Glycobiology in medicine

Glycoconjugates play important roles in biological reactions (for example sialyl Lewis^x in homing of leukocytes and mannose-6-phosphate in targeting of lysosomal enzymes) and thus aberration in carbohydrate structures in glyco-conjugates can lead to abnormal biological behaviors. In fact, glycoconjugates expressed on the surfaces of tumor and cancer cells are considerably different from those of the normal cells, at least quantitatively. There are many known carbohydrate-deficient glycoprotein syndromes. As recognition of carbohydrate groups is mostly performed by carbohydrate-binding proteins, aberration in these proteins also results in disease status (for example I-cell disease). Many pathogens use carbohydrates as recognition markers for invasion (examples are influenza virus and cholera toxin). The carbohydrate receptors in various organs can be used for targeting drugs, antibodies and even DNAs. Conjugation of polysaccharides derived from pathogenic micro-organisms with appropriate proteins provides effective vaccines against the micro-organisms.     

Cloning and Sequencing of the Gene Encoding the Soluble Fumarate Reductase from Saccharomyces cerevisiae

A gene of the soluble fumarate reductase (FRDS) that binds FADnon-covalently was cloned by polymerase chain reaction (PCR)using degenerate oligonucleotides designed from partial aminoacid sequences of highly purified enzyme. The nucleotide sequenceof a 0.99-kb amplified product was found to be nearly identicalto a partial sequence of an open reading frame (ORF) previouslyreported (EMBL database accession number S-30830). Accordingto the sequence in the EMBL database, we cloned 1.7-kb fragmentcontaining entire sequence of this ORF by PCR and found thatthis fragment contained a perfect match to the 0.99-kb sequenceamplified with the degenerate primers. From these results, weconcluded that this ORF is the FRDS gene. The amino acid sequencesof the regions involved in the non-covalent binding of FAD andthe active site, which are conserved among the flavoproteinsubunits of membrane-bound fumarate reductase and succinatedehydrogenase, were found in FRDS. However, unlike the membrane-boundenzymes, FRDS did not contain the histidine residue that covalentlybinds the isoalloxazine ring of FAD at or near the correspondingposition. FRDS showed high homology to the product of S. cerevisiaeOSM1 gene which was reported to be required for growth in hypertonicmedia.     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5′-CACTA--- -3′; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

Morphological studies of the goldfish hindgut mucosa in organ culture

Summary The morphology of the absorptive cells of the goldfish hindgut mucosa, and their capability for horseradish peroxidase (HRP) uptake, were investigated by electron microscopy after a 24-h organ culture. The columnar appearance and the fine structure of the absorptive cells were well preserved for 24 h at room temperature and 37° C with 5% CO₂ in air, in all the media used in this study. Mitoses were frequently observed in the epithelium at the bottom of cultured mucosal folds, and re-epithelization was also observed in many explants.Some structural changes were, however, noted in the cultured absorptive cells, as compared with the non-cultured absorptive cells; the deep invaginations of the surface membrane between the microvilli decreased in number; supranuclear giant vacuoles were reduced in size or almost disappeared; the distributional pattern of mitochondria in the absorptive cells was altered.The HRP uptake experiments showed that the absorptive cells cultured for 24 h could still take up HRP by endocytosis and transport it, indicating that the absorptive cells maintained their capability of macromolecule uptake and transport after 24 h of culture. In addition, HRP experiments, in which reaction product was detected within numerous cytoplasmic tubules (CT), various vacuoles and CT-vacuole complexes, suggested a close relationship between CT and vacuolar system in the apical cytoplasm during endocytotic events in the absorptive cells.     

