A novel Fe(II)/α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria has ß-hydroxylating activity of N-succinyl l-leucine.

An Fe(II)/α-ketoglutarate-dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N-substituted l-amino acids, which was especially strong with N-succinyl l-leucine. With the NMR and LC-MS analysis, SadA converted N-succinyl l-leucine into N-succinyl l-threo-ß-hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing ß-hydroxylation of aliphatic amino acid-related substances and a potent biocatalyst for the preparation of optically active ß-hydroxy amino acids.

Microbial production of conjugated gamma-linolenic acid from gamma-linolenic acid by Lactobacillus plantarum AKU 1009a.

AIMS: Optimal production conditions of conjugated gamma-linolenic acid (CGLA) from gamma-linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. METHODS AND RESULTS: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0.03% (w/v) alpha-linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml(-1)gamma-linolenic acid as a substrate in 5 -ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml(-1) dry cells] as the catalysts produced 8.8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis-6,cis-9,trans-11-octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis-6,trans-9,trans-11-octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. CONCLUSION: The practical process of CGLA production from gamma-linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.

Selective production of cis-9,trans-11 isomer of conjugated linoleic acid from trans-vaccenic acid methyl ester by Delacroixia coronata.

AIMS: Bio-process development for isomer selective and efficient production of cis-9,trans-11-octadecadienoic acid (CLA) from trans-vaccenic acid (t-VA, trans-11-octadecenoic acid) through microbial fatty acid Delta9-desaturation reaction. METHODS AND RESULTS: A total of 550 strains of fungi and yeasts were screened for CLA production from t-VA through Delta9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t-VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t-VA (t-VAME). Under the optimal conditions with 33.3 mg ml(-1) of t-VAME as substrate, 10.5 mg ml(-1) CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5.0%), tryptone (2.0%) and thiourea (0.83 micromol ml(-1)). The strain produced the cis-9,trans-11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans-9,trans-11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA). CONCLUSIONS: A practical bio-process for selective production of cis-9,trans-11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: Isomer selective bio-process for the practical production of cis-9,trans-11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.

High durability cementitious material with mineral admixtures and carbonation curing.

Nuclear waste repositories need highly durable cementitious materials to function for over thousands of years while resisting leaching and degradation. The durability of cementitious material can be effectively improved by reducing permeability and by changing cement hydrates to a less soluble matrix. This paper describes the properties of carbonated new cementitious materials containing belite-rich cement and gamma-2CaO.SiO2 as main components. In addition, the long-term leaching properties are investigated and compared with ordinary Portland cement by using a predictive leaching model.

Purification and characterization of novel transglutaminase from Bacillus subtilis spores.

Transglutaminase activity was detected in suspensions of purified spores prepared from lysozyme-treated sporulating cells of Bacillus subtilis AJ 1307. The enzyme was easily solubilized from the spores upon incubation at pH 10.5 at 37 degrees C. The transglutaminase activity was separated into two fractions upon purification by hydrophobic interaction chromatography (TG1 and TG2). Each enzyme was purified to electrophoretic homogeneity (about 1,000-fold). Both enzymes had the same molecular weight of 29,000 as estimated by SDS-PAGE, had the same N-terminal 30 amino acid sequence, and also showed the same optimal temperature (60 degrees C) and pH (8.2). The purified enzyme catalyzed formation of cross-linked epsilon-(gamma-glutamyl)lysine isopeptides, resulting in the gel-formation of protein solutions such as alphas-casein and BSA.

Study on highly diastereoselective synthesis of (2'R)-2'-deoxy[2'-2H]guanosine.

To develop an efficient method for the synthesis of a highly diasteroselective (2'R)-2'-deoxy[2'-2H]guanosine (1), studies of organic chemical conversion from 2'-bromo-2'-deoxy-N2-Isobutyryl-3',5'-O-TIPDS-guanosine (2) to 1 and a biological transdeoxyribofuranosylation of (2'R > 98% de)-2'-deoxy[2'-2H]uridine (4) were carried out. As the results, a highly diastereoselective synthesis of 1 was achieved by a biological transdeoxyribofuranosylation between 2,6-diaminopurine and 4 by the use of Enterobacter aerogenes AJ-11125, followed by treatment with adenosine deaminase. The results will be described in detail.

