Showing papers by "Kagawa University published in 1999"

Gene Duplication and Multiplicity of Collagenases in Clostridium histolyticum

Abstract: Clostridium histolyticum collagenase contains a number of different active components. Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase. We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG). We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH. Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA. These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments. The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively. Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome. C. histolyticum colG is more similar to C. perfringens colA than to colH in terms of domain structure. Both colG and colA have a homologous gene, mscL, at their 3' ends. These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C. histolyticum and C. perfringens and that further divergence of the parent gene produced colG and colA.

Combined spectral karyotyping and DAPI banding analysis of chromosome abnormalities in myelodysplastic syndrome.

Abstract: Spectral karyotyping (SKY) is a new molecular cytogenetic technique that allows simultaneous visualization of each chromosome in a different color. We have used SKY for comprehensive analysis of 20 myelodysplastic syndromes (MDSs) (13 primary MDSs, 3 therapy-related MDSs, and 4 acute leukemias developed from MDS, including 1 cell line established from a secondary leukemia), previously analyzed by G-banding. To locate the chromosomal breakpoints, DAPI-counterstained band images from all metaphases were transformed to G-band–like patterns. By using SKY, it was possible to identify the origin and organization of all clonal marker chromosomes (mar), as well as the origin of all abnormalities defined as additional material of unknown origin (add) or homogeneously staining regions (hsr) by G-banding. In total, SKY identified the chromosomal basis of 38 mar, add, and hsr, corrected 8 abnormalities misidentified by G-banding, and revealed 6 cryptic translocations in 5 cases. Total or partial chromosomal loss (mainly of -5/5q- and -7/7q-) is the most frequent cytogenetic abnormality in MDS. In 3 of 11 cases with -5/5q- and in 4 of 8 with -7/7q-, lost material was detected by SKY in unbalanced translocations. A total of 60 chromosomal losses were identified by G-banding in 16 cases with multiple chromosome abnormalities involving at least 3 chromosomes. For 26 of these losses (43%), SKY analysis suggested that the losses were not complete, but had been translocated to a variety of partner chromosomes. Moreover, SKY analysis revealed that a ring chromosome in a case of acute leukemia developed from MDS contained three to six segments that originated from chromosome 21 material. Fluorescence in situ hybridization showed the amplification of the AML1 gene on regions derived from chromosome 21, providing the first evidence of amplification involving this gene in MDS. Genes Chromosomes Cancer 26:336–345, 1999. © 1999 Wiley-Liss, Inc.

Enhancement of ultrasound-accelerated thrombolysis by echo contrast agents: dependence on microbubble structure

Abstract: The combination of ultrasound (US) exposure and ultrasound contrast agent (UCA) further increases the amount of drug-mediated thrombolysis. The aim of this study was to examine the efficacy of the combination of US and UCA on tissue plasminogen activator (tPA) thrombolysis, and the dependence on the microbubble structure. A catheter-type transducer capable of US emission (10 MHz, spatial peak temporal average intensity = 1.02 W/cm2 and peak negative pressure = 0.33 MPa) in the continuous-wave mode was employed. In 28 artificial white thrombi, serial changes in acoustic properties monitored by echography and histopathology during the tPA-mediated thrombolysis were analyzed. The thrombi were assorted to 4 groups; UCA nontreated (Control), sonicated albumin (A)-, SH-U508A (SH)- and dodecafluoropentane emulsion (DDFP)-treated groups. Persistence of microbubble opacification and thrombus weight were also measured. After the sample was suspended in a beaker with tPA (8000U) and 100 mL of saline, the UCA was administered and the mixture exposed to US for 10 min. Weight reduction of the thrombus was greatest in the DDFP Group (-49 +/- 8%), and that in the A Group (-8 +/- 5%) was not significantly different from that in the Control Group (-5 +/- 1%). The persistence of the microbubbles expressed as the decay of the time-intensity curve, was longest in the DDFP Group. The echo intensity of the superficial layer of the thrombus exposed to US was high and weight loss was marked. Multiple cavity formation was observed histopathologically. The stability of the microbubbles was an important factor of the US and UCA enhancement effect on tPA-mediated thrombolysis. This combination therapy has potential for clinical application in patients with thrombotic arterial and venous occlusion and left arterial thrombus.

