Association of free fatty acids (FFA) and tumor necrosis factor-alpha (TNF-alpha) and insulin-resistant metabolic disorder.

The roles of free fatty acids (FFA), tumor necrosis factor-alpha (TNF-alpha), and adiponectin in the development of the insulin-resistant metabolic disorder in several subjects have been studied. A total of 70 Japanese male subjects were selected according to the following three sets of criteria: subjects in group A had, (1) a fasting plasma glucose (FPG)>or=110 to <140 mg/dl, (2) a triglyceride (TG) level>or=150 mg/dl, (3) a systolic blood pressure (SBP)>or=140 and/or diastolic blood pressure (DBP)>or=90 mmHg, and (4) a body mass index (BMI)>or=25 kg/m2 (age=53.4+/-8.5 years, BMI=27.0+/-1.3 kg/m2, n=16). Subjects in group B had, (1) FPG<110 mg/dl, (2) TG<150 mg/dl, (3) SBP<140 and DBP<90 mmHg, and (4) BMI>or=25 kg/m2 (age=47.2+/-10.3 years, BMI=26.6+/-1.31 kg/m2, n=38). Subjects in group C had, (1) FPG<110 mg/dl, (2) TG<150 mg/dl, (3) SBP<140 and DBP<90 mmHg, and (4) 20>or=BMI<22 kg/m2 (age=50.4+/-9.3 years, BMI=20.9+/-0.6 kg/m2, n=16). The homeostasis model assessment of insulin resistance in group A (2.7+/-1.4) was significantly higher (p<0.0001) than in groups B (1.6+/-0.7) and C (0.9+/-0.5). FFA in group A (1.17+/-0.57 mEq/l) was significantly higher than in groups B (0.62+/-0.23 mEq/l) and C (0.48+/-0.16 mEq/l) (p<0.0001). Serum TNF-alpha in group A (1.36+/-0.62 pg/ml) was significantly higher than in groups B (0.95+/-0.35 pg/ml; p=0.003) and C (0.76+/-0.09 pg/ml; p=0.0013). No significant differences in the serum level of adiponectin were observed between groups A and B or between groups B and C. The results suggest that FFA and possibly TNF-alpha levels are closely related to the development of insulin resistance in subjects with metabolic disorders.

de Haas-van Alphen effect in ZrZn2 under pressure: crossover between two magnetic states.

We report measurements of the de Haas-van Alphen effect in ZrZn2 under hydrostatic pressures up to 21 kbar where the Curie temperature vanishes. The exchange splitting of a Fermi surface changes in behavior with increasing magnetic field, which is qualitatively consistent with the behavior of the high-field magnetization, suggesting the existence of a crossover between two distinct magnetic states. These and previously unexplained findings may be understood in terms of a p-T-B phase diagram qualitatively similar to that of the ferromagnetic superconductor UGe2.

Construction and characterization of BAC libraries for three fish species; rainbow trout, carp and tilapia.

Bacterial artificial chromosome (BAC) libraries are important tools for genomic research. We have constructed seven genomic BAC libraries from three fish species, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and tilapia (Oreochromis niloticus). The two rainbow trout BAC libraries have average insert sizes of 58 and 110 kb. The average size of inserts in the carp BAC library is 160 kb. The average insert sizes of the four tilapia BAC libraries are 65, 105, 145 and 194 kb, respectively. These libraries represent good coverage of each genome (2-64 x coverage). The libraries can be screened by conventional colony hybridization and provide a starting point for the construction of high-density filtres or polymerase chain reaction (PCR) screening approaches. These BAC libraries will facilitate the positional cloning of quantitative trait loci (QTLs) for a variety of economically important traits in these species.

Molecular cloning, expression and evolution of the Japanese flounder goose-type lysozyme gene, and the lytic activity of its recombinant protein.

