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Biomedical subjects

M Sakagami

Publications and source records attributed to M Sakagami.

At least 127 records · Page 7Linked to original sources
PubMed1981

Synthetic study on human C-peptide and its related peptides.

Syntheses of eighteen peptides related to human C-peptide and the connecting peptide are described. The syntheses were performed exclusively by the azide fragment condensation method. The immunochemical results justified the utilization of these peptides for development of human C-peptide radioimmunoassay system. The synthetic peptide fragments may be extremely important substrates for assessment of specificity of human C-peptide antisera and thus usefulness of the antisera in practical measurement of blood levels of human C-peptide immunoreactivity.

Amino Acid Sequence↗
PubMed1980

Vasoactive intestinal polypeptide-like immunoreactivity in a human neuroblastoma cell line and the coexistence of other neuropeptide immunoreactivity in the cell line.

The presence of VIP-like immunoreactivity in human neuroblastoma cell line NB-1 was demonstrated by radioimmunoassay of the crude cell extract and by immunocytochemical staining of the cells. Gel filtration profiles of VIP-like immunoreactivity in the cell extract measured by radioimmunoassays using three different region-specific antisera revealed that the immunoreactivity consists of a major molecular form corresponding to porcine VIP having 28 amino acid residues with at least two additional minor forms larger and smaller than the VIP. In addition to VIP-like immunoreactivity, the cell extract was shown to contain substance P-, neurotensin- and somatostatin-like immunoreactivity as well.

Animals↗
PubMed1978

Syntheses of C-peptides and human proinsulin.

Syntheses of human, dog, rat, and duck C-peptides and their analogues and preliminary results on the total synthesis of human proinsulin are described. In the syntheses of the C-peptides, chain elongation was performed exclusively by the azide-fragment condensation method in solution. The synthetic human, dog, rat, and duck C-peptides and their analogues were proved to be homogeneous by several analytic means. With these synthetic peptides, radioimmunoassay systems for dog, rat, and duck C-peptides were developed. For the total synthesis of human proinsulin, 10 protected peptide hydrazides were prepared, and the linearly protected hexaoctacontapeptide having the proposed sequence of human proinsulin was constructed by the azide-fragment condensation method in solution starting from the C-terminal undecapeptide (HP 75-86). After deblocking of the alpha-amino protection, the partially protected hexaoctacontapeptide was treated with sodium in liquid ammonia. The ensuing sulfhydryl form was converted to the S-sulfonate form, which was reduced and then air-oxidized. The oxidized material was purified by gel filtration on Sephadex G-50 (fine) followed by ion-exchange chromatography on DEAE-cellulose. The cross-reactivity in the insulin radioimmunoassay of the ensuing product was 62.5 per cent of porcine proinsulin on a weight basis at B/Bo = 60 per cent. Acid hydrolysis and amino acid analysis of this product gave the theoretically expected ratios. In addition, this peptide, as well as the S-sulfonate form of the hexaoctacontapeptide, showed displacement curves superimposable on that of synthetic human C-peptide on an equimolar basis in the human C-peptide radioimmunoassay (antiserum 527). These results confirm the synthesis of human proinsulin.

Amino Acid Sequence↗
PubMed1977

Synthesis of phenolic group containing analogues of porcine secretin and their immunological properties.

Syntheses by the conventional method are described of Nalpha-tyrosylsecretin, [Tyr1]secretin, and Nalpha-beta-(4-hydroxyphenyl)propionylsecretin. Secretin and [Tyr6]secretin were also prepared by the synthetic route identical with those employed for construction of the above analogues. Purification of secretin and the analogues was conducted by ion-exchange column chromatography on CM-Sephadex and gel filtration. Immunological reactivities of these analogues were examined in the radioimmunoassay system for secretin using two different antisera raised against synthetic secretin in rabbits. The tracers used in this study were [125I]-[Tyr1]secretin and [125I]-[Tyr6]secretin. The dose-response curves of Nalpha-tyrosylsecretin, [Tyr1]secretin, and Nalpha-beta-(4-hydroxphenyl)propionylsecretion were essentially superimposable upon those of natural and synthetic preparations of porcine secretin in the systems used, while [Tyr6]secretin showed discrepancy in the curve. In addition, biological activities of the synthetic polypeptides were compared with that of natural porcine secretin in term of exocrine pancreatic secretory response in anesthetized dogs.

Amino Acid Sequence↗
PubMed1977

[Studies on human proinsulin C-peptide radioimmunoassay method--preparation of antiserum and its specificity-- (author's transl)].

