Mesenteric phlebosclerosis associated with long-term oral intake of geniposide, an ingredient of herbal medicine.

BACKGROUND: Idiopathic mesenteric phlebosclerosis (IMP) is a rare disease, characterised by thickening of the wall of the right hemicolon with calcification of mesenteric veins. However, the aetiology remains unknown. AIM: To investigate the possible association of herbal medicines with IMP. METHOD: The clinical data of four of our own patients were collected. Furthermore, we searched for previous reports about similar patients with detailed descriptions of herbal prescriptions that they had taken. We compared herbal ingredients to identify the toxic agent as a possible aetiological factor. RESULTS: Clinical data on a total of 25 patients were summarised. Mean age was 61.8 years and there was female predominance (6 men and 19 women). The used Kampo prescription, the number of cases, and the mean duration of use were as follows: kamisyoyosan in 12 cases for 12.8 years, inshin-iseihaito in 5 cases for 13.4 years, orengedokuto in 4 cases for 14.3 years, inchinkoto in 1 case for 20 years, kamikihitou in 1 case for 19 years, seijobofuto in 1 case for 10 years and gorinsan in 1 case for an unknown duration. Only one ingredient, sansisi, was common to the herbal medicines of all 25 patients. This crude drug called geniposide in English is a major constituent of the Gardenia fruits. CONCLUSION: The long-term use of geniposide in herbal medicines appears to be associated with mesenteric phlebosclerosis.

Establishing nephrology nursing specialization in Japan: a matter of urgency.

This paper explores the current situation regarding the provision of renal services to patients with end-stage renal disease in Japan. The statistical data reveal that Japan is in a predicament regarding the current rate of incidence of end-stage renal disease in the population. The discussion highlights the fact that nephrology nursing practice is as yet not recognised as a speciality in its own right in Japan. Therefore Japanese nurses who work in the field of nephrology are still struggling to establish their own professional identity and to improve the professional level of their practice. This situation persists in Japan despite support from the literature, which confirms that appropriately qualified nephrology nurses are well equipped to provide specialised care. The recent decision by the Japanese Association of Nephrology Nurses is to establish the position of the specialised dialysis nurse, which does not cover the area of kidney transplantation and will start in 2004. It is expected that it will be an important challenge to improve professional skills and nephrology nursing status in Japan.

Quantitative evaluation of metastases in axillary lymph nodes of breast cancer.

We have established a highly sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect axillary lymph node metastases of breast cancer. Amplifying cytokeratin 19 (CK19) mRNA transcripts using real-time TaqMan PCR made it possible to quantify axillary metastatic burden. Metastases in 358 axillary lymph nodes obtained from 23 breast cancers of 22 patients were investigated by conventional haematoxylin and eosin (H&E) staining, immunohistochemical staining and quantitative RT-PCR assay. The detection rates of axillary lymph node metastasis using H&E staining, immunohistochemistry and RT-PCR assay were 4.5, 5.9 and 13.1%, respectively. RT-PCR assay was the most sensitive of these three methods for detecting lymph node metastases. Cytokeratin 19 mRNA expression values of both histologically and immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001), and those of histologically negative, but immunohistochemically positive lymph nodes were significantly higher than the values for lymph nodes judged to be negative by both histological and immunohistochemical methods (P<0.0001). Furthermore, metastatic rates of sentinel nodes were higher than the rates of nonsentinel lymph nodes as measured by all three methods. These results indicate that quantitative RT-PCR assay is a sensitive and reliable method for detecting lymph node metastasis. Furthermore, quantification of metastases in sentinel lymph nodes by quantitative RT-PCR assay may be useful to assess the entire axillary burden of breast cancer patients.

The SF-36: an instrument for measuring quality of life in ESRD patients.

