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Biomedical subjects

R Oyama

Publications and source records attributed to R Oyama.

At least 37 records · Page 2Linked to original sources
PubMed1991

Complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) determined by a combination of conventional and mass spectral methods.

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.

Alanine Transaminase↗
PubMed1991

Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca.

Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Artificial cell adhesive proteins engineered by grafting the Arg-Gly-Asp cell recognition signal: factors modulating the cell adhesive activity of the grafted signal.

An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.

Amino Acid Sequence↗
PubMed1991

Differential expression of beta amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP-751 and four-repeat tau.

We investigated the relationship between the differential expression of beta amyloid protein precursor (APP) and tau mRNA, and the extent of beta and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/-695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among beta plaque (-), (+), and (++(-) ) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.

Aged↗
PubMed1990

The complete amino acid sequence of the mature form of rat sepiapterin reductase.

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.

Alcohol Oxidoreductases↗
PubMed1990

Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib.

Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment. We have developed a rapid and simple method for isolation of the protein from platelet extracts using a combination of gel permeation, anion-exchange, and lectin affinity chromatography. The partial amino acid sequence was determined by analysis of peptides generated by digestion of the S-carboxyamido-methylated protein with Achromobacter protease I or cyanogen bromide. The sequence shows a remarkable periodicity of leucine residues, which is homologous to the consensus sequence of a highly diversified protein super-family with a common repetitive module. Thrombin cleavage site was determined to be located at the C-terminal region of GPV by analysis of the products separated by sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, the existence of mucin-type carbohydrate chains was indicated, as well as the existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis. From these data, we report a structural model of GPV that is analogous to glycoprotein Ib.

Amino Acid Sequence↗
PubMed1990

Purification and complete amino acid sequence of novel beta 2-microglobulin.

We have previously reported that novel beta 2-microglobulin (beta 2m) is a metabolite derived from beta 2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD). Chromatofocusing showed the presence of at least two major novel beta 2m's with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel beta 2m's and determined the complete amino acid sequence. We demonstrate herein that the novel beta 2m has the same sequence as native beta 2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native beta 2m, suggesting a possible deamidation during LT-HD.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing.

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Complete amino acid sequence of rat kidney ornithine aminotransferase: identity with liver ornithine aminotransferase.

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

Alcaligenes↗
PubMed1990

Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs.

The complete amino acid sequence and the location of disulfide bonds of a lectin from Japanese frog (Rana japonica) eggs, which specifically agglutinates transformed cells, are presented. The sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with Achromobacter protease I, or chymotrypsin, and by chemical cleavage with BNPS-skatole or cyanogen bromide. The lectin is a single-chain protein consisting of 111 residues, with a pyroglutamyl residue at the amino terminus. Four disulfide bonds link half-cystinyl residue 19 to 72, 34 to 82, 52 to 97, and 94 to 111. The sequence and the location of the disulfide bonds are highly homologous to those of bull frog (Rana catesbeiana) egg S-lectin. They are also homologous to human angiogenin, a tumor angiogenesis factor, and a family of pancreatic ribonucleases.

Amino Acid Sequence↗
PubMed1989

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide.

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate. Cell adhesion mediated by the mutagenized protein was inhibitable by a synthetic peptide Gly-Arg-Gly-Asp-Ser but not by a related peptide Gly-Arg-Gly-Glu-Ser, confirming that the inserted RGDS tetrapeptide served as a recognition signal for cell adhesion. Furthermore, the RGDS-containing protein was capable of adhering cells onto an immunoglobulin-coated surface which could not by itself support cell adhesion. Thus, the cell adhesive and immunoglobulin binding activities of the mutagenized protein appear to function coordinately. The protocol described here is essentially applicable to any protein and, therefore, provides a general principle in tailoring novel multifunctional proteins having cell adhesive activity.

Amino Acid Sequence↗
PubMed1989

Subsensitivity to substance P in SART-stressed mice.

The sensitivity of SART-stressed (repeated cold-stressed) mice to substance P (SP) was studied. The behavioral responses to intrathecal SP in the stressed mice were less marked than those in non-stressed mice. The 50% effective dose of SP, however, did not differ between the 2 groups. Also, the antagonistic effect of intrathecal [D-Pro2, D-Trp7,9]-substance P against SP was not different. These results suggest that SART-stressed mice are subsensitive to exogenous SP and that the number but not the affinity of SP receptors may be altered.

Animals↗
PubMed1989

[Transcatheter arterial embolization with hepatic arterial induction of endogenous TNF in hepatocellular carcinoma].