Mycotoxins (trichothecenes, zearalenone and fumonisins) in cereals associated with human red-mold intoxications stored since 1989 and 1991 in China.

Abstract: Two corn powder samples implicated in the human food poisoning that occurred in Guangxi province in 1989, and eight wheat and two barley samples linked to an episode that involved about 130,000 people in gastrointestinal disorders in Anhui province in 1991 were analyzed for trichothecenes including deoxynivalenol (DON), nivalenol (NIV) and their esters, zearalenone (ZEA) and fumonisins (FMs) by gas chromatography/mass spectroscopy and high performance liquid chromatography, and T-2 toxin by enzyme-linked immunosorbent assays. DON was detected in all samples as a major trichothecene (16-51,450 microg kg(-1)), and NIV was in one corn, one barley and all wheat at relatively low levels (10-6935 microg kg(-1)). ZEA was found in all corn and barley, and six wheat samples (46-3079 microg kg(-1)). In addition, 3-acetyl-DON (2544 microg kg(-1)) and 15-acetyl-DON (2537 microg kg(-1)) were detected separately in one corn and one wheat sample. The highest levels of these mycotoxins were found in one wheat sample associated with the human intoxication in Anhui province. FMs in corn were below 1000 microg kg(-1). Risks of DON and ZEA on the people who consumed the causative cereals were assessed.

Effect of droplet size on oxidation of docosahexaenoic acid in emulsion system

Abstract: We investigated the effect of oil droplet size on the oxidation rate of DHA monodispersed emulsion with xanthan. Xanthan-free emulsions creamed rapidly but no creaming was observed in the emulsions containing xanthan over 8 weeks. While the peroxide value (POV) of the xanthan-free emulsion reached 10meq/ kg at 20 days, that of both emulsion systems containing xanthan changed little during the first 20 days. The POV for the emulsion with small droplet was higher than that for the emulsion with large droplet.

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives

Abstract: A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 µg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 µg/ml; for another, 2.9 µg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 µg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8-21 µg/ml.

The hydA gene encoding the H2‐evolving hydrogenase of Clostridium perfringens: molecular characterization and expression of the gene

Abstract: A putative hydrogenase (hydA) gene of Clostridium perfringens encodes a protein with strong identity to Clostridium pasteurianum hydrogenase I. Disruption of the hydA gene abolished H2 productivity, confirming its function. A putative butyrate kinase gene (buk) is adjacent to the hydA gene. When cultures were grown in medium with glucose, 1.8-kb hydA and 2.1-kb buk transcripts and a 3.9-kb transcript hybridized with both hydA and buk-probe were detectable in all the exponential growth phases. In medium without glucose, these transcripts were decreased rapidly after the mid-exponential phase. These results suggest that the transcription of these two genes is probably regulated by a similar mechanism in response to glucose availability.

Fossil Flowers and Associated Plant Fossils from the Kamikitaba Locality (Ashizawa Formation, Futaba Group, Lower Coniacian, Upper Cretaceous) of Northeast Japan

Abstract: in situ spores, and megaspores also document the presence of Selaginellaceae and Schizaeaceae.

