Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.     

Cloning and Characterization of the Murine GPI Anchor Synthesis GenePigf,a Homologue of the HumanPIGFGene

Many eukaryotic proteins are bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. Its core backbone, which is conserved in different organisms, is synthesized in the endoplasmic reticulum by the sequential addition of glycan components to phosphatidylinositol. One of the human GPI synthesis genes,PIGF(phosphatidylinositol glycan complementation class F), which is involved late in the synthesis pathway, has been cloned. In this study, we isolated complementary and genomic clones ofPigf,a murine counterpart ofPIGF. Pigfencodes a 219 amino acid protein that complements a class F mutation. ThePigfgene consists of six exons spanning 30 kb and was mapped to chromosome 17 at 17E4–E5. These features are very similar toPIGF,thus demonstrating the interspecies conservation of structure, function, gene organization, and genetic locus between these GPI synthesis genes. The results also extend a region in murine distal chromosome 17 that is syntenic to human chromosome 2p16–p22.     

The phylogenetic relationships ofEeniella nana Smith,Batenburg-van der Vegte et Scheffers based on the partial sequences of 18S and 26S ribosomal RNAs (Candidaceae)

Summary Two strains ofEeniella nana were examined for their partial base sequences of 18S and 26S rRNAs. In the partial base sequences of 18S rRNA (prositions 1451 through 1618, 168 bases) the strains ofE. nana have five, five, four and eleven base differences with those ofDekkera bruxellensis (type species).D. anomala (andBrettanomyces anomalus),D. naardenensis andD. custersiana, respectively. In the 26S rRNA partial base sequencings (positions 1611 through 1835, 225 bases and positions 493 through 622, 130 bases) the base differences were 46, 43, 34 and 40 and the percent similarities were 53–54, 51–54, 56–57 and 51–53, respectively. The sequence data obtained are discussed phylogenetically and taxonomically, especially on retention of the generic nameEeniella.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms. Part LVIII. For part LVII, see ref. [20].     

Evaluation of an automated DNA sequencing system developed in RIKEN

For the advancement of Human Genome Project, we have developed an automated DNA sequencing system HUGA-I. It is composed of several automated instruments and transfer robots connecting them. In this paper we describe the results of the performance evaluation test of HUGA-I. Although some of the system units showed good performances, the total performance of the HUGA-I was about 1/6 of the designed value. By revealing principal reasons of this poor performance, we would like to contribute to the automation in genome analysis, particularly in human genome analysis.Since the sequence technology advanced remarkably in these years, the system units of HUGA-I become older than those which are now commercially available and the throughput of it is out of our expectations. Nevertheless, we believe that it is meaningful to introduce the exact performance of HUGA-I and present the bottle neck points in the automating sequencing processes. Because, automation in the gene analysis is ultimately important, in particular for the analysis of large genomes such as the human genome. The aims of this paper are to introduce the results in performance evaluation of HUGA-I and to elucidate the bottle neck points in the automation of sequencing processes.The authors express their sincere thanks to Mr. Morisada Hayakawa and Mrs. Nobuko Kato for their technical asistance.     

An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide