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Biomedical subjects

M Tsuda

Publications and source records attributed to M Tsuda.

At least 379 records · Page 21Linked to original sources
PubMed1991

Inhibition of homoserine dehydrogenase I by L-serine in Escherichia coli.

We have reported that a major cause of growth inhibition of Escherichia coli by L-serine is its inhibition of homoserine dehydrogenase I (HDH I), which is involved in the biosyntheses of threonine and isoleucine [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. However, Patte et al. reported that L-serine does not inhibit HDH I [Patte, J.-C., Truffa-Bachi, P., & Cohen, G.N. (1966) Biochim. Biophys. Acta 128, 426-439]. In studies on the reason for these discrepant results, we found that the concentration of K+ and the pH in the assay mixture strongly influenced the inhibitory effect of L-serine. L-Serine strongly inhibited the HDH I activities in both the forward and reverse reactions between aspartate semialdehyde and homoserine at a physiological K+ concentration (100 to 200 mM) and physiological pH (7.5) for E. coli cells. On the other hand, two well-known inhibitors of HDH I, L-threonine and L-cysteine, strongly inhibited the activity regardless of the K+ concentration and pH.

Cations, Monovalent↗
PubMed1991

Cloning and nucleotide sequence of the gene (citA) encoding a citrate carrier from Salmonella typhimurium.

A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.

Amino Acid Sequence↗
PubMed1991

Total synthesis of a gene for octopus rhodopsin and its preliminary expression.

To carry out systematic structure-function studies of octopus rhodopsin, photoreceptor protein of octopus visual cells, by means of specific amino-acid replacements, we have totally synthesized a DNA duplex of 1,365 base pairs that encodes the entire octopus rhodopsin of 455 amino acids [Ovchinnikov et al. (1988) FEBS Lett. 232, 69-72] by introducing codons preferred in Escherichia coli. Total synthesis simplifies site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their newly synthesized counterparts containing the required nucleotide alterations. Thirty unique restriction sites were introduced in the octopus rhodopsin gene, which was assembled on a plasmid in two steps. Five cartridge genes of 344, 296, 320, 212, and 317 base pairs capable of being expressed independently were first constructed by using 48 synthetic oligonucleotides ranging in size from 54 to 73 nucleotides. The entire gene was constructed by consecutive linkage of cartridge genes. These cartridge genes were designed to correspond to the transmembrane helical unit of octopus rhodopsin, resulting in easy construction of various chimeric rhodopsins. The nucleotide sequences were confirmed by sequencing the cartridges as well as the entire gene. These synthetic genes were cloned into an expression vector carrying the trp promoter of E. coli, and were preliminarily expressed in vitro and in vivo.

Amino Acid Sequence↗
PubMed1991

Sandwich-enzyme immunoassays for endothelin family peptides.

Sandwich-enzyme immunoassays (sandwich-EIAs) for endothelin-1 (ET-1), endothelin-3 (ET-3), big ET-1, big endothelin-2 (big ET-2), and big endothelin-3 (big ET-3) have been established using two antibodies directed against the N-terminal and C-terminal portions of each ET and big ET. These sandwich-EIAs are sensitive enough to detect 0.1-0.4 pg/well of each ET or big ET with cross-reactivity of less than 1% with other ETs and big ETs (except in the case of the EIA for ET-1, which detects immunoreactivity of ET-2 as well as ET-1). Plasma levels of immunoreactive (ir-) ET-1, big ET-1, and ET-3 in normal humans were approximately 2.0, 5.1, and 0.9 pg/ml, respectively, when corrected for recovery efficiency. In reverse-phase high-performance liquid chromatography (HPLC) analysis, immunoreactivities detected by these sandwich-EIAs were eluted at the positions of authentic ET-1, big ET-1, and ET-3, indicating that plasma levels of ET-1, big ET-1, and ET-3 were precisely measured by our methods. Levels of ir-big ET-2 and ir-big ET-3 were also detected in normal human plasma; however, further studies are needed to determine their precise values. The establishment of reliable sandwich-EIAs and the measurement of specific ETs and big ETs in normal human plasma will contribute to clarifying the biological and clinical significance of ET family peptides.

Adult↗
PubMed1991

Immunological abnormalities in Kawasaki disease with coronary artery lesions.

A review of our previous immunological studies on Kawasaki disease (KD) was undertaken. The results showed that peripheral blood macrophages/monocytes, T-cells and B-cells become activated during acute KD in terms of numerical changes in immunocompetent cells, expression of activated antigens on the cell surfaces and cytokine production. Also, during acute KD with coronary artery lesions (CALs) the numbers of macrophages/monocytes are increased. In addition, both the increased levels of tumor necrosis factor-alpha and shed intercellular adhesion molecule-1 in serum are more evident in KD patients with CALs than in those without. Our results further suggest that the main characteristics of the pathogenesis of KD are increased numbers of peripheral blood macrophages/monocytes with the secretion of monokines by these activated cells, and the expression of adhesion molecules on immunocompetent cells. These immune responses develop more vigorously in KD patients with CALs.