Al binding in the epidermis cell wall inhibits cell elongation of okra hypocotyl

Abstract: Al inhibits root elongation at micromolar concentrations, but the mechanisms leading to this process are unknown. In these studies, Al-induced inhibition of cell elongation was examined using hypocotyl of okra (Abelmoschus esculentus Moench cv. Clemson Spineless) as an experimental model. One-h exposure to Al (0.5 mM A1CI3) in the presence of 10 /uM auxin in 0.5 mM CaCI2, pH 4.0 significantly inhibited auxin-induced cell elongation of okra hypocotyl segments. Elongation was further suppressed with increasing Al concentration s up to 1 mM. Treatment of the hypocotyl with 1 mM citrate for 10 minutes after 2-h exposure to Al resulted in significant recovery of elongation. The amount of Al in the cell wall relative to the total in the tissue was 96.0,96.2, and 85.4%, respectively, following 1-, 2-, and 3-h exposure to the Al solution. The total and cell wall Al content was decreased by half after the citrate desorption treatment. Furthermore, 95% of Al was found in the epidermis, and 95% of the Al in the epidermis was associated with the cell wall. Experiments using split hypocotyl segments showed that Al exposure increased the outward bending of hypocotyl segments, suggesting that the epidermis elongation was specifically inhibited by Al. Al inhibited the autolysis of epidermis by about 20%, but had little effect on the autolysis of core tissue. Taken together, these results suggest that Al binding in the epidermal cell wall inhibits critical components in cell wall loosening mechanism, resulting in inhibition of cell elongation.

Evaluation and application of a simple and rapid method for the analysis of aflatoxins in commercial foods from Malaysia and the Philippines.

Abstract: For application to the analysis of aflatoxins (AF) in commercial peanut and corn products, the ISOLUTE multimode column (IMC, solid phase multifunctional column) method was validated by comparing with the modified Florisil column (MFC) method. Twenty-two peanut and eight corn products from Malaysia and the Philippines were analysed for AFB1, AFB2, AFG1 and AFG2 firstly by the MFC method and then by the IMC method. For peanut products, 14 out of 22 samples were positive by the two methods in the range of 1-378 micrograms/kg of AF, and correlation coefficients (r) for AFB1 and AFB2 were 0.987 and 0.997, respectively. For corn and corn products, all the samples were positive in the range of 1-130 micrograms/kg, and r values were 0.992 and 0.805 for AFB1 and AFB2 respectively. Thus, the results were significantly (p < 0.01) in close agreement, particularly for lower range of 1-50 micrograms/kg of AF concentrations in all the samples. For the occurrence of AF, 11 (65%) of peanut products from Malaysia were contaminated with AF at a mean level of 50 micrograms/kg (maximum 180 micrograms/kg) and two (40% products from the Philippines were contaminated with as high as 375 micrograms/kg and 177 micrograms/kg of AF, respectively. All the corn products from the Philippines were contaminated with AF at a mean level of 44 micrograms/kg (maximum 130 micrograms/kg). Contamination of commercial foods with high levels of AF is a very important issue to both the countries since these foods are very popular among children.

Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner

Abstract: The phospholipase C gene (plc )o fClostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP–plc promoter complex through extension of the contact region. The upstream (UP) element of the EscherichiacolirrnBP1promoterstimulatedthedownstream promoter activity temperature independently, differing from the phased A-tracts. When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts. These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature.

Relationship between myocardial tissue density measured by microgravimetry and sound speed measured by acoustic microscopy.

Abstract: If myocardial tissue can be assumed to be fluid-like, myocardial tissue elasticity can be estimated by the sound speed of tissue based on the equation K=ρc2, where K is the elastic bulk modulus, ρ is density, and c is the sound speed of tissue. However, little data exist regarding the relationship between the sound speed of tissue and tissue density. The purpose of the present study was to evaluate the relationship between the sound speed of tissue and tissue density of various diseased myocardia. Myocardial tissue specimens at autopsy were obtained from 10 control patients without cardiovascular disease, 8 patients with pressure overload left ventricular hypertrophy (POLVH), and 8 patients with cardiac amyloidosis (AMD). Myocardial tissue sound speed was measured using a scanning acoustic microscope operating in the frequency of 450 MHz, and tissue density was measured by microgravimetry. The sound speed in POLVH (1639 ± 17 m/s) was higher and that in AMD (1565 ± 11 m/s) was lower than that in control patients (1615 ± 15 m/s) (p < 0.001) at the temperature of 20–22°C. The density in POLVH (1.087 ± 0.004 g/cm3) was higher and that in AMD (1.072 ± 0.003 g/cm3) was lower than that in control patients (1.082 ± 0.003 g/cm3) (p < 0.001). Tissue density correlated with sound speed in all three groups (r = 0.96, p < 0.001). Therefore, myocardial tissue sound speed data obtained by acoustic microscopy enabled us to evaluate tissue elasticity without measuring tissue density directly.

