Search PubMed⌕ Search

Biomedical subjects

J Sakata

Publications and source records attributed to J Sakata.

52 records · Page 3Linked to original sources
PubMed1994

Experimental tracheal replacement using tissue-engineered cartilage.

The authors tested the feasibility of using tissue-engineered cartilage, grown in the shape of cylinders, for replacing large circumferential defects of the cervical trachea in rats. Chondrocytes obtained from the shoulder of newborn calves were seeded onto a synthetic nonwoven mesh, 100 microns thick, of polyglycolic acid fibers 15 microns in diameter, cut into pieces of 2.5 x 4 cm. Twenty cell-polymer constructs were wrapped around silastic tubes and implanted into 10 nude mice for 4 weeks. Specimens were then excised and evaluated grossly and histologically for the presence of new cartilage, and biomechanically for their ability to resist collapse upon application of negative pressure. Six cylinders of tissue-engineered cartilage were then sutured into large circumferential defects created in the cervical tracheas of nude rats to replace the excised trachea. Implantation of cell-polymer constructs resulted in the formation of cylinders of hyaline cartilage. When placed within the lumen of a segment of bowel denuded of its mucosal lining, the hollow cylinders resisted collapse in all instances upon administration of negative 200 mm Hg pressure. The cartilage was grossly and histologically identical to that from which the cells had been initially isolated. Four of the six animals receiving these cartilage cylinders as tracheal replacements survived the procedure and were able to breathe in an unassisted fashion. Three of these animals never recovered fully from the anesthetic and the operation, and expired at 24, 48, and 72 hours. The fourth animal fully recovered from the procedure, and breathed spontaneously for 1 week, with no apparent limitations. Increasing respiratory distress then developed, and the animal died.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Molecular cloning and biological activities of rat adrenomedullin, a hypotensive peptide.

Adrenomedullin is a new hypotensive peptide recently identified in human pheochromocytoma arising from adrenal medulla. We report here the cDNA encoding a rat adrenomedullin precursor. The precursor is 185 amino acids in length, including a 21 residue putative signal peptide at the N-terminus. Rat adrenomedullin consists of 50 amino acids similar to, but distinct from human adrenomedullin; 2 residues were deleted and 6 residues were substituted compared to those in the human peptide. In the proadrenomedullin N-terminal 20 peptide, whose amino acid sequence is present in adrenomedullin precursor, 3 amino acids were substituted. RNA blot analysis showed that rat adrenomedullin mRNA was expressed in adrenal glands, lung, kidney, heart, spleen, duodenum and submandibular glands. Synthetic rat adrenomedullin elicits a potent and long-lasting hypotensive activity in anesthetized rats.

Adrenal Glands↗
PubMed1993

Cloning and characterization of cDNA encoding a precursor for human adrenomedullin.

Adrenomedullin is a novel hypotensive peptide recently isolated from human pheochromocytoma. Since a high concentration of immunoreactive adrenomedullin was found in pheochromocytoma tissue, the cDNA library of pheochromocytoma was constructed, and the cDNA clone encoding an adrenomedullin precursor was isolated and sequenced. The precursor for human adrenomedullin (human preproadrenomedullin) is 185 amino acids in length, including an adrenomedullin sequence. Proadrenomedullin (proAM) contains a unique twenty amino acid sequence followed by Gly-Lys-Arg in the N-terminal region. It is possible that a novel 20 residues peptide, termed "proadrenomedullin N-terminal 20 peptide" (proAM-N20) whose carboxy terminus may be Arg-NH2, is processed from proadrenomedullin. By RNA blot analysis, human adrenomedullin mRNA was found to be highly expressed in several tissues including adrenal medulla, ventricle, lung and kidney as well as pheochromocytoma.

Adrenal Gland Neoplasms↗
PubMed1992

Renal microangiography and correlated histopathological observation of cows with nephropathy.

Seven Holstein-Friesian cows showing chronic nephropathy were studied by renal microangiography and its correlated histopathology. In cases of pyelonephritis associated with severe pathological lesions such as thickening of arterial walls, narrowing of the arterial and arteriolar lumen, and interstitial inflammation and abscess formation, patchy loss of the peritubular capillary plexus from the cortex to the medulla was clearly demonstrated by microangiography. Interlobular arteries were tortuous and attenuated or truncated. Opacification in the vasa rectae and interstitial capillaries was increased. Extensive non-perfused regions could be detected in the cortex. In cases of mild interstitial nephritis and moderate pyelonephritis, microangiography showed focal changes in the renal vasculature. Microangiography is thus shown to clearly demonstrate changes in the renal vasculature corresponding to the severity of the histopathological lesions.

Angiography↗
PubMed1991

Characterization of two isozymic forms of heart fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Occurrence of two isozymic forms of fructose 6-P, 2-kinase: fructose 2,6-bisphosphatase in bovine heart was investigated by transcribing mRNAs and amplifying the cDNAs with polymerase chain reactions. Analysis of the PCR products revealed 1.7 Kb and 1.5 Kb DNAs, and the determination of their nucleotide sequences showed that these DNAs are identical except for the lack of 180 base pairs near the 3' of the bovine heart enzyme DNA previously reported (6). This missing nucleotide sequence encodes Asn451-Gln510 and contains the phosphorylation sites for cAMP dependent protein kinase and protein kinase C.