Cell Adhesion Molecules↗
PubMed1991

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.

Animals↗
PubMed1991

Formycin A resistant mutants due to defect in adenosine transport system in Vibrio parahaemolyticus.

An antibiotic formycin A inhibited growth of Vibrio parahaemolyticus under certain conditions, which suggested that formycin A was taken up by cells under these conditions. We found that formycin A was transported via the adenosine transport system which we previously reported as a Na(+)-coupled cotransport system. We isolated many formycin A resistant mutants, and about half of them grew very poorly on adenosine as a sole source of carbon. Judging from their reversion frequencies, these mutants seemed to have single mutations. Respiration driven uptake of 14C-adenosine was not observed in such mutants; also, Na+ uptake induced by the addition of adenosine or formycin A to a cell suspension was completely abolished in them. Thus we conclude that these mutants possess a defect in the Na+/adenosine cotransport system, and have become formycin A resistant.

Adenosine↗
PubMed1991

[A case report of childhood multiple sclerosis with periodic synchronous discharge on EEG].

A 12-year-old girl with multiple sclerosis (MS) with periodic synchronous discharge (PSD) on electroencephalogram (EEG) is reported. The patient developed clonic seizure of both arms at the age of 10. The muscle strength of left hand and both legs were decreased and her school records were declined at the age of 12. On neurological examination, the patient showed mild intellectual disturbance, mild weakness of face and extremities and bilateral decreased DTRs. Her gait was slightly ataxic. Cerebrospinal fluid (CSF) revealed an oligoclonal band. Serial CT scans disclosed ring enhancements in the regions corresponding to clinical symptoms (right central gyrus on Sep. '85, right lower and middle temporal gyri, and mesial site of occipital lobe on Oct. '85, right cerebellum on Jan. '86). The same lesions were visualized as a high signal intensity on T2 weight images and as a low signal intensity on T1 weight images on MRI. The attack of seizure occurs more frequently in children with than adults with MS. However, as far as we know in the cases of children with MS, there were no reports of PSD which was usually seen in the cases of subacute sclerosing panencephalitis. The pathomechanism of PSD is speculated to be suffered from subcortical damage.

Age Factors↗
PubMed1991

Nitrite-trapping capacity of thioproline in the human body.

Human nitrosating capacity has been monitored using proline; however, N-nitrosothiazolidine 4-carboxylic acid (NTCA; N-nitrosothioproline), one of the predominant N-nitroso compounds in human urine, is also nonmutagenic and, presumably, noncarcinogenic. Thioproline is nitrosated about 1000 times faster than proline in vitro, and NTCA is excreted into the urine without being metabolized. We have therefore proposed thioproline as an effective nitrite-trapping agent in the human body. Recently, we found thioproline in various cooked foods, including cod and dried shiitake mushrooms. In the study reported here, we evaluate the nitrite trapping capacity of thioproline in a male nonsmoking volunteer ingesting NO3- and eating a controlled diet. The highest level of NTCA excreted, 5.89 mumol, was measured after the subject ingested 6 mmol NO3- followed by 0.45 mmol (60 mg) thioproline. We estimated the effective amount of nitrite, defined as the actual amount of nitrite participating in nitrosation in the stomach, to be 0.3% of the NO3- ingested. Thus, the effective amount of NO2- for 6 mmol NO3- ingested was calculated to be 18 mumol, and 33% of this nitrite was trapped by ingestion of 0.45 mmol thioproline. We conclude that thioproline is a most sensitive probe for evaluating human nitrosating capacity and an effective nitrite-trapping agent.

Ascorbic Acid↗
PubMed1991

[Cystinuria with symptoms of cerebellar atrophy--a case report].

We report cystinuria and symptoms of cerebellar atrophy in a 45-year-old man. His parents were first cousins, and many members of his family had stones of urinary tract or gait impairment. Neurological examination disclosed cerebellar signs resembling those of spinocerebellar degeneration. Urinalysis disclosed high cystine, lysine, ornitine and arginine output. Cystine was 1153.8 micro mol/day (normal range, 22-170); lysine, 3443.9 (normal range, 44-1000); ornitine, 283.8 (normal range, 7-40); and arginine, 154.0 (normal range, 9-50). Neurological complications reported to be associated with cystinuria include mental retardation, muscular dystrophy, hypotonia and dwarfism, mongolism, paroxysmal dyskinesia, myopathy, migraine, spastic paraplegia, multiple sclerosis, subacute combined degeneration and cranial polyneuropathy. Cerebellar signs have been reported in only two cases, and to our knowledge, this is the first case of cystinuria with cerebellar atrophy ever reported. Some common metabolic errors may have caused both disorders, although they also may have developed independently.

