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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 469 records · Page 26Linked to original sources
PubMed1985

Molecular cloning of the human thyrotropin-beta subunit gene.

Genomic DNA fragments that carried a gene for human thyrotropin-beta (hTSH beta) subunit were isolated. Nucleotide sequence analysis of the gene showed that the hTSH beta subunit precursor consists of 138 amino acid residues. There is an N-terminal sequence of 20 amino acids as a signal peptide, followed by 112 amino acids, whose sequence is in agreement with that known for the secretory form of hTSH beta subunit. This is followed by an additional stretch of 6 hydrophobic amino acids, which may be eliminated post-translationally. The coding region is separated by an intron of about 460 bp. Genomic Southern blot hybridization analysis suggested that the hTSH beta gene is a unique single copy gene.

Amino Acid Sequence↗
PubMed1985

An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions.

Two deoxyoligonucleotide probes (23-mer and 26-mer) carrying deoxyinosine residues (I) at positions corresponding to ambiguous nucleotides derived from amino acid sequence have been synthesized by the phosphotriester method using a polymer support. The 23-mer and 26-mer corresponded to the mRNA for 8 amino acids from gastrin and 9 amino acids from cholecystokinin, respectively. The dIs have been used where the base in the third position of the amino acid codon is ambiguous. These deoxyoligonucleotides were used as probes for hybridization with colonies containing the corresponding cDNAs or genes. The hybrid formed between a gastrin clone and the 23-mer that harbors 5 dIs was dissociated at 50-55 degrees C, suggesting that deoxyinosine did not significantly effect the stabilization or destabilization of the DNA duplex. A similar result was obtained using the 26-mer that contains 5 dIs and a phage clone DNA of the cholecystokinin gene. Thus oligonucleotide probes with deoxyinosine residues at ambiguous points seem to be useful as hybridization probes for cloning genes for proteins containing amino acids with degenerate codons.

Base Sequence↗
PubMed1985

Origin and initiation sites of lambda dv DNA replication in vitro.

The sequence of lambda DNA essential for the unique initiation of replication was analyzed in an in vitro replication system. Fragments of lambda DNA were inserted into pBR322 and used as templates or were circularized in vitro in the absence of pBR322 and employed in the same tests. A 165-bp region left of the EcoRI site in the O gene of the lambda genome was defined as the functional origin. This region, which we defined as the ori region, carries, in order, the 4 19-bp repeat sequences where the O protein binds (ori-repeats), an A+T-rich stretch, and a region that constitutes part of a large palindromic structure. Regions that have long been suspected to participate in lambda DNA replication initiation, ice and oop were not required for the O, P-dependent lambda-specific replication initiation. The lambda dv and the "ori region plus" recombinant plasmids initiated DNA synthesis at or around this region, and the reaction depended on the presence of lambda-coded initiators, O and P proteins. Early replicative intermediates of lambda dv were prepared in an in vitro replication system in the presence of ddCTP, an inhibitor of DNA chain elongation. This system allows only that DNA synthesis that is a result of replication initiation. Using this system, the initiation site(s) of the DNA synthesis was finely analyzed by mapping the transition sites from primer RNA to DNA synthesis. Short-chain DNAs produced from regions near the ori region were purified from the intermediates. A fraction of the short-chain DNAs was covalently linked to primer RNA. The 5'-ends that had been linked to RNA (transition sites) were exposed by alkaline hydrolysis, labeled with 32P, and the transition sites were mapped along the nucleotide sequence of the genome. Two striking features emerged from this analysis: (i) The transition sites are located on both sides of the ori region, and no transition arose within the 165-bp ori region; (ii) The transition sites on both sides are not unique, but multiple, and are clustered in one of the 2 strands. Furthermore, their orientation demonstrates that the DNA synthesis in initiation of replication from the 2 sides of the ori region converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis. These results reflect asymmetric bidirectional replication of lambda dv DNA, and may also reflect replication of lambda phage DNA.

Bacteriophage lambda↗
PubMed1985

Restriction fragment length polymorphism detected by human salivary amylase cDNA.

