Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas.

BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

PCR-RFLP genotypes associated with quinolone resistance in isolates of Flavobacterium psychrophilum.

A novel genotyping method for epizootiological studies of bacterial cold-water disease caused by Flavobacterium psychrophilum and associated with quinolone resistance was developed. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was performed on 244 F. psychrophilum isolates from various fish species. PCR was performed with primer pair GYRA-FP1F and GYRA-FP1R amplifying the A subunit of the DNA gyrase (GyrA) gene, which contained the quinolone resistance determining region. Digestion of PCR products with the restriction enzyme Mph1103I showed two genotypes, QR and QS. The difference between these genotypes was amino acid substitutions at position 83 of GyrA (Escherichia coli numbering). The genotype QR indicated an alanine residue at this position associated with quinolone resistance in F. psychrophilum isolates. Of the 244 isolates tested in this study, the number of QR genotype isolates was 153 (62.7%). In isolates from ayu (n=177), 146 (82.5%) were genotype QR. With combination of this technique and previously reported PCR-RFLP genotyping, eight genotypes were observed in F. psychrophilum isolates. Using this genotyping system, the relationships between genotype and host fish species, or locality of isolation, were analysed and are discussed.

A hairy root culture of melon produces aroma compounds.

Musk melon is the favorite fruit with a high market value in Japan, and the fragrance is one of the major factors determining the fruit quality of melon. In this study, mutant melon hairy roots which had been induced by means of the T-DNA insertion mutagenesis were found to produce volatile compounds with the fruity fragrance of mature melon. The volatile compounds were extracted and identified by GLC-mass spectrometry. Some essential oils such as (Z)-3-hexenol, (E)-2-hexenal, 1-nonanol, and (Z)-6-nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. The productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits.

Structural analyses of a precursory substance of bitterness: new polyisoprenepolyols isolated from an edible mushroom (Hypsizigusmarmoreus) by fast atom bombardment mass spectrometry.

New polyisoprenepolyols (hypsiziprenol AA and BA) were isolated from an edible mushroom (Hypsizigus marmoreus). These polyols occur as a mixture of homologous polyisoprene derivatives with 40-70 carbon atoms. Analyses by FAB/MS in the positive and negative ion modes are complementary with each other in that the former provides information on the number of hydroxy groups present while the latter specifies the isoprenoid sequence, and thus become a powerful tool for analyzing the structures of polyisoprenepolyols. No polyisoprenepolyols obtained here were found to have antitumor activity on NCI-H292 and EL-4 cell lines.

[Mitral valve replacement for three cases of hypertrophic obstructive cardiomyopathy--surgical treatment].

Three patients with obstructive cardiomyopathy underwent surgical treatment. Mitral valve replacement was performed in all three cases and myectomy of hypertrophic septal muscle was performed in one case. The pressure gradients between the left ventricle and the aorta was less than 10 mmHg in all cases after surgery, Clinical symptoms strikingly improved in three cases. An accurate surgical treatment could be achieved by choosing either myotomy-myectomy, mitral valve replacement or both in the setting of individual condition of each patients.

Characterization and distribution of O-glycosylated carbohydrates in the cell adhesion molecule, contact site A, from Dictyostelium discoideum.

This paper presents further investigation of the properties of carbohydrate II in the cell adhesion molecule, contact site A, from Dictyostelium discoideum. A purified contact site A was digested with Achromobacter protease I to produce a 31-kDa fragment to which carbohydrate II was mainly bound and a 21-kDa fragment containing the NH2 terminus of contact site A, which was identified as Ala-Pro-Thr-Ile-Thr-Ala. The NH2 terminus of the 31-kDa fragment was Thr-Glu-Ala-Thr-Thr-Ser. It was estimated from the cDNA sequence data of contact site A that more than 20 Ser/Thr residues exist as target sites for the O-linked oligosaccharides in the 31-kDa fragment, but not for the N-linked oligosaccharides. These results suggest that carbohydrate II exists as clustered O-linked oligosaccharides in the COOH terminus of contact site A. The results of two-dimensional electrophoresis confirm that oligosaccharides of contact site A contain sialic acids. Immunoelectron microscopy was carried out to define the organelle in which O-glycosylation by carbohydrate II occurs and how carbohydrate II antigens are distributed on the cell surface. The results show that O-glycosylation can occur in the Golgi apparatus in D. discoideum as observed in other cells, although this O-glycosylation was inhibited by tunicamycin. Furthermore, gold particles were densely concentrated in cell-cell contact regions but sparsely distributed in noncontact regions.

In situ PCR technique based on pricking microinjection for cDNA cloning in single cells of barley coleoptile and powdery mildew pathogen.