This paper provides a review of four studies, which use the generic Medical Outcome Study Questionnaire 36-Item Short Form Survey (SF-36) to investigate the quality of life of patients with end-stage renal disease. Both English and translated versions of this instrument have been demonstrated to be valid. It is difficult however, to compare results from western and eastern populations as cultural differences may influence how people rate their quality of life. The SF-36 has been demonstrated to be a valid instrument when used to measure the quality of life of ESRD patients. Research findings from Europe and the United Kingdom demonstrate that patients who are treated with a successful renal transplant can experience a QOL which, is superior to that achieved with any dialysis modality and which is very close to the QOL scores of the general population. It is acknowledged that while the studies cited in this article all used the SF-36 the research design employed in each instance is not exactly comparable, and any conclusions drawn here require further testing.

[Four cases of multiple liver metastasis of colorectal cancer treated with hepatic resection after chemotherapy].

Four cases of multiple liver metastasis of colorectal cancer treated with hepatic resection after chemotherapy were investigated. The 4 patients consisted of 3 males and 1 female with a mean age of 67.8 years. Two patients underwent systemic chemotherapy, and the other 2 received hepatic arterial infusion chemotherapy. The effect was PR in all patients, and mean duration until resection was 8.5 months. One patient had lung metastasis and one had recurrence in a local lesion and residual liver after hepatic resection, but all patients remain alive. The longest survival periods in 4 patients who underwent hepatic resection and 13 who did not were 32 and 24 months, respectively. It is suggested that hepatic resection after chemotherapy for multiple liver metastasis of colorectal cancer may be useful for improvement of outcome.

Mass production of bacterial alpha 2,6-sialyltransferase and enzymatic syntheses of sialyloligosaccharides.

To supply alpha 2,6-sialyltransferase for the large-scale synthesis of sialoside, we investigated culture conditions for the production of sialyltransferase 0160. The addition of galactose and beef extract, and control of the pH of the culture medium were effective on the production of sialyltransferase 0160. The maximal enzyme productivity reached 550 units/L. Using a crude extract of Photobacterium damsela JT0160 cells as an enzyme source, enzymatic syntheses were performed with mono- and di-saccharides as the sialyl acceptors. It was clarified that a crude extract of P. damsela JT0160 cells can be used as an synthetic catalyst for the enzymatic synthesis of sialyloligosaccharides. Furthermore, the enzyme assay showed that sialyltransferase 0160 could transfer NeuAc to not only N-linked but also O-linked carbohydrate chains. These results indicated that an abundant supply of sialyltransferase 0160 and its broad specificity make possible the synthesis of sialoside on a large scale.

Cloning and expression of a marine bacterial beta-galactoside alpha2,6-sialyltransferase gene from Photobacterium damsela JT0160.

Sialyltransferase 0160, a bacterial sialyltransferase which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6, is produced by Photobacterium damsela JT0160. The gene coding for sialyltransferase 0160 (bst) was cloned, sequenced, and expressed in Escherichia coli. The sialyltransferase 0160 gene contains an open reading frame of 2,028 base pairs encoding a protein of 675 amino acid residues. The deduced amino acid sequence of sialyltransferase 0160 did not contain the sialylmotif and had no significant similarity to mammalian sialyltransferases. Crude extracts of cultured E. coli MV1184 cells carrying an expression plasmid for the sialyltransferase 0160 gene showed sialyltransferase activity, which was identified as beta-galactoside alpha2,6-sialyltransferase activity by enzymatic reaction product analysis. In addition, when mutant genes, lacking 3'-coding regions for COOH-terminal portions of the protein, which are thought to form alpha-helix structures, were expressed in E. coli MV1184, soluble-form enzymes were obtained. This implies that the COOH-terminal portion of sialyltransferase 0160 is required for membrane binding.

A carboxy-terminal pro-sequence of aqualysin I prevents proper folding of the protease domain on its secretion by Saccharomyces cerevisiae.