Superinfection of TT virus and hepatitis C virus among chronic haemodialysis patients.

BACKGROUND: The TT virus (TTV), a new DNA virus found in Japan from a patient with post-transfusion hepatitis non-A-non-G, is frequently positive in the sera of patients with liver disease. It is not established whether this virus causes liver damage. We studied the frequency of superinfection of this virus and hepatitis C virus (HCV) known to be endemic among haemodialysis patients, and the possible deleterious effect of TTV on HCV-induced chronic liver disease. METHODS: We used primers from a conservative region in the TTV genome (Okamoto, 1998) to detect TTV. Sera from 163 dialysis patients positive for anti-HCV and 77 dialysis patients negative for anti-HCV (control) were tested. RESULTS: TT Virus positivity was 35% among HCV antibody (anti-HCV)-positive patients and 45.4% among anti-HCV-negative patients. TT Virus positivity was unrelated to the length of haemodialysis or amounts of blood the patients had received in the past. More anti-HCV-positive patients had a history of transfusion, but TTV positivity was not as closely associated with transfusion as anti-HCV positivity. The severity of chronic liver disease was estimated from peak serum alanine aminotransferase levels in the preceding 6 months. Among anti-HCV positives, TTV-positive patients tended to have less active disease; at least there was no indication that TTV superinfection aggravated chronic hepatitic C in long-term dialysis patients. Four of 35 anti-HCV-negative, TTV-positive patients had chronic active liver disease, while none of the anti-HCV-negative and TTV-negative patients did. CONCLUSIONS: TT Virus infection is prevalent among haemodialysis patients. Its transmission occurs not only by blood transfusion, but also by non-parenteral infection. Superinfection of TTV does not exert deleterious effects on the liver disease induced by HCV. However, it may cause chronic hepatitis in a limited number of patients, but remains dormant most of the time. Triple infection, HCV and TTV plus HBV or HGV (one case each), did not cause severe liver disease.

Acellular calcified columns in the normal growth plate of mouse vertebrae.

The present study aims to demonstrate the calcified columnar structures of the growth plate of mouse vertebrae and to show their age related changes. For light microscopy, paraffin sections of decalcified lumbar spines were stained with hematoxylin-eosin or toluidine blue; methacrylate sections of undecalcified specimens were stained to detect calcium precipitate. For scanning electron microscopy, lumbar spines treated with 5% NaClO solution were dehydrated by acetone and metal-coated. Light microscopy of hematoxylin-eosin stained sections revealed that the acellular columnar structures appeared between the chondrocyte stacks in the growth plate. These structures were stained more densely by toluidine blue. The methacrylate-embedded sections for calcium staining showed calcium deposition in the columns. Scanning electron microscopy of NaClO-treated specimens enabled the direct observation of the columns on both the epiphyseal and diaphyseal bone surfaces facing the cartilaginous growth plate. Numerous projections on each surface were distributed in mirror images ; the corresponding projections were similar in size and shape, indicating that the projections bonded with each other and formed calcified columns in the cartilaginous growth plate. Longitudinal sections of the spine confirmed these findings. The calcified columns first appeared about 2 or 3 weeks of age and increased in number with time. While increasing in number, they also grew in size fusing with the neighboring ones. The proportional area of the columns occupying the surface facing the growth plate also increased with age. These findings indicate that the calcified column ultimately concerns the cessation of the bone growth.

Acute hepatitis C among renal failure patients on chronic haemodialysis.

