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Biomedical subjects

K Sanada

Publications and source records attributed to K Sanada.

At least 37 records · Page 2Linked to original sources
PubMed1992

Characterization of two members (CST4 and CST5) of the cystatin gene family and molecular evolution of cystatin genes.

Two members (CST4 and CST5) of the cystatin gene family have been characterized partially by DNA analysis. The CST4 clone contained the gene coding for the precursor form(141 amino acids) of cystatin S, and its exon-intron organization is the same as that of other members (the cystatin SN gene at the CST1 locus, the cystatin SA gene at the CST2 locus, the cystatin C gene at the CST3 locus and a cystatin pseudogene at the CSTP1 locus). The second cystatin pseudogene was elucidated in the clone, CST5, and it was assigned to the CSTP2 locus. Alignment of DNA sequences of cystatin genes with other genes suggested that the genes for cystatins, kininogens, and Bowman-Birk type inhibitors have evolved from an ancient ribonuclease-like gene.

Amino Acid Sequence↗
PubMed1992

Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease.

Volatile sulfur compounds (VSC) in mouth air were estimated by gas chromatography. The amount of VSC and the methyl mercaptan/hydrogen sulfide ratio were significantly increased in patients with periodontal disease. These two parameters also increased in proportion to the bleeding index and probing depth. A study was also done on the effect of removal of tongue coating on VSC concentrations in mouth air from patients with periodontal involvement. VSC and the methyl mercaptan/hydrogen sulfide ratio were reduced to 49% and 35%, respectively, by removal of the tongue coating. The average amount of tongue coating removed from patients with periodontal disease was significantly higher than from controls (90.1 mg vs. 14.6 mg, p less than 0.01). Estimated production of VSC from tongue coating was 4 times higher than the control value, and the methyl mercaptan/hydrogen sulfide ratio was also markedly increased. However, a saliva putrefaction study suggested that saliva does not contribute to the elevated ratio of methyl mercaptan in mouth air. These results strongly suggest that, in addition to periodontal pockets, tongue coating has an important role in VSC production, in particular leading to an elevated concentration of methyl mercaptan, which is more pathogenic than hydrogen sulfide.

Adult↗
PubMed1992

Biochemical and clinical factors influencing oral malodor in periodontal patients.

The amounts of volatile sulfur compounds (VSC) and methyl mercaptan/hydrogen sulfide ratio in mouth air from patients with periodontal involvement were 8 times greater than those of control subjects. Our studies demonstrated that, in patients with periodontal disease: 1) the concentration of disulfide, which is converted to VSC, increased in proportion to the total pocket depth; 2) 60% of the VSC was produced from the tongue surface; 3) the amount of tongue coating was 4 times greater than in control subjects; and 4) VSC production and the methyl mercaptan/hydrogen sulfide ratio of the tongue coating were increased. 2-Ketobutyrate, which is a byproduct of the metabolism of methionine to methyl mercaptan, was higher in the saliva of patients with periodontal disease. This implies that metabolism of methionine to methyl mercaptan increases in the oral cavity of patients with periodontal pockets. Since free L-methionine, rather than protein, is the main source for methyl mercaptan, we estimated the methionine supply from the gingival fluid into the oral cavity of patients with periodontal involvement. The results showed that the ratio of methionine to whole free amino acids was significantly higher than that of cysteine. Our studies suggest that not only microorganisms, but also the tongue coating and gingival fluid are factors which enhance VSC production in patients with periodontal disease.

Halitosis↗
PubMed1991

Cystatins of family II are harboring two domains which retain inhibitory activities against the proteinases.

Two cyclic peptides, Ac-CTKSQPNLDTC-NH2 (SA-LOOP1) and Ac-CSFQIYEVPWE DRMSLVNSRC-NH2 (SA-LOOP2) were prepared. These sequences are respectively found in the second and third exons of cystatin SA and are well conserved among the cystatins of family II. In addition, these sequences are extremely homologous to the inhibitory regions of several serine-proteinase inhibitors. The peptides were assayed for their inhibiting properties towards serine- and cysteine-proteinases. SA-LOOP1 inhibited porcine pancreatic trypsin (Ki = 370 microM), but did not inhibit cysteine-proteinases. SA-LOOP2 inhibited not only porcine pancreatic alpha-chymotrypsin (Ki = 23 microM) but also papain (Ki = 24 microM) and ficin (Ki = 52 microM). These data indicate that the exon-intron organization of the cystatin genes coinside with the structural and/or functional domains of the protein, and may have significant implications for understanding the active sites of cystatins.