The precursor of aqualysin I, a subtilisin-type protease secreted by Thermus aquaticus, consists of four domains, an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence. A non-covalent N-terminal pro-sequence facilitates the production of active aqualsin I, when the C-terminal pro-sequence is deleted. The role of the C-terminal pro-sequence in protein secretion was analyzed using a Saccharomyces cerevisiae expression system. Deletion of the C-terminal pro-sequence resulted in increased secretion of aqualysin I, i.e., about three times as much as that in the case of the wild type. In the case of the wild-type precursor, non-secreted aqualysin I with the C-terminal pro-sequence was retained in the endoplasmic reticulum in an inactive form, suggesting that the C-terminal pro-sequence prevents the protease domain from taking on a properly folded structure, unlike the N-terminal pro-sequence.

Participation of c-met in the progression of human gastric cancers: anti-c-met oligonucleotides inhibit proliferation or invasiveness of gastric cancer cells.

The protooncogene c-met encodes the receptor for hepatocyte growth factor (HGF), a potent epithelial cell mitogen with scattering activity. In this study, we first screened c-met expression in human gastric carcinomas. Twenty-eight of 154 tumors (18%) were positively stained for MET proteins. The incidence of c-met expression increased with higher histopathological stages of the cancer. Second, we examined functional roles of c-met in cultured gastric carcinoma cells, using an antisense strategy. Cell lines used were MKN-45, TMK-1, and MKN-28. Among them, MKN-45 cells exhibited the highest c-met expression and grew in response to HGF, whereas TMK-1 cells had an ability to invade in an HGF-dependent manner. When antisense oligodeoxyribonucleotides complementary to c-met messenger RNA (mRNA) were administered to the culture medium, the content of MET protein was selectively decreased in either MKN-45 or TMK-1 cells, indicating that the antisense molecules did inhibit the translation of c-met mRNA. The growth of MKN-45 cells was markedly inhibited by the antisense c-met oligonucleotides in a dose-dependent manner, but not by sense or scrambled controls. The antisense oligonucleotides also effectively inhibited the migration of TMK-1 cells. These results indicate that c-met gene products may be causally related to the proliferation or invasion of gastric cancer cells, and that antisense c-met DNA has the potential to help circumvent the progression of gastric cancers.

Purification and characterization of a marine bacterial beta-galactoside alpha 2,6-sialyltransferase from Photobacterium damsela JT0160.

A bacterial sialyltransferase, named sialyltransferase 0160, was purified from a marine bacterium that had been isolated from seawater from Sagami Bay, Kanagawa. This strain has been identified as Photobacterium damsela, and named P. damsela JT0160. Sialyltransferase 0160 was purified 688-fold to homogeneity from the crude extract of the cells with a yield of 19% using a combination of anion exchange chromatography, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography. The purified enzyme migrated as a single band (61 kDa) on sodium dodecyl sulfate-polyacrylamide gel. This sialyltransferase was found to be a beta-galactoside alpha 2,6-sialyltransferase [EC 2.4.99.1] which catalyzes the incorporation of NeuAc from CMP-NeuAc into the galactose residue of the carbohydrate chain at position 6 on the basis of an analysis of the enzymatic reaction products with HPLC, 1H-, 13C-NMR spectroscopy, and fast atom bombardment mass spectroscopy.

Efficient production of Thermus protease aqualysin I in Escherichia coli: effects of cloned gene structure and two-stage culture.

The DNA sequence encoding Thermus protease aqualysin I was inserted downstream from a bacteriophage T7 promoter in an expression vector. In the T7 expression system, using a strain lacking an F' episome, aqualysin I was produced in soluble form without chemical induction. The deletions of part (30 amino acid residues) or all (105 residues) of the C-terminal pro-sequence from the C terminus significantly affected both cellular growth and the production of the enzyme. Complete deletion adversely affected both. In contrast, the 30-residue deletion markedly improved productivity by approximately four times compared to non-deletion, and shortened the time needed for the activation of a precursor to active enzyme. The concentration of inducer isopropyl beta-D-thiogalactopyrano-side (IPTG) was varied to examine its effects, and it was found that a low concentration of IPTG improved aqualysin I production. To avoid the inhibitory effects of acetic acid accumulation in the culture medium, the use of other carbon sources besides glucose was examined. When cells were cultivated with glycerol, the acetic acid level remained relatively low, and both good cellular growth and a high level of production were attained. The aqualysin I productivity for a fed-batch culture using two carbon sources, glucose and glycerol, reached more than 150 kU/ml enzymatically active aqualysin I.

Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter.

The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.

Fermentation conditions for efficient production of thermophilic protease in Escherichia coli harboring a plasmid.

Escherichia coli TG1, transformed with an expression plasmid pAQN carrying the aqualysin I (AQI) gene derived from Thermus aquaticus YT-1 under the control of the tac promoter, was cultivated under various conditions in order to find fermentation conditions for the efficient production of the thermophilic protease, AQI. The amount of AQI produced was closely related to the growth phase at the time of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, and the highest production was obtained when it was added during the exponential growth phase. The addition of yeast extract had a greater effect on AQI production than did Polypeptone or casamino acids, and AQI productivity increased from 1.1 x 10(3) kU/g to 2.7 x 10(3) kU/g cells when 2 g/l yeast extract was supplied. Furthermore, the specific growth rate improved from 0.35 h-1 to 0.89 h-1 when 5 g/l yeast extract was supplied. The culture temperature also affected AQI gene expression. When the temperature was shifted from 37 degrees C to 34 degrees C at the time of IPTG induction, 19 kU/ml enzymatically active AQI was obtained, corresponding to a 28% increase over the amount produced in a batch culture without a shift. This is about a 44-fold higher yield than was obtained from the original strain, T. aquaticus YT-1.

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.

Purification and characterization of a new agarase from a marine bacterium, Vibrio sp. strain JT0107.

A marine bacterial strain that decomposes the cell walls of some seaweeds, including a Laminaria sp. and Undaria pinnatifida, has been isolated from seawater. This strain has been classified to the genus Vibrio. One of the enzymes which the bacteria secreted into the culture medium was isolated and purified 45-fold from the culture fluid by a combination of ammonium sulfate precipitation and successive rounds of anion-exchange column chromatography. Purified protein migrated as a single band (M(r), 107,000) on sodium dodecyl sulfate-polyacrylamide gels. By amino acid sequence analysis, it was determined that this protein had a single N-terminal sequence that did not exhibit identity with the sequences of other agarases from marine bacteria. This novel enzyme was found to be an endo-type beta-agarase (EC 3.2.1.81) which hydrolyzes the beta-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopy ranosyl (1-->3)-D-galactose] and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose] at a pH of around 8. The optimum temperature was 30 degrees C. This enzyme did not decompose sodium alginate or lambda-, iota-, or kappa-carrageenan. This enzyme may be of practical application in gene technology in the isolation of DNA fragments from agarose gels after electrophoresis.

Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli.

Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1. The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence. In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65 degrees C and mature enzyme is detected. In the case of mutant genes containing partial deletions in the NH2-terminal pro-sequence, no proteolytic activity was detected and the precursor protein was found to be unstable in E. coli. These results indicate that the NH2-terminal pro-sequence is required to produce the active enzyme by stabilizing the precursor structure. Amino acid substitutions in the conserved sequence of the NH2-terminal pro-sequence found among subtilisin-type proteases made the processing faster compared with the wild type.

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli.

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.

[A case of pneumonitis due to serrapeptase].

A case of pneumonitis due to Serrapeptase was described. A 69-year-old man was treated with Serrapeptase for 16 days because of common cold, then fever, nonproductive cough and dyspnea developed and chest X-ray revealed diffuse fine granular shadows in bilateral lung fields. Once the administration of Serrapeptase was halted, symptoms, chest X-ray abnormalities and laboratory data improved markedly. The fraction of lymphocytes increased in bronchoalveolar lavage fluid and OKT4/T8 decreased. Microscopic examination of transbronchial lung biopsy showed interstitial pneumonia. Both leukocyte migration inhibition test and sensitized hemagglutination test were positive for Serrapeptase. Based on these findings, we diagnosed this case as Serrapeptase-induced pneumonitis.

Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme.

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.