Amino Acid Sequence↗
PubMed1991

Molecular cloning of the DNA and expression and characterization of rat testes fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase.

We have isolated and sequenced two overlapping cDNA fragments which could encode the complete amino acid sequence of rat testis fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Northern blot analysis revealed that the major 2-kilobase mRNA isolated from rat testis hybridized with a cDNA fragment. A full length cDNA, which encoded a protein of 468 amino acids, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a Mr of 55,000 by gel electrophoresis under denaturing conditions, compared to the deduced Mr of 54,023. Fru-6-P,2-kinase:Fru-2,6-bisphosphatase with the same Mr 55,000 was also present in rat testis extract. The active enzyme was a dimer as judged by molecular sieve filtration. The expressed enzyme was bifunctional with specific activities of 90 and 22 milliunits/mg of the kinase and the phosphatase activities, respectively. Various kinetic constants of the expressed fructose 6-P,2-kinase were KmFru 6-P = 85 microM and KmATP = 270 microM, and those of fructose 2,6-bisphosphatase were KmFru 2,6-P2 = 21 microM and KiFru 6-P = 3.4 microM. The enzyme was phosphorylated by Fru-2,6[2-32P]P2 and also by protein kinase C, but not by cAMP-dependent protein kinase, which is in contrast to the liver and heart isozymes.

Amino Acid Sequence↗
PubMed1991

Application of renal microangiography to normal and diseased kidneys of cattle and mice.

Use of microangiography is now essential for the study of microcirculation in various organs. Renal microangiographic studies have been reported in rats, rabbits, dogs, human beings, and mice. However, we could not find any report on use of the technique in cattle, despite high incidence of renal disease in that species. The perfusion technique used in mice was improved over that of our previous report, and was applied to normal and diseased bovine kidneys. For the microangiographic technique, composition of the contrast medium, pressure of the injection, duration of perfusion, and washing of kidneys with heparinized saline solution before perfusion are important. In cattle, 1- to 2-mm-thick sections of the kidneys were generally necessary to observe renal vasculature: arcuate and interlobular arteries, afferent arterioles, and glomerular capillaries. In normal bovine kidneys, the angiographic and microangiographic findings were easily recognized as normal, compared with those of normal mice. In affected bovine kidneys, which histologically represented glomerulonephritis and pyelonephritis, angiography and microangiography revealed corresponding findings.

Angiography↗
PubMed1990

Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.

We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities.

Amino Acid Sequence↗
PubMed1988

Identification of new atrial natriuretic peptides in frog heart.

It has been observed that mammalian atrial natriuretic peptide (ANP)-like immunoreactivity is found in frog heart, but to date the natriuretic factors have not yet been identified. Isolation from bull-frog heart extract was performed mainly by immunoaffinity chromatography on a column linked with anti-hANP IgG. From the low molecular weight fraction, 24- and 21-amino acid peptides were purified to homogeneity. Both peptides were found to elicit diuretic-natriuretic as well as vasorelaxant activity, and were named "frog ANP-24" and "frog ANP-21", respectively. Complete amino acid sequences of the peptides were established by microsequencing and confirmed by syntheses. Frog ANP-21 was identified as an N-terminally three amino acid deleted form of frog ANP-24. Remarkable sequence homology was observed between frog ANP and mammalian ANP, especially in the regions flanked by two half-cystine residues.

Animals↗
PubMed1986

Tissue distribution and characterization of peptide C-terminal alpha-amidating activity in rat.

The C-terminal alpha-amide formation of peptides is one of the most important events in prohormone processing. Recently, we developed a simple and sensitive assay for detecting alpha-amidating activity in tissues by using (125I)-Ac-Tyr-Phe-Gly as a substrate. Using this assay method, we have determined the tissue distribution of alpha-amidating enzyme activity in adult male rat. High concentrations of alpha-amidating activity were found in pituitary, brain, thyroid, gastrointestinal tract, pancreas, heart, submaxillary glands and parotid glands. Alpha-amidating enzyme activities in all tissues examined exhibit very similar copper and ascorbate requirements, pH dependence, and behavior on gel-filtration.

Amides↗
PubMed1986

Peptide C-terminal alpha-amidating enzyme purified to homogeneity from Xenopus laevis skin.

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. In this study, we have developed a simple and sensitive assay for monitoring alpha-amidating activity by using radioiodinated Ac-Tyr-Phe-Gly as a substrate. By utilizing this assay, an alpha-amidating enzyme was first purified to homogeneity from Xenopus laevis skin. The purified enzyme has a single polypeptide chain with an apparent molecular weight of 39,000 and its N-terminal sequence was determined as Ser-Leu-Ser-. The enzyme converts several synthetic peptides with C-terminal glycine to the corresponding des-glycine peptide alpha-amides. The enzyme activity, with an optimal pH 6-7, was dependent on the copper ion and ascorbate. In the presence of 0.25 mM ascorbate, the enzyme exhibited a Km of 0.35 microM and a Vmax of 1.9 nmol/microgram/h for Ac-Tyr-Phe-Gly.

Amino Acid Sequence↗