Hepatitis C virus (HCV) infection is common in haemodialysis units, yet little information is available about the clinical feature of acute hepatitis C among renal failure patients. The present study is based on 49 cases of acute hepatitis C seen at a haemodialysis centre where sporadic nosocomial infection was occurring up to June 1993. Liver function tests were done at 4 weekly intervals on all dialysis patients, anti-HCV antibodies were tested by the C-100 and second generation tests and serum HCV-RNA was determined by the branched DNA and Amplicore tests. Diagnosis of acute hepatitis C was made on the basis of an acute rise in alanine aminotransferase (ALT) and seroconversion to positive anti-HCV antibodies. Clinical presentation of acute hepatitis was generally mild with rare overt jaundice and the diagnosis was possible only from increased ALT, which was generally low. Spontaneous resolution of acute hepatitis within 8 months with clearance of viral RNA occurred in only four cases, 91.8% of patients developing chronic hepatitis. Biopsy in 12 cases with high ALT levels showed mild to moderate inflammatory activities. In conclusion, the clinical presentation of acute hepatitis C is generally mild in chronic haemodialysis patients, but spontaneous resolution is infrequent. A longer follow-up period is required for defining the long-term prognosis.

Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli.

We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the sigma K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.

Cryoglobulinaemia among maintenance haemodialysis patients and its relation to hepatitis C infection.

It has been shown that hepatitis C virus (HCV) infection is closely associated with mixed type cryoglobulinaemia. It is also known that HCV infection is rampant among chronic haemodialysis patients. We studied 531 renal failure patients on maintenance dialysis including 170 with positive HCV antibodies for cryoglobulinaemia, and its incidence was compared with controls which consisted of 242 chronic hepatitis C patients without renal failure and 183 healthy adults. Cryoglobulinaemia was present in 30.6% of dialysis patients with HCV infection, 10.8% of dialysis patients without HCV infection, 29.8% of patients with chronic hepatitis C without renal failure, and 0% of healthy adults. Among the 30 new renal failure patients who were started on dialysis within 6 months, four were positive for HCV antibodies, and one of them had cryoglobulinaemia; of the 26 HCV-negative patients, four (15%) were cryoglobulinaemic. The cryocrit values among dialysis patients were much lower than those of the control cases and other reports on non-dialysis cases. Patients with cryoglobulinaemia were generally younger compared with patients negative for this condition. There was no correlation between cryoglobulinaemia and past blood transfusion, underlying disease or length of dialysis. Cryoglobulinaemic patients seem to develop renal failure at relatively young ages and a considerable proportion of cryoglobulinaemic dialysis patients may have already had cryoglobulinaemia at the time of the start of haemodialysis. There was no indication that the presence of cryoglobulin in serum adversely affects the liver disease nor increases serum virus load in HCV-infected dialysis patients. Thus, it was concluded that although HCV infection has a certain role in the development of cryoglobulinaemia in dialysis patients, they develop cryoglobulinaemia less frequently and produce cryoglobulin to a lesser degree in the presence of HCV infection as compared with non-dialysis patients.

Adsorption of hepatitis C virus particles onto the dialyzer membrane.

It was recently found that the blood level of hepatitis C virus (HCV) RNA is significantly reduced after each dialysis procedure in patients on chronic hemodialysis. This study was designed to elucidate the mechanism for this phenomenon. In two patients with high serum levels of HCV RNA, the filtrate through the dialyzer (TF-alpha, Teijin Co., Tokyo, Japan) was analyzed for viral RNA using the polymerase chain reaction. At the end of dialysis, the filter was washed with saline, and during the saline washing, aliquots were taken for quantification of RNA by the branched DNA method. The HCV core antigen was quantified as a measure of viral particles, and hemoglobin was also measured for correcting for blood contamination. After the clearance of the blood, the filter was washed with guanidinium isothiocyanate, and the recovery of RNA was measured. The filtrate did not contain detectable RNA. The saline washing of the filter after dialysis contained a significant amount of RNA. Washing with guanidinium isothiocyanate of the thoroughly saline washed filter also recovered a significant amount of RNA. During saline washing, the recovery of RNA in the washing was much delayed behind that of hemoglobin, suggesting the adsorption of the former onto the filter membrane. There was a discordant recovery of RNA and HCV core antigen in the washing, the recovery of the former being delayed behind that of the latter. These results indicate that viral particles are adsorbed onto the inner surface of the filter membrane during dialysis. Some of these adsorbed viral particles are perhaps destroyed by hydraulic pressure applied to blood for dialysis.