Amino Acid Sequence↗
PubMed1991

Identification of full-sized forms of salivary (S-type) cystatins (cystatin SN, cystatin SA, cystatin S, and two phosphorylated forms of cystatin S) in human whole saliva and determination of phosphorylation sites of cystatin S.

Our recent work on the gene structures for human salivary (S-type) cystatins [Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the full-sized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophosphorylated cystatin S and Ser1(P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.

Amino Acid Sequence↗
PubMed1991

The human cystatin gene family: cloning of three members and evolutionary relationship between cystatins and Bowman-Birk type proteinase inhibitors.

Three genes from the human cystatin gene family have been isolated from a bacteriophage lambda library containing Hind III digests of human genomic DNA. The cloned genes were identified with three DNA probes each containing exon 1, exon 2 and exon 3 of the CST1 gene for cystatin SN. The genes, which we name CST2B, CST4, and CST5, are 6.8 kb, 5.4 kb and 12.5 kb in size, respectively. Statistical analysis of DNA sequence homology elucidated that the second and third exons of cystatin (family II) genes and three cystatin (family II) gene like segments in the kininogen (family III) genes are significantly homologous to the gene segments coding for the inhibitory domains of Bowman-Birk type proteinase inhibitors.

Amino Acid Sequence↗
PubMed1989

The human cystatin C gene (CST3) is a member of the cystatin gene family which is localized on chromosome 20.

The fourth gene from the human cystatin gene family of salivary-type cysteine-proteinase inhibitors has been isolated and partially characterized by DNA analysis. The gene, which we name CST3, codes for human cystatin C, and has the same organization as the CST1 gene for cystatin SN and the CST2 gene for cystatin SA. Southern analysis of EcoR I digested DNAs from 32 independent somatic cell hybrid clones hybridized to a probe from CST1 demonstrated that all members of the cystatin gene family segregate with human chromosome 20. These results indicate that the genes for salivary-type cystatins and cystatin C are members of a multigene family--the cystatin gene family.

Amino Acid Sequence↗
PubMed1989

'Salivary peptide P-C' of human pancreatic B-cells shares only partly immunoreactivity with salivary peptide P-C indicating a new B-cell protein which is different from insulin.

Salivary peptide P-C like immunoreactivity, originally isolated from human whole saliva has later been found in the human pancreatic B-cells. In the present work an indirect immunofluorescence technique using monoclonal antibodies against isolated salivary peptide P-C was applied to Bouin fixed pancreas and parotid glands to study the possible identity of the two substances. Positive P-C immunofluorescence was found in the serous cells of parotid glands but not in pancreatic B-cells, suggesting that pancreatic P-C substance is not salivary peptide P-C itself, but a substance sharing the common antigenic site with salivary peptide P-C. To examine this, an indirect immunofluorescence technique using polyclonal P-C antisera pre-absorbed with six kinds of synthetic fragments (1-22, 23-44, 23-29, 30-44, 30-38 and 38-44) of salivary peptide P-C was applied to the human pancreas. The result showed that pancreatic P-C substance was a substance which shares the common antigenic site with the 38-44 amino acid residue of salivary peptide P-C. Western blot analysis using extracts of human pancreata further showed that pancreatic P-C substance is not a precursor of insulin but a protein with molecular weight of 11,500 dalton, indicating the presence of a new protein in the insulin secretory granules of human pancreatic B-cells.

Amino Acid Sequence↗
PubMed1988

Cystatin superfamily. Evidence that family II cystatin genes are evolutionarily related to family III cystatin genes.

Human saliva contains at least three molecular species of cystatin S-type cysteine proteinase inhibitor (cystatin S, cystatin SN and cystatin SA), which have similar but distinct amino-acid sequences. The nucleotide sequences of the CST 1 gene for cystatin SN and the CST 2 gene for cystatin SA are highly homologous to each other and to the corresponding regions of the cDNA for cystatin C and the EcoRI-PstI fragment from the cystatin C gene. Three cystatin-like domains in the kininogen gene and the salivary cystatin genes share the same gene organizations. These data demonstrate that family II cystatin genes are evolutionarily related to family III cystatin genes.

Amino Acid Sequence↗