In this study, we cloned the goose-type (g-type) lysozyme gene from the Japanese flounder genomic DNA library, the first such data in fish and only the second after the chicken g-type lysozyme gene. The Japanese flounder g-type lysozyme gene was 1252 bp in length from the transcription site to the polyadenylation site, coded for 758 bp of mRNA and 195 deduced amino acids, which contain five exons and four introns. A phylogenetic analysis based on amino acid sequences showed that the flounder gene was closer to g-type lysozyme, followed by phage-type lysozyme and then chicken-type (c-type) lysozyme. Although exon 1 of the flounder gene differs from exons 1 and 2 of the chicken g-type lysozyme gene, three catalytic residues, as well as their neighboring amino acids were conserved between the Japanese flounder and the four avian g-type lysozymes. In a Southern blot analysis using the genomic DNA of homo-cloned Japanese flounder, the flounder g-type lysozyme gene showed a simple pattern, suggesting that it is encoded by a single copy gene. A Northern blot analysis showed that this gene was expressed in all tissues of Japanese flounder that we examined in this study and showed major differences from those expressed tissues of the chicken g-type gene. Japanese flounder g-type lysozyme mRNA levels in the intestine, heart and whole blood increased after injecting the fish with Edwardsiella tarda. Recombinant flounder g-type lysozyme, which has an optimal pH and temperature of pH 6.0 and 25 degrees C, possessed lytic activity against Micrococcus lysodeikticus and several fish pathogenic bacteria. This is the first report of a g-type lysozyme gene other than for reported avian species.

Expression of Japanese flounder c-type lysozyme cDNA in insect cells.

Lysozyme is a widely distributed hydrolase which likely plays an important role in bio-defense systems. In the current study, we constructed a baculovirus expression system for the c-type lysozyme cDNA of Japanese flounder (Paralichthys olivaceus) and evaluated the activity of the recombinant protein. This activity was estimated, by turbidimetric assay, to be 7.7U/mg, a value five times higher than that of hen egg white (HEW) c-type lysozyme examined under the same conditions. The optimum pH and temperature for the lytic activity of the Japanese flounder c-type recombinant lysozyme were found to be 5.0-6.5 and 40 degrees C, respectively. Two groupings for fish lysozyme activity are proposed; the first has an optimum pH of approximately 6.0 and the second an optimum pH of above 8.0. Flounder c-type lysozyme was found to possess little lytic activities against Edwardsiella tarda and Streptococcus sp. This latter characteristic may help explain the fact that these two bacterial species are responsible for significant disease problems in cultured Japanese flounder.

Induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cells.

5-Bromodeoxyuridine (BrdU) universally induces a senescence-like phenomenon in mammalian cells. To assess this phenomenon at the level of gene expression, we constructed a PCR-based subtractive cDNA library enriched for mRNA species that immediately increase by administration of BrdU to HeLa cells. Candidate cDNA clones were isolated by differential colony hybridization, and then positive clones were identified by Northern blot analysis. Sequencing analysis revealed that the identified cDNA species were classified into three groups: widely used senescence-markers, known species whose relevance to senescence is yet to be reported, and known or novel ESTs. As expected, the majority of them showed an increase in expression in senescent human diploid fibroblasts. These results suggest that similar mechanisms operate in the regulation of BrdU-induced genes and senescence-associated genes.

Antibacterial agents that inhibit histidine protein kinase YycG of Bacillus subtilis.

We demonstrated in vitro that YycG-YycF of Bacillus subtilis constitutes a two-component system and shows a specificity of the sensor protein for the cognate phosphorylation partner. Based on inhibition of such an autophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial agents such as NH125, NH126, NH127, and NH0891.

Tumor stage in the proximal colon under conditions of a proximal shift of colorectal cancer with age.

BACKGROUND/AIMS: Although the incidence of proximal colon cancer is known to increase with age, the prognosis of proximal colon cancer remains unclear. METHODOLOGY: One thousand two hundred and five patients with colorectal cancer were analyzed with regard to tumor location, age and Duke's stage. RESULTS: When Duke's stage was compared with tumor location, an advanced Duke's stage was found more frequently in the proximal colon than in the distal colorectum (p<0.01). In Duke's A colorectal cancer, the incidence of tumors limited to within the submucosal layer decreased from 73.1% in patients under 65 years of age to 60.9% in patients over 65 years of age. CONCLUSIONS: Since advanced stage cancer was more frequently found in the proximal colon than in the distal colorectum, careful examination for the early detection of proximal colon cancer, particularly in the elderly, is called for.

Possibility of different cancer development between the proximal and distal colon: comparison of the distribution between adenomatous polyps and cancer.

BACKGROUND/AIM: The aim of this study was to compare the distribution of adenomatous polyps with that of cancer in the colon by using adenomatous polyps removed by colonoscopy. METHODOLOGY: 1223 adenomatous polyps removed by an endoscopic polypectomy were compared with 226 colon cancers with regard to their distribution, size and age. RESULTS: With patients over 65 years of age, the incidence of adenomatous polyps was lower in the proximal colon than in the distal colon, however, the incidence of cancer was higher in the proximal colon than in the distal colon (p<0.05). The distribution of adenomatous polyps according to size was also significantly different between the proximal and distal colon (p<0.05). CONCLUSION: These results thus suggest that some difference may exist in cancer development between the proximal and distal colon.