Antitumor effect and reduction of tumor size by some cytokines as Biological Response Modifier have been demonstrated by various studies. Endogenous tumor necrosis factor is produced from macrophage. To increase the antitumor effect of transcatheter arterial embolization (TAE) in hepatocellular carcinoma (HCC), we treated 7 HCC patients with endogenous tumor necrosis factor (ETNF) which was induced by hepatic arterial injection of gamma-IFN (1.0-3.0 X 10(6) IU) as priming agent and OK-432 (2-5 KE) as triggering agent. TAE was performed with Lipiodol, ADM and gelatin sponge on 3-10 days after the induction of ETNF. TNF activity was detected in 2 cases and suspected to depend on the dose of gamma-IFN and OK-432. Serum alpha-Fetoprotein levels after the injection of ETNF began to decrease from 3-30 days in 5 patients and remained unchanged in 2 cases. Serum alpha-Fetoprotein levels after TAE with the induction of ETNF were decreased 1-5 months in 5 cases. Reduced size and low-density area on CT scan in 3 advanced cases after these procedures were no different from those of HCC patients treated with TAE alone. In one of two inoperable cases with a single mass lesion in the liver, CT scan after one more added TAE following these procedures showed a low-density area around the Lipiodol uptaking tumor, indicating obstruction of the peripheral portal vein. CT scan of another case revealed low density around Lipiodol in the tumor, which showed complete necrotic change. In all cases, middle-grade fever and hypotension were seen transiently, but these subsided by symptomatic treatment. The antitumor effect of TAE in HCC might be enhanced with ETNF induced by hepatic arterial injection of a low dose of gamma-INF and OK-432.

Aged↗
PubMed1989

Determination of partial amino acid sequence of lipoate acetyltransferase of rat pyruvate dehydrogenase complex.

The partial amino acid sequence of rat lipoate acetyltransferase was determined using the intact protein and the peptides derived from a digest with Achromobacter protease I. The results showed the amino-terminal sequence of the mature enzyme to be (N) Ser-Leu-Pro-Pro-His-Gln-Lys-Val-Pro-Leu-Pro-Ser- Leu-Ser-Pro-Thr-Met-Gln-Ala-Gly-Thr-Ile-Ala-Arg-Trp-Glu-Lys. In addition, the sequences of two possible lipoyl-binding sites in the subunit, which are very similar to each other, were established.

Acetyltransferases↗
PubMed1988

Mechanism of the analgesic effect of neurotropin.

Neurotropin, an extract from the inflamed skin of vaccinia virus-inoculated rabbits, has been observed clinically to be effective for treating pain in patients with lumbago, SMON and other neuropathies. In the present study, we examined the mechanism of the antinociceptive effect of neurotropin in mice in relation to administration routes, opioids, and noradrenergic or GABAergic drugs, by the tail pressure method. The antinociceptive effects of neurotropin were large when administered by the i.p. and intracisternal (i.cist.) routes, but comparatively small in the case of the intrathecal (i.th.) route. Neurotropin may thus act at the supraspinal level rather than on the spinal cord. The antinociceptive effect of neurotropin was not blocked by naloxone, and no cross-tolerance developed between neurotropin and morphine. The effect of neurotropin was blocked by phentolamine and reserpine, but not by atropine. Its effect was enhanced by GABA, muscimol, aminooxyacetic acid and diaminobutyric acid, but not by baclofen, and blocked by bicuculline methiodide. From these results, the antinociceptive action of neurotropin appears to be non-opioid in nature, and may possible be mediated by the noradrenergic and GABAergic systems, but unrelated to the cholinergic system.

Analgesics↗
PubMed1981

[Direct action of timepidium bromide on the gastric mucosa and its inhibitory effect on pepsin secretion (author's transl)].

As quaternary ammonium compounds are generally not absorbed from gastric mucosa, the direct action of these compounds on the gastric mucosa can be investigated by means of intragastric (oral) administration to pylorus ligated animals. Effects of timepidium bromide (TB), an anti-cholinergic quaternary ammonium compound, on gastric secretion of pylorus ligated rats were investigated. When TB (100 mg/Kg) was administered orally, the secretion volume of gastric juice decreased slightly, while the concentrations of free acid and pepsin in gastric juice decreased remarkably. When TB was administered intraduodenally (100 mg/kg) or subcutaneously (10 mg/kg), secretion of gastric juice and free acid was strongly inhibited, but the pepsin concentration in gastric juice was not influenced (i.d.) or slightly increased (s.c.). In the oral administration of various drugs such as atropine, hyoscine-N-butylbromide and oxethazaine (Ox), only Ox caused a decrease in pepsin concentration in gastric juice. Similar effects of TB on the pepsin concentration in the gastric juice were also observed in experiments using stomach perfused rats. TB has no effect on enzymatic activity of pepsin in in vitro experiments. From these results, it is assumed that TB inhibits acid and pepsin secretion mainly by a direct action on the gastric mucosa.

Animals↗