To clone cDNAs of mRNA specifically expressed at the infection sites, we applied the polymerase chain reaction (PCR) combined with pricking microinjection to barley coleoptile epidermis inoculated with powdery mildew pathogen. In essence, first-strand cDNAs were synthesized in situ the needle-pricked epidermal cells in which fungal haustoria had formed, and were subsequently amplified by PCR with synthetic primers. The amplified DNAs were subcloned into a plasmid vector for the construction of a cDNA library. The antisense RNAs were in vitro-transcribed from subcloned DNAs, labelled, and introduced into pathogen-invaded coleoptile epidermal cells by pricking microinjection. Target cell-specific cDNAs were identified by a specific in situ hybridization in the pathogen-invaded cells. This technique was also applied to the amplification and identification of cDNAs which were reverse-transcribed from mRNAs of targeted infection structures of the powdery mildew pathogens inoculated onto barley coleoptile epidermis.

Chitinous component of the cell wall of Fusarium oxysporum, its structure deduced from chitosanase digestion.

The cell of Fusarium oxysporum was digested with commercial Bacillus chitosanase. The chitosanase produced low molecular weight heterooligosaccharides consisting of GlcN and GlcNAc from the cell wall. A main component of the digestion products was identified as 2-amino-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-D-glucopyranose. The chitosanase appeared to be more effective than Streptomyces griseus chitinase for cell wall digestion. Moreover, maltose was unexpectedly found in the digestion products, indicating that the cell wall contains alpha-1,4-linked glucan chain as a polysaccharide component.

Fast atom bombardment mass spectrometry and linked scan analyses at constant B/E in the structural characterization of new polyisoprenepolyols isolated from an edible mushroom (Hypsizigus marmoreus).

Fast atom bombardment (FAB) mass spectrometry is a powerful tool for analyzing the structure of polyisoprenepolyols. Analyses in the positive and negative ion mode are complementary in that the former provides data on the number of hydroxy groups present while the latter provides data on the isoprenoid sequence. Some of this information is already available from routine FAB mass spectra. More detailed information is contained in the linked scan spectra.

Carbohydrate structures of the cell adhesion molecule, contact site A, from Dictyostelium discoideum.

We determined the carbohydrate structures of contact site A from Dictyostelium discoideum. The carbohydrate moieties of contact site A were released by hydrazinolysis. Fractionation of the deacidified oligosaccharide mixture by Bio-Gel P-4 column chromatography revealed that it was composed of four major oligosaccharides. Their respective structures were determined by sequential exoglycosidase digestion. It is known that contact site A consists of two kinds of carbohydrates, I and II. Taking together the previous and the present results, it was deduced that carbohydrate I comprises N-linked oligosaccharides and carbohydrate II O-linked ones. Furthermore, the relative molar contents of GalNAc and GlcNAc in reducing terminal suggested that contact site A contains 67% of N-linked and 33% of O-linked oligosaccharides.

Chitinous components of the cell wall of Fusarium oxysporum.

The cell wall of Fusarium oxysporum f. sp. lycopersici was digested with chitinase to analyze the structure of its chitinous components. In spite of a similar acetylation degree of the cell wall components to that of 25-35% acetylated chitosan, only N-acetylglucosamine disaccharide [(GlcNAc)2] was obtained from chitinase hydrolyzate of the fungal cell wall by CM-Sephadex C-25 column chromatography, while (GlcNAc)2 and several types of deacetylated chitooligosaccharides were separated from that of 25-35% acetylated chitosan. The results indicate that N-acetylglucosamine residues in the polysaccharide chains of the fungal cell wall are most likely condensed into some region, while acetylated residues are more scattered in 25-35% acetylated chitosan.

Identification of sialic acids in cell adhesion molecule, contact site A from Dictyostelium discoideum.

It has been believed that Dictyostelium discoideum cell membranes contain no sialic acid. In this study, however, we found that contact site A, the cell adhesion molecule of D. discoideum, is a major glycoprotein containing sialic acids. This suggests that sialic acid in non-reducing terminal plays an important role in the cell adhesion in which contact site A is involved.

Molecular cloning and characterization of the fusaric acid-resistance gene from Pseudomonas cepacia.

Fusaric acid-resistance genes (fus) were isolated from Pseudomonas cepacia. The nucleotides of the 5437 base pairs containing the fus genes were sequenced.

Selection for Fusarium wilt disease resistance from regenerants derived from leaf callus of strawberry.

Resistant lines of strawberry to the fungal wilt disease caused by Fusarium oxysporum f. sp. fragariae were selected strawberry plants regenerated from leaf-derived callus tissues. Regenerants were transplanted to a field heavily infested with this pathogen, and normally growing plants were selected as the putative resistant lines. Daughter plants produced vegetatively through runner formation of the lines were similarly tested in the pathogen-infested field over an additional three generations. Finally, two resistant lines were obtained from a total of 1,225 regenerants. The stable propagation of disease resistance in these lines was confirmed by directly inoculating the daughter plants with the pathogen and planting in a pathogen-infested soil. All of the control plants were efficiently infected and died within one month. The isolated plant lines grew and developed runners even after direct inoculation and produced daughter plants in this soil. Thus, the present study demonstrated the existence of somaclonal variation for disease resistance against a soil-borne fngal pathogen.