The antisera using at final dilution of 1 : 10,000 have been prepared by immunizing synthetic human proinsulin connecting peptide to rabbits for human proinsulin C-peptide radioimmunoassay. The cross reactivities of human proinsulin C-peptide derivatives with the prepared antisera were reduced by leaving amino acid residues from N terminal, although this phenomenon was a little different among antisera. Those results suggested that main antigen determinant in N terminal 31-38 of human proinsulin connecting peptide. The cross reactivities of other animal proinsulin C-peptide and other peptide hormones with the prepared antissera were not recognized at 10(3) p mole/ml.

C-Peptide↗
PubMed1977

Immunological aspects of secretin, substance P, and VIP.

Secretin, substance P, and vasoactive intestinal peptide (VIP) were studied from the immunological point of view using synthetic hormones and their related peptides which were prepared by the conventional method for peptide synthesis. Immunological properties of these hormones were characterized by radioimmunoassays specific to the respective hormones. Antisecretin antisera (NCC-R-1 and R-801) were generated in rabbits with synthetic porcine secretin absorbed on polyvinylpyrrolidone. Antiserum to substance P (R-400) was produced in a rabbit with synthetic substance P-human alpha-globulin conjugate. Generation of anti-VIP antiserum (R-502) was carried out by immunizing rabbits with synthetic VIP absorbed on polyvinylpyrrolidone. Synthetic polypeptides related to the three hormones that were examined in this study include secretin(4-27), secretin(5-27), secretin(7-27), secretin(11-27), secretin(14-27), secretin(18-27), secretin(1-22)amide, secretin(7-22)amide, Nalpha-tyrosyl-secretin, [1-Tyr]secretin, [4-Ala]secretin, [4-D-Ala]secretin, [4-Ala,5-Val]secretin, [6-Tyr]secretin, substance P(2-11), substance P (3-11), substance P(4-11), substance P(5-11), substance P(6-11), Nalpha-tyrosyl-substance P, [1-Tyr]substance P, [8-Tyr]substance P, [11-Leu]substance P, des-11-Met-substance P, VIP(7-28), VIP(11-28), VIP(18-28), VIP(1-18)amide, and VIP(1-22)AMIDE. The results revealed two antigenic regions at the amino- and carboxylterminal portions of the secretin and VIP molecules. As to substance P, the major antigenic region was located within the 3 to 11 sequence. The proline residue in position 4 and methionine in position 11 seemed to be of special importance. The immunoassays demonstrated the existence of immunoreactivities of these hormones in hot water extracts from various porcine tissues. In the pituitary, VIP and substance P immunoreactivities were detected, whereas secretin was not. Secretin, VIP, and substance P were found in the pancreas, but at low concentrations. Distributions of these hormones in various sites of the gastrointestinal tract were also demonstrated.

Animals↗
PubMed1976

Substance P radioimmunoassay using Nalpha-tyrosyl-substance P and demonstration of the presence of substance P-like immunoreactivities in human blood and porcine tissue extracts.

Sensitive and specific radioimmunoassay for substance P was developed using synthetic substance P and 125I-Nalpha-tyrosyl-substance P. Substance P-human alpha-globulin conjugate was used for production of anti-substance P antisera in rabbits. Synthetic substance P was used as a standard and the dextran-coated charcoal method was employed to separate the free peptide from that bound to antibodies. No cross-reactions by physalaemin and eledoisin observed in this system proved its high specificity to substance P. Nalpha-Tyrosyl-substance P and [Tyr1]-substance P showed the displacement curves indistinguishable from that of the standard substance P. Neither substance P5-11 nor substance P6-11 competed with the tracer at the concentration used. The minimum measurable dose of substance P by the assay system was 2.5-5 pg/incubate. Utilizing the system, human plasma samples from 42 healthy volunteers of both sexes were shown to contain immunoreactive substance P in amounts that averaged 298 pg/ml in male and 251 pg/ml in female. Substance P-like immunoreactivity was also demonstrated in hot-water extracts of porcine duodenum, jejunum, ileum, cecum, middle colon, rectum, pancreas, stomach and pituitary. The highest concentration (379 ng/g wet weight of organ) was found in the pituitary, and the ileum (7.9 ng/g wet weight of organ) and jejunum (1.9 ng/g wet weight of organ) were rich in the contents.

Animals↗
PubMed1976

Radioimmunoassay for secretin using Nalpha-tyrosylsecretin and [Tyr1]-secretin.

Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.

Animals↗