Elevation of anti-cytokeratin 19 antibody in sera of the patients with idiopathic pulmonary fibrosis and pulmonary fibrosis associated with collagen vascular disorders.

Abstract: It has been suggested that cytokeratin 19 is expressed in regenerated bronchoepithelial cells in patients with pulmonary fibrosis, and serum cytokeratin 19 fragment is elevated in patients with pulmonary fibrosis. We hypothesized that serum antibodies to cytokeratin 19 may be formed in patients with pulmonary fibrosis. To prove the existence of anti-cytokeratin 19 antibodies in patients' sera, human recombinant cytokeratin 19 was stained with patients' sera by a Western immunoblot. Then, we tried to establish an enzyme-linked immunosorbent assay to quantitate anti-cytokeratin 19 antibody in the sera of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis associated with collagen vascular disorders (PF-CVD). We demonstrated the anti-cytokeratin 19 antibody in patient' sera by a Western immunoblot. In patients with IPF and PF-CVD, significantly high anti-cytokeratin 19 antibody was demonstrated compared with normal volunteers, patients with chronic bronchitis, and patients with pneumonia. These results suggest that anti-cytokeratin 19 antibody may have played a role in the process of lung injury in pulmonary fibrosis.

Effect of water temperature on the mass emergence of the mayfly, Ephoron shigae, in a Japanese river (Ephemeroptera: Polymitarcyidae)

Abstract: Summary 1. Winged stages of Ephoron shigae were collected every day during their emergence period during a 6-year period from 1989 to 1994, by net sweeps or a light trap along the Asahi-gawa River in western Japan. Emergence occurred mainly in September. 2. Coefficients of determination (r2) were calculated for the regression of the mean date of emergence against cumulative degree-days during various periods from late March to early September. 3. In both sexes the highest values of r2 were obtained for regression with degree-days from late June to late August. This indicates that thermal conditions during the late instars affected the emergence timing most strongly. The timing of emergence can be well predicted from the cumulative degree-days during summer.

Imprinted polymer catalysts for the hydrolysis of p-nitrophenyl acetate

Abstract: Network polymers imprinted with a transition state analogue of hydrolysis were prepared by co-polymerization of vinylimidazole assembled around p-nitrophenyl phosphate (transition state analogue) with divinylbenzene as a cross-linker. After removal of the template, the resulting polymers efficiently catalyzed the hydrolysis of p-nitrophenyl acetate. The inhibition effect of the template was also examined.

CD38-Mediated Signaling Events in Murine Pro-B Cells Expressing Human CD38 With or Without Its Cytoplasmic Domain

Abstract: To elucidate the signaling mechanism of CD38 (a transmembrane molecule highly expressed in immature hemopoietic cells), we transfected Ba/F3 murine pro-B cells with a cDNA encoding human CD38. CD38 ligation with anti-CD38 Abs caused rapid, transient, dose-dependent tyrosine phosphorylation of several proteins, including the tyrosine kinase TEC and the adaptor molecule CBL, and association of tyrosine-phosphorylated proteins with phosphatidylinositol 3-kinase p85. Exposure to anti-CD38 Abs or their F(ab')2 and Fab also induced tight aggregation of CD38-transfected Ba/F3 cells, which appeared to be Ca2+ and Mg2+ independent and did not involved LFA-1. Aggregation was abrogated by addition of the tyrosine kinase inhibitor herbimycin A and was delayed by the phosphatidylinositol 3-kinase inhibitor wortmannin, suggesting a link between biochemical events and cellular effects induced by CD38. Cell aggregation was accompanied by a decrease in cell recovery. After 3 days of culture on bone marrow-derived stroma, the mean (+/-SD) cell recovery in the presence of anti-CD38 (T16) was 10.5 +/- 9.2% (n = 7) of that in parallel cultures with an isotype-matched nonreactive Ab. Finally, CD38 ligation in Ba/F3 cells expressing a mutant human CD38 lacking the cytoplasmic domain induced tyrosine phosphorylation with intensity and kinetics similar to those seen with the entire protein. It also induced cell aggregation and decreased cell recovery. We conclude that CD38 triggers remarkably similar signaling pathways in human and murine immature B cells. This signaling is independent of the CD38 cytoplasmic domain, suggesting the existence of accessory transmembrane molecules associated with CD38.