GB virus-C infection among chronic haemodialysis patients: clinical implications.

It is known that patients on chronic haemodialysis are frequently infected with hepatitis C virus (HCV). It has recently been found that GB virus-C (GBV-C) and hepatitis G virus frequently coinfect patients with HCV. This study aimed at elucidating the clinical implications of GBV-C infection among haemodialysis patients who have and do not have HCV infection. GBV-C RNA was detected in sera of randomly selected 98 anti-HCV-positive and 85 -negative patients on dialysis by reverse transcription-polymerase chain reaction using two sets of amplification primers made from the reported sequences of the non-structural protein 3 and 5' untranslated regions. In these patients, liver function tests were carried out at regular intervals. There were six patients who were coinfected with HCV and GBV-C and three who had only GBV-C RNA. All had a history of past blood transfusion. The onset of mild hepatitis was identified in three HCV-negative patients; elevation of alanine aminotransferase (ALT) following blood transfusion was very mild but recognizable, and aspartate aminotransferase (AST) was higher than ALT. In two of six coinfected patients, the onset of liver disease was recognized with a peak ALT of 72 and 90 IU/L, respectively. Two of these six were Amplicore (HCV-RNA) negative and asymptomatic, two had low-grade HCV viraemia and two moderate-grade HCV viraemia. Of the 98 anti-HCV-positive cases, 41 were thought to have had nosocomial infection of HCV or non-A, non-B virus; none of them had GBV-C. GBV-C RNA was negative in nine patients who had chronic non-A-E hepatitis. GBV-C infection was detected in 6.1% of anti-HCV-positive and in 3.5% of -negative dialysis patients. All had blood transfusion in the past, and there was no evidence of patient-to-patient spread of GBV-C in hospital. The liver disease was very mild and self-limited in GBV-C infection alone. The natural history of coinfected patients may be similar to that of those with chronic HCV infection, but the liver disease appears to be milder.

Galacto-oligosaccharide production from lactose by Sirobasidium magnum CBS6803.

Galacto-oligosaccharide (Gal-OS) was produced from lactose by a yeast, Sirobasidium magnum CBS6803. With toluene-treated resting cells, 136 mg ml-1 of Gal-OS was produced from 360 mg ml-1 of lactose at 50 degrees C for 42 h. Then, the yield of Gal-OS was increased by a culture method in which cell growth followed the enzymatic reaction: 224 mg ml-1 of Gal-OS was produced at 30 degrees C for 60 h. Finally, combination of the toluene-treated resting cells and glucose oxidase plus catalase was applied to improve productivity by the removal of a by-product, glucose, which inhibits the Gal-OS production, from the reaction mixture. In this case, 242 mg ml-1 of Gal-OS was produced at 50 degrees C for 42 h without cell growth. The structure of the major product was identified as 4'-galactosyl-lactose.

Production of Galacto-Oligosaccharide from Lactose by Sterigmatomyces elviae CBS8119.

Volume 61, no. 11, p. 4022, column 2, line 10: "K(inf2)HSO(inf4)" and "KH(inf2)SO(inf4)" should read "K(inf2)HPO(inf4)" and "KH(inf2)PO(inf4)," respectively. Line 13: "K(inf2)HSO(inf4)" should read "K(inf2)HPO(inf4)." Line 14: "KH(inf2)SO(inf4)" and "0.2 g of CaCO(inf3)" should read "KH(inf2)PO(inf4)" and "1 g of CaCO(inf3)," respectively. [This corrects the article on p. 4022 in vol. 61.].