The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7 kb and 6.5 kb fragment alleles, are 0.55 and 0.45, respectively.

Alleles↗
PubMed1985

Expression of a synthetic human growth hormone gene in yeast.

A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 10(6) molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.

Alkaline Phosphatase↗
PubMed1985

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Animals↗
PubMed1985

Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine.

A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.

Amino Acid Sequence↗
PubMed1985

Mini-F protein that binds to a unique region for partition of mini-F plasmid DNA.

A mini-F-coded protein, named F2 protein, binds specifically to mini-F DNA. This protein has a molecular weight of 37,000 and is coded by the A2 segment of the mini-F genome (47.3 to 49.4 kilobases on the F coordinate map). The binding site is located also in the A2 segment of mini-F. This binding site is lost by spontaneous deletion when the A2 segment alone, but not A2 together with its neighboring segment, is cloned in a multicopy plasmid pBR322. These data are discussed in connection with incompatibility and plasmid stability.

Bacterial Proteins↗
PubMed1985

Analysis of the long-lasting antagonistic effect of caerulein on amphetamine hyperactivity in rats.

Caerulein (CLN), which is chemically related to cholecystokinin octapeptide (CCK-8) and produces a short-lasting pharmacological effect when administered peripherally, caused a long-lasting antagonistic effect on amphetamine (AMP) hyperactivity in rats when given in combination with haloperidol (HLP). Briefly, rats were treated with a combination of CLN (0.3-40 micrograms/kg, s.c.) and HLP (0.1 mg/kg, s.c.) and exposed to AMP on the first day. The animals became less sensitive to AMP for 24 hr to about 2 weeks depending on the CLN dose, according to measurements of their ambulatory activities in an open field or with an Animex activity meter at low sensitivity. Examination of the properties of this long-lasting effect revealed that: 1) in animals treated with CLN and HLP, but without AMP on the first day, the susceptibility to AMP was not influenced on the next day; 2) in substitution experiments, the antagonistic effect of CLN could be reproduced by higher doses of CCK-8 (160 micrograms/kg) but not by nonsulfated CLN; 3) in the regimen of the treatment schedule, HLP could be replaced by chlorpromazine or sulpiride, but not by alpha-blocking agents like phenoxybenzamine or yohimbine; 4) apomorphine, nomifensine and tranylcypromine could not substitute for AMP. Thus, injection of CLN together with HLP and AMP on the first day might be necessary to produce the long-lasting anti-AMP effect. The possible mechanism of this CLN effect is discussed.

Amphetamine↗
PubMed1985

Possible involvement of the central nervous system in long-term effect of caerulein on amphetamine-induced hyperactivity in rats.

The present experiments were performed in order to ascertain whether the long-term antagonistic effect of caerulein on amphetamine-induced hyperactivity is produced in the central nervous system. In the vagotomized rats, animals treated with a combination of caerulein and haloperidol and exposed to amphetamine on the 1st day became less sensitive to amphetamine for 24 hr to 1 week when their ambulatory activities were measured in an open field apparatus. The same results were also observed in the experiments where caerulein was applied directly into the lateral ventricle. However, the animals given caerulein in the nucleus accumbens showed no reduced susceptibility to amphetamine. Thus, the present results suggest that caerulein may indirectly modulate some dopaminergic activity in the central nervous system. One possible mechanism involved in the antagonistic effect of caerulein on amphetamine-induced hyperactivity is changes in activities of endocrine systems.

Amphetamine↗
PubMed1985

[Prognostic factors influencing the improvement of allogeneic bone marrow transplantation in acute leukemia based on a national survey in Japan].

The records of 115 patients with acute leukemia who underwent allogeneic bone marrow transplantation from 1975 through August 1984 were collected by a national survey in Japan. One-year survival significantly improved from 9% in group I (before 1980) to 52% in group II (after 1981). Multivariate analysis indicated that selection of the patients in remission without infection at the time of transplant, as well as progress in technology of total body irradiation and selection of a platelet donor with negative cytomegalovirus titer resulted in the significant improvement in survival after 1981.

Acute Disease↗