Primary structure of a potassium channel toxin from the sea anemone Actinia equina.

A potassium channel toxin (AeK) was isolated from the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. AeK and alpha-dendrotoxin inhibited the binding of 125I-alpha-dendrotoxin to rat synaptosomal membranes with IC50 of 22 and 0.34 nM, respectively, indicating that AeK is about sixty-five times less toxic than alpha-dendrotoxin. The complete amino acid sequence of AeK was elucidated; it is composed of 36 amino acid residues including six half-Cys residues. The determined sequence showed that AeK is analogous to the three potassium channel toxins from sea anemones (BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS from Anemonia sulcata), with an especially high sequence homology (86%) with AsKS.

Esophagojejunostomy with manual single layer suturing after a total gastrectomy for gastric cancer.

BACKGROUND: We wished to verify the clinical usefulness of manually performed single layer suturing for an esophagojejunostomy after a total gastrectomy versus stapled suturing. METHODS: We compared retrospectively 24 patients who underwent manual single layer suturing with 38 patients who underwent stapled suturing. RESULTS: Anastomotic leakage was seen in one patient (4%) with single layer suturing and one patient (3%) with stapled suturing. No anastomotic stenosis was seen in the patients with single layer suturing. There was no difference in the operative time, blood loss, postoperative days for oral intake, or the length of hospital stay between the patients with single layer suturing and those with stapled suturing. CONCLUSIONS: Manual single layer suturing is considered to be as safe as stapled suturing and is also thought to be clinically useful in reducing anastomotic failure for esophagojejunostomy.

Isolation and amino acid sequences of two Kunitz-type protease inhibitors from the sea anemone Anthopleura aff. xanthogrammica.

Two protease inhibitors (AXPI-I and -II) were isolated from the sea anemone Anthopleura aff. xanthogrammica by a combination of acetone precipitation, gel filtration on Sephadex G-75, cation-exchange fast protein liquid chromatography (FPLC) on Mono S and reverse-phase HPLC on TSKgel ODS-120T. Both inhibitors are basic polypeptides, and their amino acid compositions are characterized by the presence of six half-Cys residues and the absence of Met and Trp. They are potently active against trypsin; inhibition of other serine proteases (alpha-chymotrypsin and elastase) is also displayed by only AXPI-I. However, the inhibitors show no affinity for metallo-proteases and cysteine proteases. Analyses of the N-terminal portion and enzymatic fragments established their complete amino acid sequences comprising 58 residues. The overall sequence homology and the conserved location of all half-Cys residues confirmed that the A. aff. xanthogrammica inhibitors belong to the Kunitz-type family.

Increased incidence of proximal colon cancer in the elderly.

To define the characteristics of colorectal cancer with regard to site distribution, age, and sex, due to the significantly increased incidence of colorectal cancer in Japan, we investigated 1,205 Japanese patients with colorectal cancer between 1975 and 1994. When we compared site distribution for age and sex in the entire 20-year period, tumors in the proximal colon were more frequent in elderly patients than in young ones (p < 0.05); this tendency was true only for females (p < 0.01), but not for males. The increased incidence of proximal colon cancer in elderly patients was also found in the second 10-year period (p < 0.05). Because elderly patients are characterized by an increasingly high incidence of proximal colon cancer in recent years, more concerted efforts for the early detection of proximal colon cancer, particularly in the elderly, are called for.

Prognostic value of combination assays for CEA and CA 19-9 in gastric cancer.

Preoperative serum CEA and CA 19-9 levels in 158 patients with gastric cancer were analyzed with respect to prognostic factors, using univariate and multivariate analysis. The incidence of high preoperative levels of both CEA and CA 19-9 was 10.1% (16/158). 13.9% (22/158) showed high CEA levels and normal CA 19-9 levels, whereas the reverse was true in 16.5% (26/158). Neither marker showed a high level in 59.5% (94/158). The multivariate analysis showed that in addition to tumor stage, the depth of invasion, liver metastasis and peritoneal dissemination, combination assays of preoperative serum CEA and CA 19-9 levels were an independent prognostic factor. Combination assays of preoperative serum CEA and CA 19-9 will allow us to conduct a more careful postoperative follow-up of high-risk patients, and also help determine the optimum adjuvant chemotherapy.