Effects of osmotic pressure on prolactin and growth hormone secretion from organ-cultured eel pituitary

Summary Effects of medium osmotic pressure on the release of prolactin (PRL) and growth hormone (GH) from the pituitary of the Japanese eel, Anguilla japonica, were examined during long-term organ culture in a defined medium. Prolactin and GH release, as measured by homologous radioimmunoassays, increased gradually for 7 days during incubation in isosmotic medium (295 mOsmolal). On day 7, 3 to 5 times more PRL and GH were released than on day 1. The amount of GH released was about 100 times greater than that of PRL. Electron microscopic observation revealed that both PRL and GH cells were in good condition after 7 days incubation. The reduction of medium osmotic pressure from 295 (isosmotic) to 235 or 260 mOsmolal significantly stimulated PRL release for 4 days. By contrast, an increase in medium osmolality from 295 to 360 mOsmolal was without effect. These treatments produced no significant alterations in GH release. The stimulatory effect of hyposmotic medium (235 mOsmolal) was no longer evident by 12 h after the pituitaries were returned to isosmotic medium. The isosmotic but low-sodium medium, prepared by adding mannitol to the hyposmotic medium, did not stimulate PRL release from the pituitary. These results indicate that plasma osmolality may be an important physiological factor controlling PRL release during freshwater adaptation of the eel.Abbreviations GH growth hormone - OAPBS PBS with 1% ovalbumin - PAGE polyacrylamide gel electrophoresis - PBS phosphatebuffered saline - PRL prolactin - rER rough endoplasmic reticulum     

Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids.

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.     

Evidence That More than 90% of beta-Glucuronidase-Expressing Cells after Particle Bombardment Directly Receive the Foreign Gene in their Nucleus

Plasmid DNA harboring the β-glucuronidase (GUS) gene, coated on gold particles, was delivered into cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells using a pneumatic particle gun. Cytological analyses of intracellular location of the introduced gold particles before and after GUS expression assay indicated that more than 90% of GUS-expressing cells after bombardment received a DNA-coated particle in their nucleus.     

Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity.

Ca(2+)-sensitive mutants of the yeast Saccharomyces cerevisiae showing a Pet- phenotype (cls7-cls11) have lesions in a system for maintaining intracellular Ca2+ homeostasis (Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132, 979-988). Genetic and biochemical studies have demonstrated that these Pet- cls mutants are related to defects in vacuolar membrane H(+)-ATPase. CLS7 and CLS8 were found to be identical with the structural genes encoding subunit c (VMA3) and subunit a (VMA1), respectively, of the enzyme. In addition, these five mutants all had vma defects; no vacuolar membrane ATPase activity was detected in the cls cells, and the cls mutants showed a loss of ability to acidify the vacuole in vivo. Measurements of the cytosolic free Ca2+ concentration [( Ca2+]i) in individual cells showed that the average [Ca2+]i in wild-type cells was 150 +/- 80 nM, whereas that in five Pet- cls cells was 900 +/- 100 nM. These data are consistent with the observation that vacuolar membrane vesicles prepared from the Pet- cls cells have lost ATP-dependent Ca2+ uptake activities. The cls defects of vacuolar membrane H(+)-ATPase resulted in pleiotropic effects on several cellular activities, including Ca2+ homeostasis, glycerol metabolism, and phospholipid metabolism. The mutants showed an inositol-dependent phenotype, possibly due to alteration in regulation of phospholipid biosynthesis; the phosphatidylserine decarboxylase activities of the mutants were 15-50% of that of the wild-type cells and were not repressed by the addition of inositol. In contrast to the majority of previously isolated pet mutants (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225), the Pet- cls mutants showed no detectable mitochondrial defects. Taking all these findings into account, we suggest that at least six genes, VMA1 (CLS8, subunit a), VMA2 (subunit b), VMA3 (CLS7, subunit c), VMA11 (CLS9), VMA12 (CLS10), and VMA13 (CLS11), are required for expression of the vacuolar membrane H(+)-ATPase activity.     

Spotted fever group rickettsia in dogs in Japan

Prevalence of antibody against spotted fever group-rickettsia in dogs (14/134) from the northern part of Shikoku Island, where spotted fever group rickettsia infection in human is endemic, is significantly higher than that in dogs (4/189) from nonendemic areas.