Elevated serum and BAL cytokeratin 19 fragment in pulmonary fibrosis and acute interstitial pneumonia.

Abstract: Cytokeratin 19 fragment (CK19) levels in serum have already been documented as a useful tumour marker for lung cancer. In the present study, it was hypothesized that CK19 may be increased in the serum and epithelial lining fluid of the respiratory tract from patients with pulmonary fibrosis. CK19 was measured in the serum and bronchoalveolar lavage fluid (BALF) of patients with pulmonary fibrosis and the correlation between CK19 levels and clinical parameters evaluated. Nineteen patients diagnosed with idiopathic pulmonary fibrosis (IPF), eight with pulmonary fibrosis associated with a collagen vascular disorder (PF-CVD), seven patients with acute interstitial pneumonia (AIP), and 10 normal smokers as a control group were studied. CK19 levels in sera of patients with IPF and patients with PF-CVD were significantly increased compared to those of normal smokers. CK19 levels in sera of patients with AIP were significantly increased compared to those of other groups. CK19 values in the BALF of patients with pulmonary fibrosis were significantly elevated compared to those of normal smokers. CK19 values in sera charged according to the progression or improvement of the acute lung injury. Immunohistochemical study using pulmonary tissues obtained from patients with AIP demonstrated that the hyaline membrane and proliferating type II pneumocytes were stained by anti-human cytokeratin 19 antibody. These data demonstrated that the measurement of cytokeratin 19 fragment is a useful parameter to evaluate the activity of lung epithelial cell damage and repair.

Myeloid- and lymphoid-specific breakpoint cluster regions in chromosome band 13q14 in acute leukemia.

Abstract: Abnormalities of chromosome band 13q14 occur in hematologic malignancies of all lineages and at all stages of differentiation. Unlike other chromosomal translocations, which are usually specific for a given lineage, the chromosomal translocation t(12;13)(p12;q14) has been observed in both B-cell and T-cell precursor acute lymphoblastic leukemia (BCP-, TCP-ALL), in differentiated and undifferentiated acute myeloblastic leukemia (AML), and in chronic myeloid leukemia (CML) at progression to blast crisis. The nature of these translocations and their pathologic consequences remain unknown. To begin to define the gene(s) involved on chromosome 13, we have performed fluorescence in situ hybridization (FISH) using a panel of YACs from the region, on a series of 10 cases of acute leukemia with t(12;13)(p12;q14) and 1 case each with "variant" translocations including t(12;13)(q21;q14), t(10;13)(q24;q14) and t(9;13)(p21;q14). In 8/13 cases/cell lines, the 13q14 break fell within a single 1.4 Mb CEPH MegaYAC. This YAC fell immediately telomeric of the forkhead (FKHR) gene, which is disrupted in the t(2;13)(q35;q14) seen in pediatric alveolar rhabdomyosarcoma. Seven of the 8 cases with breaks in this YAC were AML. In 4/13 cases, the 13q14 break fell within a 1.7-Mb YAC located about 3 Mb telomeric of the retinoblastoma (RB1) gene: all 4 cases were ALL. One case of myelodysplastic syndrome exhibited a break within 13q12, adjacent to the BRCA2 gene. These data indicate the presence of myeloid- and lymphoid-specific breakpoint cluster regions within chromosome band 13q14 in acute leukemia.