Atrophy↗
PubMed1990

Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli.

The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174. Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated. The original point mutation at codon 174 was intact in these strains. Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing. These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells. The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400. The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function. Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent. The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0).

Amino Acid Sequence↗
PubMed1990

Structural organization of the human S-antigen gene. cDNA, amino acid, intron, exon, promoter, in vitro transcription, retina, and pineal gland.

S-Antigen (S-Ag) is a major soluble photoreceptor protein involved in the visual transduction cascade. Several S-Ag cDNAs and a gene coding for human S-Ag were isolated from cDNA and gene libraries. The gene sequences of the coding, noncoding, and 5'-flanking regions of the gene were determined. The S-Ag gene was approximately 50 kbp (kilobase pairs) in length and contained 16 exons and 15 introns. The length of most exons was less than 100 base pairs (bp) and the smallest one was only 10 bp. In contrast, the length of most introns was larger than 2 kbp, and the gene comprised 97% intron and 3% exon. The splice sites for donor and acceptor were in good agreement with the GT/AG rule. The S-Ag protein of 403 amino acid residues was translated from a mRNA of 1.9 kbp, and the mRNA was transcribed from a gene of 50 kbp. The 5'-flanking region of the gene, approximately 1.1 kbp long, had no known regulatory elements for transcription such as TATA, GC, and CCAAT boxes. Interestingly, the 5'-flanking region had promoter activity in an in vitro transcription assay using a nuclear extract of rat brain. A major transcription start site was found at 387 bp upstream from the translation start site ATG. Our results indicate that the sequence of S-Ag promoter differs from other known promoters and may, perhaps, be specific for photoreceptor rod cells and pinealocytes.

Amino Acid Sequence↗
PubMed1990

Adrenal pheochromocytoma PC12h cells respond to pituitary adenylate cyclase activating polypeptide.

An adrenal pheochromocytoma cell line, PC12h, was found to respond to a novel hypothalamic neuropeptide, Pituitary Adenylate Cyclase Activating Polypeptide (PACAP). The cells elevated both intracellular and extracellular cAMP levels on stimulation by PACAP, whereas they showed little response to VIP which is structurally related to PACAP. Using [125I]PACAP27 (a shorter form of the peptide) and [125I]VIP, we found large amounts of specific binding sites for PACAP but few binding sites for VIP in PC12h cells. These results indicate that PC12h cells respond to PACAP via a specific PACAP receptor.

Adrenal Gland Neoplasms↗
PubMed1990

Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture.

Since we found relatively high concentrations of immunoreactive (ir-) ET-3 in the rat pituitary gland (190 pg/g tissue), we have investigated the possible ET-3 secretion from the primary culture of anterior pituitary cells and the effects of various growth factors on the ET-3 secretion. The ir-ET-3 was detected in the incubation medium within 2 h, and 24 h of culture attained the concentrations of 1.15 +/- 0.26 pg/well/6 x 10(5) cells. The ir-ET-3 secretion was stimulated by insulin, insulin like growth factor-II (IGF-II), and most effectively by insulin like growth factor-I (IGF-I) in a dose- and time-dependent manner, whereas the production of ir-ET-1 and ir-big ET-1 was slightly inhibited by IGF-I and IGF-II. In reverse-phase HPLC, the ir-ET-3 released into the culture media showed identical retention time with authentic ET-3. Although ir-ET-1 and ir-big ET-1 secretion was stimulated by transforming growth factor-beta (TGF-beta), ir-ET-3 secretion was inhibited. These results indicate that the anterior pituitary cells secrete ET-3 and the secretion is stimulated by IGF-I.

Animals↗
PubMed1990

Solubilization of receptor for pituitary adenylate cyclase activating polypeptide from bovine brain.

A receptor for pituitary adenylate cyclase activating polypeptide (PACAP) was solubilized from bovine brain membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Saturable binding experiments using [125I]PACAP27 revealed that the solubilized extract contained a single class of binding sites with a Kd of 200 pM and a Bmax of 0.9 pmol/mg protein. The Kd did not change significantly after the solubilization. Competitive binding experiments confirmed that the solubilized receptor retained a specificity for PACAP. Thus, the solubilization of the receptor in an active form was successfully achieved by the present procedure.

Animals↗