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Biomedical subjects

G Kobayashi

Publications and source records attributed to G Kobayashi.

At least 19 recordsLinked to original sources
PubMed2005

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean.

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.

Amino Acid Sequence↗
PubMed2001

Differential diagnosis of large-sized pedunculated polypoid lesions of the gallbladder by endoscopic ultrasonography: a prospective study.

PURPOSE: We have previously reported the effectiveness of endoscopic ultrasonography (EUS) for the differential diagnosis of pedunculated polypoid lesions of the gallbladder, based on retrospective studies using resected specimens. We proposed the following diagnostic criteria for EUS findings: when the contour of a lesion is nodular or smooth, the lesion is diagnosed as a neoplasm, and when a lesion has a granular contour, it is diagnosed as a nonneoplasm. The present study was designed to verify the clinical utility of our EUS ctiteria prospectively. METHODS: Forty-six consecutive patients with pedunculated polypoid lesions of the gallbladder 10mm or greater in size diagnosed as nonneoplasms at the initial EUS, all of whom underwent follow-up examinations, were enrolled in this study. The occurrence of changes in these lesions during the observation period was examined. RESULTS: No evident changes in lesions were observed in 43 of the 46 patients. Spontaneous self-detachment of lesions was recognized during the observation period in the other 3 patients. CONCLUSIONS: EUS is useful for determining treatment indications for pedunculated polypoid lesions of the gallbladder, even when the lesions are large, and contributes to avoiding unnecessary surgery.

Adenomatous Polyps↗
PubMed2001

Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition.

GTP-binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G-1 to G-5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross-over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature-sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA-binding ability.

Amino Acid Sequence↗
PubMed2001

Distribution and significance of epithelial types in columnar-lined esophagus.

An abnormal columnar-lined esophagus (CLE) is characterized by the presence of cardiac mucosa (CM) oxynto-cardiac mucosa (OCM), and intestinal metaplastic epithelium (IM) between gastric oxyntic mucosa and esophageal squamous epithelium. Thirty-two patients with CLE measuring 2-16 cm long had 5-37 biopsies per patient that showed CM, OCM, or IM for a total of 424 biopsies. Detailed mapping of the distribution of epithelial types within the CLE showed a distinct zonation of epithelial types; CM was present throughout the CLE, whereas OCM and IM tended to occur in the distal and proximal part of the CLE, respectively. All 32 patients (64 of 68 biopsies) showed IM at the most proximal level, compared with 22 of 32 patients (40 of 102 biopsies) in the most distal level biopsies. The density of goblet cells was highest in the most proximal level. The differences in prevalence and density of goblet cells between most proximal and most distal level biopsies were highly significant. These data suggest that for a given number of biopsies within the CLE, the likelihood of finding IM is greatest when the biopsies are concentrated in the most proximal area of the CLE. We suggest that glandular transformation of squamous epithelium results in CM. which evolves into OCM and IM by development of specialized parietal cells and goblet cells, respectively. The severity and nature of reflux cause these epithelial transformations in a constant and predictable manner. Recognition of these changes permits the development of morphologic definitions of reflux disease and the characterization of the sequence of epithelial changes that represent the reflux-adenocarcinoma sequence.

Barrett Esophagus↗
PubMed2001

[Clinical efficacy of magnetic resonance cholangiopancreatography in the diagnosis of biliary tract diseases].

This study was carried out to clarify the efficacy of magnetic resonance cholangiopancreatography (MRCP) in the diagnosis of biliary tract diseases, Three hundred and eleven patients who underwent MRCP and ERCP during the period from January 1999 to December 1999 at our institution were included in the study. Two gastroenterologists who were blinded to results of direct cholangiography and final diagnoses interpreted MRCP images prospectively. The biliary duct was visualized by MRCP and ERCP in 99.4% and 89.5% of the cases, respectively. The sensitivity, specificity, and accuracy of MRCP in the diagnosis of choledocholithiasis were 95%, 97%, and 97%, respectively. MRCP depicted the presence of strictures with a sensitivity, specificity, and accuracy of 97%, 96%, and 96%, respectively. There were three false-positive cases because of artifacts from arterial compression, The accuracy of MRCP and ERCP in differentiation of benign strictures from malignant ones was 85% and 96%, respectively. Based on these results, it is concluded that MRCP has high capability in visualization of the biliary tree, and in detection of stones and strictures, To avoid over and under-diagnosis, awareness of some pitfalls inherent in, MRCP is important. We suggest that MRCP should be recommended for patients with suspicion of biliary tract diseases in the initial stage of making diagnosis.