Suppression of the powdery mildew pathogen by chitinase microinjected into barley coleoptile epidermal cells.

An exogenous chitinase from Streptomyces griseus was introduced into coleoptile epidermal cells of barley (Hordeum vulgare) by microinjection, and the effect of injected chitinase on the growth or development of the powdery mildew pathogen (Erysiphe graminis f. sp. hordei) was examined. Prior to microinjection, an enzymatic degradation of fungal haustorium, the organ taking nutrients from host plant cells, was examined by treating fixed coleoptile epidermis harboring haustoria with this enzyme. The result showed that haustoria were effectively digested by chitinase, suggesting the effectiveness of chitinase treatment for suppressing the fungal development. Microinjection of chitinase was conducted using living coleoptile tissues inoculated with the pathogen. Epidermal cells in which the haustorial primordia had been formed, or in which the haustoria had matured, were selected as targets for injection. The result clearly indicated that injection at the stage of primordium formation was effective in completely digesting haustoria and suppressing the subsequent formation of secondary hyphae of the pathogen. In microinjection after haustorial maturation, hyphal elongation was considerably suppressed though there was no detectable morphological change in the haustoria. Thus, the present study provides the experimental basis for genetically manipulating barley to produce transgenic plants resistant to the powdery mildew disease.

Transient expression of the ß-glucuronidase gene introduced into barley coleoptile cells by microinjection.

A ß-glucuronidase gene was introduced directly into barley (Hordeum vulgare L. cv. Kobinkatagi) coleoptile cells by microinjection and transient expression of the gene was examined. Inner epidermis tissue of coleoptiles was excised and injected with plasmid DNA, pBI221, carrying cauliflower mosaic virus 35S promoter, ß-glucuronidase gene, and a nopaline synthase polyadenylation region. Histochemical assay for ß-glucuronidase production showed positive enzyme activity only in coleoptile cells injected with plasmid DNA. Expression of the ß-glucuronidase gene was examined chronologically using honogenates of injected coleoptile tissues. Glucuronidase activity first appeared after 6 hr, reached the maximum level 24 hr after injection, and decreased afterwards. These results suggest that microinjection of coleoptile tissues may be a useful approach for the genetic engineering of Gramineae plants in which protoplast regeneration is difficult.

Selection of bacterial wilt-resistant tomato through tissue culture.

Bacterial wilt-resistant plants were obtained using a tomato tissue culture system. A virulent strain ofPseudomonas solanacearum secreted some toxic substances into the culture medium. Leaf explant-derived callus tissues which were resistant to these toxic substances in the culture filtrate were selectedin vitro and regenerated into plants. These plants expressed bacterial wilt resistance at the early infection stage to suppress or delay the growth of the inoculated bacteria. On the other hand, complete resistance was obtained in self-pollinated progeny of regenerants derived from non-selected callus tissues. These plants showed a high resistance when inoculated with this strain, and were also resistant when planted in a field infested with a different strain of the pathogen.

Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance.

Tobacco mosaic virus-resistant tobacco was selected in vitro using callus tissues induced from axillary buds of systemically infected tobacco plants. Callus lines in which the virus was continuously multiplying were first isolated and redifferentiated into shoots. By the procedure, non-diseased, healthy shoots were successfully isolated from diseased shoots, which showed typical mosaic symptoms of the virus, and regenerated into intact plants.These regenerated plants showed resistance to virus inoculation, and selfed progeny of virus-resistant regenerants segregated the resistance and susceptibility according to the Mendelian system.

[Respiratory function of mitochondria fractionated from isolated hepatocytes of rats with obstructive jaundice].

Obstructive jaundice model was created using rats by the ligation of bile duct. One, 2, 3 weeks later, the respiratory function and morphology of the hepatic mitochondria were comparatively evaluated between mitochondria directly fractionated from liver tissue and mitochondria from isolated hepatocytes. Respiratory function of the former deteriorated with the duration of jaundice. ATP synthesis decreased to 75% of the control at 1 and 2 weeks, and 58% at 3 weeks after ligation. On the contrary, it was 97%, 88% and 87% of the control. On the contrary, it was 97%, 88% and 87% of the control at 1, 2 and 3 weeks in the latter. By electron microscopic examination, the size of mitochondria of the jaundiced liver is inconsistent and smaller in general than the control. These data suggest that the deterioration of mitochondrial function in the jaundiced liver is not caused mainly by the disturbance of mitochondria themselves, but caused by the respiratory inhibitory factors which exist around the mitochondria.

Intranuclear microinjection for transformation of tomato callus cells.

An efficient method, called the culture plate method, was devised for microinjection of foreign materials into nuclei of tomato callus cells. The culture plate method, used in this study, is advantageous because cells suitable for microinjection can be selected microscopically and the injected cells subsequently cultured in the same plate. With this microinjection system, some foreign materials were injected into nuclei of callus cells without causing detrimental effects. Kanamycin-resistant callus clones were obtained 1 month after injection from single cells whose nuclei were microinjected with a NPT II DNA fragment of the pE2KX plasmid.