Production of galacto-oligosaccharide from lactose by Sterigmatomyces elviae CBS8119.

Our stock cultures were screened for microorganisms that can produce galacto-oligosaccharide (Gal-OS) from lactose. Of the 574 strains of bacteria and yeasts tested, Sterigmatomyces elviae CBS8119, Rhodotorula minuta IFO879, and Sirobasidium magnum CBS6803 were found to be efficient producers of Gal-OS from lactose and S. elviae CBS8119 was selected as a representative, high-level producing strain. With toluene-treated resting S. elviae CBS8119 cells, 135 mg of Gal-OS per ml was produced from 360-mg/ml lactose. During this reaction, the by-product glucose was found to inhibit Gal-OS production. Therefore, in order to remove the glucose from the reaction mixture, a culture method in which cell growth followed the enzymatic reaction was devised, which increased the yield of Gal-OS considerably because of the consumption of glucose for cell growth. Under such conditions, 232 mg of Gal-OS per ml was produced from 360-mg/ml lactose after incubation at 30 degrees for 60 h. The structure of the major product was identified as O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-galactopyranosyl-(1-->4)-D- glucopyranose (4'-galactosyl-lactose) by 13C nuclear magnetic resonance spectroscopy.

Hypoaminotransferasemia in patients undergoing long-term hemodialysis: clinical and biochemical appraisal.

BACKGROUND & AIMS: It has been reported that patients with chronic renal failure have low serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels as a result of vitamin B6 deficiency. Chronic hepatitis C is common among patients undergoing hemodialysis, and low aminotransferase levels cause diagnostic problems. The aim of this study was to determine the cause of hypoaminotransferasemia. METHODS: Serum levels of vitamin B6 and its coenzyme were reassessed in relation to AST and ALT levels in patients undergoing long-term hemodialysis using high-performance liquid chromatography. RESULTS: The mean (+/- SD) serum AST and ALT levels in 304 patients negative for hepatitis B surface antigen and anti-hepatitis C virus were 9.2 +/- 2.4 and 7.4 +/- 1.7 IU/L, respectively. In 556 normal adults, they were 22.7 +/- 5.4 and 18.0 +/- 4.0 IU/L, respectively (P < 0.001). Mean serum vitamin B6 and pyridoxal-5'-phosphate levels in patients undergoing dialysis were not reduced compared with the control, although occasionally patients had low levels. The AST and ALT assay reagents in Japan do not contain added pyridoxal-5'-phosphate; addition of pyridoxal-5'-phosphate (0.1 mmol/L) to the reagent significantly increased measurements to similar extents in both groups. CONCLUSIONS: Serum AST and ALT levels in patients undergoing dialysis are very low, but they are not a result of vitamin B6 deficiency. The upper normal limits of AST and ALT levels in patients undergoing dialysis should be reduced considerably, and these levels should be interpreted with caution in the diagnosis of liver disease.

Interferon treatment for chronic hepatitis C in haemodialysis patients: suggestions based on a small series.

Chronic hepatitis C is endemic among chronic haemodialysis patients. There have been a number of reports on hepatocellular carcinoma developing in such patients in Japan. The present study reports on the treatment of 15 patients who showed elevated ALT levels due to biopsy proven chronic hepatitis C with interferon alpha-2a (IFN). The dose schedule was 6 mega units (MU) daily for the first two weeks followed by 3 doses per week for 5.5 months. Side effects were so severe that IFN treatment was discontinued early in one patient, the dosage reduced in 11 and only tolerated in the original schedule by three patients. Excluding one patient who only recently completed the therapy, 13 were able to be evaluated for therapy efficacy by assessment of serum ALT and viral RNA. The overall results showed that IFN was effective in eight of 13 patients, a rate somewhat higher than the reported figures in this country. It is concluded that IFN therapy is indicated in haemodialysis patients with progressive chronic hepatitis C, but the dose administered should be lower and the dose schedule more flexible, perhaps 3 MU three times a week, in order to minimize untoward side effects.