Adult↗
PubMed2000

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli.

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.

Amino Acid Sequence↗
PubMed2000

DNA phase transition promoted by replication initiator.

DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.

Adenosine Triphosphate↗
PubMed2000

Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants.

A recombinant lipase, CWB-LipB, localized on the Bacillus subtilis cell surface and retaining lipase activity was unstable and not accumulated in a high yield. To improve the accumulation, we examined cell wall binding protease (wprA)- and/or sigma D (sigD)-deficient mutants, and also a NprE and AprA protease-deficient mutant as host strains. The nprE aprA mutation did not lead to a significant increase in the CWB-LipB accumulation. The wprA mutant accumulated a greater amount than the wild-type only in the stationary phase, but the sigD mutant accumulated a greater amount in both the exponential and stationary phases. The double mutant exhibited great accumulation of CWB-LipB, the amount being 36% of the total proteins extracted from the cell surface.

Bacillus subtilis↗
PubMed2000

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

Acyltransferases↗
PubMed2000

A case of amelanotic spindle-cell melanoma presenting as metastases to breast and axillary lymph node: diagnosis by FNA cytology.

Metastatic neoplasms to the breast are relatively rare. Spindle-cell lesions of the breast are also uncommon. Here we present a case of fine-needle aspiration (FNA) of an amelanotic, spindle-cell melanoma metastatic to the breast and axillary lymph node. The patient was a 47-yr-old female who presented with a right breast mass, left axillary adenopathy, and a pigmented skin lesion on the back. FNA of the right breast and left axilla showed malignant, nonpigmented spindle cells that were weakly positive for HMB-45 on immunocytochemistry. The skin biopsy showed a pigmented malignant melanoma with epithelioid features, and also weak positivity for HMB-45. Although malignant melanoma is one of the more common tumors to metastasize to the breast, this is the first known case that showed exclusive spindle-cell morphology. History and physical examination were crucial in making the correct FNA diagnosis. The cytologic differential diagnosis of spindle-cell tumors of breast and the discordant morphology between the primary and metastatic melanotic lesions observed in this case are discussed.

Axilla↗
PubMed2000

A phase II study of combined chemoradiotherapy for limited disease-small-cell lung cancer.

A study to evaluate the efficacy of cisplatin, doxorubicin, and etoposide chemotherapy with combined radiotherapy was undertaken in 26 patients with limited disease-small-cell lung cancer. Patients were treated with cisplatin (80 mg/m2) intravenously (i.v.) on day 1, doxorubicin (30 mg/m2) i.v. on day 1, and etoposide (80 mg/m2) i.v. on days 1, 3, and 5, every 4 weeks for four cycles. Thoracic irradiation of 40 Gy in 20 fractions was delivered during 4 weeks to the primary site starting on day 8 of the second cycle of chemotherapy. The objective response rate was 100%. A complete response was observed in 10 patients (38%). The median survival time was 23 months, and the 3-year survival rate was 42%. Seven patients (27%) continued to survive at least 8 years and remain free from disease. Grade III/IV leukopenia was observed in 25 patients (96%). Grade III/IV thrombocytopenia developed in 19 patients (73%). Grade III/IV esophagitis was not seen. Interstitial pneumonitis occurred in two patients. This regimen is effective and has acceptable toxicity for use in the treatment of limited disease-small-cell lung cancer.

Aged↗
PubMed2000

[Pulmonary hypertrophic osteoarthropathy associated with primary lung cancer].

The patient was a 61-year-old man admitted with the complaints of cough, arthralgia, and swelling of the legs. A chest roentgenogram and chest computed tomographic scan revealed a giant mass in the right upper lobe. Transperitoneal lung biopsy was performed, and a diagnosis of poorly differentiated adenocarcinoma was made. Physical examination confirmed swelling of the legs and clubbing of fingers on both hands. Bone scintigrams showed marked accumulation of 99 m-Tc-MDP in the long bones, bones of the hands, and patellae. These findings yielded a diagnosis of pulmonary hypertrophic osteoarthropathy associated with primary lung cancer. Although a high serum level of growth hormone was also detected, immunohistochemical analysis did not find growth hormone in the tumor itself. Chemotherapy and radiotherapy were performed but did not stop progression of the disease. The patient subsequently experienced worsening arthralgia and swelling of the legs. Steroid therapy rapidly alleviated the arthralgia and swelling, but not the clubbing of the fingers. Thereafter, the patient's serum CRP and ICTP dropped to normal levels, and the abnormal findings of bone scintigrams subsequently disappeared. The pulmonary hypertrophic osteoarthropathy was not clearly attributable to growth hormone. Steroid therapy was effective in this case. Bone scintigrams and serum CRP and ICTP may be useful indicators in the therapeutic follow-up and monitoring of patients with pulmonary hypertrophic osteoarthropathy.

