time ratio, hepatocyte growth factor, and hyaluronic acid that reflected graft viability. Deterioration of postperfusion energy metabolism in the steatotic grafts may be involved in the development of irreversible graft damage.

Simultaneous quantitative analysis of prostaglandins and thromboxane after low-dose X irradiation.

The appearance of prostaglandins and thromboxane in mouse serum after X irradiation was observed by simultaneous quantitative analysis using gas chromatography/mass spectrometry/selected ion monitoring with stable isotope dilution methods. Mice of two strains (C57BL/CN Jcl and BALB/c) showed similar responses to X irradiation. In C57BL/6N Jcl mice, 0.2 Gy irradiation elicited a significant increase in generation of prostanoids: Immediately after irradiation, the 6-keto PGF1 alpha:TXB2 ratio and the level of PGE2 increased, after 20 min 6-keto PGF1 alpha and PGE2 increased, and after 4 h PGE1 and PGE2 increased. In BALB/c mice, generation of prostanoids was increased significantly immediately after irradiation (6-keto PGF1 alpha, 6-keto PGF1 alpha:TXB2 ratio, PGE2), and the increase was maintained from 20 min to 4 h (PGE1, PGE2) after 0.2 Gy irradiation. In C57BL/6N Jcl mice, a significant increase in production of 9alpha,11beta-PGF2 was observed at 20 min after irradiation. In BALB/c mice, a significant increase in 9alpha,11beta-PGF2 was seen immediately after irradiation and was maintained for 20 min. In C57BL/6N Jcl mice, the level of 8-epi PGF2 alpha was clearly increased 4 h after 4 Gy irradiation. A slight and slow increase was also seen after 0.2 Gy irradiation. In BALB/c mice, 8-epi PGF2 alpha was increased significantly at 20 min and 4 h after 4 Gy irradiation. These results show that 0.2 Gy irradiation stimulates production of prostanoids related to the inflammatory response in mice.

Demonstration of microcystic disease of the pancreas by magnetic resonance cholangiopancreatography: report of a case.

A case of microcystic disease of the pancreas which was clearly demonstrated by magnetic resonance cholangiopancreatography (MRCP) is reported herein. Cystic dilatation of the pancreatic duct was recognized by computed tomography scanning and endoscopic retrograde cholangiopancreatography (ERCP). Furthermore, the existence of microcystic clusters surrounding the dilated pancreatic duct were clearly visualized by MRCP. These microcystic clusters were strongly suspected preoperatively of having caused dilatation of the major pancreatic duct. Based on these findings, a distal pancreatectomy was performed. The operative specimen showed no accumulation of mucin and no evident lesions in the dilated pancreatic duct, being inconsistent with the entity of a mucus-producing tumor. Pathological examination revealed that the inner parts of microcysts constituted columnar epithelium with mucus production and papillary growth. Thus, a final histological diagnosis of intraductal papillary adenoma with idiopathic pancreatic duct ectasia was confirmed. In conclusion, MRCP, being a less aggressive diagnostic procedure than ERCP, proved extremely useful for obtaining precise information on cystic lesions of the pancreas in this patient.

Analysis of tenascin mRNA expression in the murine mammary gland from embryogenesis to carcinogenesis: an in situ hybridization study.

The expression of tenascin gene during murine mammary gland development was analyzed by in situ hybridization with non-radioactive cRNA probes. The aim was to identify whether cells that synthesize tenascin are mesenchymal or epithelial. During embryogenesis, tenascin mRNAs were demonstrated in the epithelial cells of the mammary bud on the 14th and 15th day of gestation, and in the mesenchymal cells from the 14th day to the 17th day, at the epithelial-mesenchymal border of the growing bud. However, cells displaying tenascin mRNAs were not found beyond the bifurcation of the mammary sprout at the beginning of the branching morphogenesis. In post-natal development, tenascin mRNAs were demonstrated in mesenchymal cells surrounding end buds in juvenile mice, in mesenchymal cells surrounding the epithelial cells of plaques, in epithelial cells of the lactating mammary gland, in malignant epithelial cells and in the mesenchymal cells surrounding cancer nests. By immunohistochemistry, tenascin immunoreactivity was shown to have the same spatiotemporal distribution as that of tenascin mRNAs, but was observed to be restricted to the stroma, except in the lactating mammary gland where tenascin was demonstrated in the milk by Western blot. The present study thus showed that both epithelial and mesenchymal cells are sources of tenascin at different stages of murine mammary gland development.

Glycogen content of the donor liver and its relation to postreperfusion hepatic energy metabolism.

OBJECTIVES: Many studies have suggested that glycogen in donor livers is an important fuel during cold ischemic time and at reperfusion. However, it remains unclear as to whether the depression of glycogen content in the graft results in a critical derangement of energy metabolism after reperfusion. The purpose of this study was to assess the possible implications of the glycogen concentration of donor livers for the hepatic energy metabolism after reperfusion. METHODS: The glycogen content of 28 donor livers and the plasma concentrations of metabolic substrates were measured during liver transplantation. RESULTS: Gluconeogenesis was maintained even in the glycogen-depleted graft at reperfusion. However, glycogen-depleted grafts produced more ketone bodies until 24 h after reperfusion. Free carnitine concentrations in these patients were significantly higher than those in the patients with glycogen-nondepleated grafts until 48 h after reperfusion. CONCLUSIONS: A glycogen-depleted liver graft may restore essential metabolic function by producing energy substrates through enhanced ketogenesis in the postreperfusion period. The enhanced production of carnitine by the graft provides a substrate for the production of ketone bodies and thus may be relevant to the enhanced ketogenesis.

Plasma carnitine kinetics during orthotopic liver transplantation.

BACKGROUND: Carnitine is synthesized mainly in the liver and plays an essential role in the transport of fatty acids in liver mitochondria for subsequent oxidation and energy production. METHODS: The plasma concentrations of free carnitine, acylcarnitine, total ketone bodies, lactate, pyruvate, and hepatocyte growth factor (HGF) were measured during liver transplantation. RESULTS: The plasma free carnitine and acylcarnitine concentrations and the lactate to pyruvate ratio in patients with compromised grafts (group A) were significantly higher than those in patients with well-functioning grafts (group B) after reperfusion. The acylcarnitine concentration in group B decreased after incision, but it remained at a high level in group A. Significant correlations were found between the concentrations of HGF and free and acylcarnitine after reperfusion. CONCLUSION: The accelerated flux of carnitine in the graft may be associated with deterioration of energy metabolism in the graft. An increased acylcarnitine concentration may reflect impaired liver regeneration.