Hyogo Sinohara

Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin

We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin (bone sialoprotein) (nucleotides 265-886 and 1183-1424). Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that osteopontin is presumably involved in stone formation as stone matrix.

Does catalytic activity of bence-jones proteins contribute to the pathogenesis of multiple myeloma?

Some Bence-Jones proteins have been found to be capable of hydrolyzing DNA, chromogenic amide substrates, such as benzoylarginine p-nitroanilide, and natural oligopeptides, such as arginine vasopressin. Patients who excrete Bence-Jones protein with the DNA-nicking activity have shown moderately severe symptoms. When incubated with LLC-PK1 (porcine kidney proximal tu bule) cells, some Bence Jones proteins penetrated the cytoplasm, and en tered the nucleus with little or no degradation of epitopes. Intranuclear Bence Jones proteins sultimately induced DNA fragmentation in situ and cell death. This cytocidal activity was not directly associated with the DNA-nicking activity, since Bence Jones proteins with no detectable DNase activity also produced cell death. These results, however, suggest that the biological activities of Bence Jones proteins described here makes a significant contribution to the development and/or deterioration of multiple myeloma.

Glycopeptides isolated from sericin of the silkworm, Bombyx mori

1. 1. Sericin of the silkworm, Bombyx mori, was extensively digested with pronase, and three glycopeptides were isolated by chromatography on ion-exchange resins, Bio-Gel, and charcoal. 2. 2. Structural analyses of these glycopeptides with digestion by specific glycosidases and Smith degradation indicate that sericin contains two types of carbohydrate units. 3. 3. One type contains the carbohydrate units which consist of either N-acetylgalactosamine alone or a disaccharide, β-galactosyl(l → 3)-N-acetylgalactosamine, and which are linked to peptide core with an alkali-labile O-glycosidic bond between N-acetylgalactosamine and serine or threonine. 4. 4. The other type contains the carbohydrate units which consist of several mannose residues and two N-acetylglucosamine residues, and which are linked to peptide core with an alkali-stable N-glycosidic bond between N-acetylglucosamine and asparagine.

Glycopeptides from silk fibroin of Bombyx mori

Abstract Fibroin of the silkworm, Bombyx mori, was subjected to sequential digestion with trypsin and pronase. The small quantity of glucosamine and mannose present in the fibroin could be detected in the digest largely as two glycopeptides, the structure of which was studied with sequential periodate oxidation, Edman degradation, and alkaline treatment. One of the glycopeptides contained two residues of glucosamine and five residues of mannose, whereas the other which occured in a lower concentration contained two residues of glucosamine and three residues of mannose. Both glycopeptides had the same amino acid sequence, SerAsnThr, to which oligosaccharide unit was attached by a glucosaminylasparagine linkage.

The Target Antigen of Anti-Tubular Basement Membrane Antibody-Mediated Interstitial Nephritis

Our previous studies showed that 54 kD and 48 kD tubular basement membrane (TBM) proteins were the major form of the target antigen involved in anti-TBM antibody-mediated tubulo-interstitial nephritis in humans. In those studies, we isolated the 54 kD glycoprotein (named gp54) from collagenase-digested bovine TBM. NH2-terminal amino acid sequencing indicated that gp54 represented a newly defined glycoprotein. In this study, we further characterized the target antigen, using mouse monoclonal antibodies to gp54 and polyclonal anti-gp54 peptide antibody. Two monoclonal antibodies (H79 and H80) were established, and they reacted, by immunofluorescence, predominantly with the proximal TBM of humans, rabbits, and Wistar, Sprague-Dawley, and Brown-Norway rats, but not with that of Lewis rats. They were also fixed by blotting intensely to the 54 kD component and weakly to the 48 kD component of collagenase-digested human TBM. In vivo transfer of H79 to Wistar rats showed extensive linear binding of mouse IgG to the TBM and the basal membrane of the small intestine; however, no pathologic changes were seen by light microscopy. The anti-gp54 peptide antibody reacted with both the 54 kD and 48 kD TBM components of human TBM. mRNA was prepared from rabbit kidneys, and fractionated to enrich mRNA encoding the 54 kD and 48 kD peptides. On in vitro translation experiments with the mRNA fraction, the 54 kD and 48 kD peptides were immunoprecipitated with anti-gp54 antibodies. These findings indicate that the 54 kD and 48 kD components are encoded with different mRNA, but that they share the same antigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation by sex hormones of serum levels of contrapsin and α1-antiprotease in the mouse

Abstract Serum contrapsin and α1-antiprotease levels were higher in males than in females in ten inbred mouse strains tested. The levels of both proteins were diminished by orchiectomy but increased by testosterone administration to females. Estradiol administration to intact females caused a marked elevation in contrapsin level but a decrease in α1-antiprotease level.

Carbohydrate content of various silk fibroins

Abstract All the silk fibroins isolated from 9 species of silkworm belonging to the Bombycidae and Saturniidae contained small amounts of mannose. In fibroins from the Saturniidae, additional sugars such as galactose, glucose, fucose, or xylose were also present.

Amidolytic and peptidolytic activities of immunoglobulin G present in sera from patients with rheumatoid arthritis, Sjogren’s syndrome and systemic lupus erythematosus

Polyclonal immunoglobulin G (IgG) from healthy subjects was found to be capable of hydrolyzing carbobenzoxy–Val–Gly–Arg–p‐nitroanilide (a synthetic chromogenic substrate for trypsin) and d–Pro–Phe–Arg–p‐nitroanilide (a substrate for plasma kallikrein). Statistically significant elevation of activity against the former substrate was found in patients with rheumatoid arthritis (RA), but not in patients with Sjogren’s syndrome (SjS) or systemic lupus erythematosus (SLE). On the other hand, IgG samples from the patients with these three autoimmune diseases showed reduced activity against d–Pro–Phe–Arg methylcoumarinamide, although the differences were not statistically significant. Preliminary studies have shown that two out of three IgG samples from RA patients exhibited the activity of cleaving a pentapeptide, Gln–Arg–Arg–Ala–Ala, whereas virtually no cleavage of the same peptide was observed with IgG from healthy controls or from patients with SjS or SLE.

Guinea pig α1-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase

cDNA encoding alpha 1-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavia porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gln rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change.

Cloning and sequencing of cDNA encoding haptoglobin, an acute phase protein in Syrian hamster, Mesacricetus auratus.

One of the most prominent acute phase proteins in Syrian hamster (Mesacricetus auratus) was identified as haptoglobin and cDNA encoding this protein was sequenced. The deduced amino acid sequence of the mature protein is 83.6, 80.5, 79.6, and 76.1% identical to those of mouse, rat, human (1 s isoform), and dog homologues, respectively. As compared with six known members of this family, including human haptoglobin-related protein, hamster haptoglobin had 11 unique substitutions and one unique codon deletion, that is, the corresponding residues have been conserved in all other members. This indicates that hamster haptoglobin gene has accumulated these unique mutations after the time of cricetid-murid split while the ancestral sequence has been conserved in all other species examined. Hamster haptoglobin, however, contains nine cysteine residues, all of which are found in conserved positions in primate and rodent homologues. Molecular phylogenetic trees of alpha- and beta-chains show that the alpha-chain is more divergent than the beta-chain and that the difference in genetic distance between canine and hamster alpha-chains is much greater than that of corresponding beta-chains.