Adenocarcinoma↗
PubMed2000

[IFOBT-positive gastric cancer].

Among 888 patients who underwent operation or endoscopic resection for gastric cancer (1994-1998), 75 patients, who had no colorectal disease or only small polyps 5 mm or less in diameter, were positive on the immunologic fecal occult blood test (IFOBT) (the positive group). They are compared with the other 813 patients (the negative group) as to the following 6 points: symptoms, presence of anemia, depth of invasion including macroscopic appearance, location, maximum diameter of lesions, and microscopic findings. The rate of positive-IFOBT gastric cancer was 8.4%. The average blood hemoglobin concentration was significantly lower in the positive group than in the negative group. Advanced cancers, especially type 2 and 3, were significantly more frequent in the positive group than in the negative group. The size of the lesions tended to be larger in the positive group than in the negative group. There was no difference between the groups as to symptoms, location, depth of invasion and microscopic findings. In conclusion, IFOBT-positive patients who have no colorectal disease or only small polyps 5 mm or less in diameter should be recommended to undergo upper gastrointestinal endoscopy.

Female↗
PubMed2000

[The significance of magnetic resonance cholangiopancreatography in acute cholecystitis].

To clarify the significance of magnetic renonance cholangiopancreatography (MRCP) in the acute phase of acute cholecystitis, MRCP was carried out in forty-five patients with acute cholecystitis in their acute phase. The MR pericholecystic high signal was observed in 38 of the 45 patients (84%). Enlargement of the gallbladder, presence of gallstones, and impacted stones was seen in 71%, 53%, and 18%, respectively. The MR pericholecystic high signal was classified into four categories: type 0, not observed; type 1, a liner high signal; type 2, a band-like high signal; type 3, a radiating high signal. In patients who showed a type 3 MR pericholecystic high signal, 91% required percutaneous transhepatic gallbladder drainage, and most of the gallbladders were diagnosed as necrotic cholecystitis by histology. The accuracy of MRCP for the diagnosis of choledocholithiasis was 96%. It was suggested that MRCP for patients with acute cholecystitis in the acute phase provides useful information for planning the treatment.

Acute Disease↗
PubMed2000

Accumulation of a recombinant Aspergillus oryzae lipase artificially localized on the Bacillus subtilis cell surface.

cutL cDNA encoding an extracellular lipase, L1, from Aspergillus oryzae was fused to the cell wall-binding domain (CWB) region of a plasmid, pHCB3R. SDS-polyacrylamide gel electrophoresis (PAGE) and zymography of proteins extracted from the cell surface of Bacillus subtilis 168 harboring a fused lipase plasmid (pHCB3RCL) revealed that the fused gene product, CWB-CutL, was localized in the B. subtilis cell wall and retained lipase activity. B. subtilis WASD (wprA sigD), recently used for the accumulation of CWB-LipB (the CWB protein fused with B. subtilis lipase B), was also a suitable host for the accumulation of CWB-CutL, the amount being 10% of the total proteins extracted from the cell surface.

Journal Article↗
PubMed1999

Co-expression of polyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonas caviae establishes copolyester biosynthesis pathway in Escherichia coli.

Polyhydroxyalkanoate biosynthesis genes of Aeromonas caviae were expressed in Escherichia coli LS5218 (fadR atoC(Con)), and the polyhydroxyalkanoate-producing ability of the recombinants was investigated. A LS5218 strain harboring only phaCAc (polyhydroxyalkanoate synthase gene) did not accumulate any polyhydroxyalkanoate from dodecanoate in spite of the existence of translated polyhydroxyalkanoate synthase protein, whereas co-expression phaCAc and phaJAc ((R)-specific enoyl-CoA hydratase gene) resulted in the accumulation of P(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer up to 7-11 wt% of dry cell weight from octanoate and dodecanoate. These results indicated that both phaCAc and phaJAc are essential for E. coli LS5218 to establish the polyhydroxyalkanoate biosynthesis pathway from alkanoic acids. The copolyester content in the strain expressing both the genes under the lac promoter control reached to 38 wt% from dodecanoate. Enzyme assays suggest that efficient monomer formation via beta-oxidation by a high level expression of phaJAc was important to achieve a high polyhydroxyalkanoate content in the recombinant E. coli.

Acyltransferases↗