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Biomedical subjects

K Matsubara

Publications and source records attributed to K Matsubara.

At least 451 records · Page 25Linked to original sources
PubMed1986

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene.

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.

Bacterial Proteins↗
PubMed1986

Expression of human salivary alpha-amylase gene in Saccharomyces cerevisiae and its secretion using the mammalian signal sequence.

A cDNA fragment coding for human salivary alpha-amylase precursor was joined to the promoter of the Saccharomyces cerevisiae PHO5 gene, and the recombinant gene was inserted into a vector plasmid capable of autonomous replication in yeast. Yeast cells transformed with this recombinant plasmid synthesized about 5 X 10(5) molecules of the enzyme per cell when synthesis was induced by deprivation of inorganic phosphate and released about half of the synthesized enzyme into the medium. The enzyme is stable, and exhibited the same specific activity as alpha-amylase in human saliva. The amylase-producing yeast grew on starch and produced alcohol.

Acid Phosphatase↗
PubMed1986

Expression of the human salivary alpha-amylase gene in yeast and characterization of the secreted protein.

Recombinant plasmids were constructed in which the human salivary alpha-amylase gene, with or without the N-terminal signal sequence for secretion, was placed under control of the APase (PHO5) promoter of Saccharomyces cerevisiae. In yeast cells transformed with the alpha-amylase gene having the human signal sequence for secretion, the gene was expressed and the enzyme was secreted into the medium in three different glycosylated forms. The amylase gene without the signal sequence was also expressed in yeast, but the products were neither secreted nor glycosylated. Determination of the N-terminal amino acid (aa) sequence revealed that the 15-aa signal sequence had been cleaved from the secreted enzyme, and that the N-terminal residue, glutamine, had been modified into pyroglutamate, as is commonly observed with the mammalian salivary alpha-amylase. Thus, the human salivary alpha-amylase signal sequence for secretion was correctly recognized and processed by the yeast secretory pathway. The C-terminal residue was identified as leucine, which is predicted from the nucleotide sequence data to be located at position 511 in front of the termination codon. Therefore, there is no post-translational processing in formation of the C terminus.

Acid Phosphatase↗
PubMed1986

Expression of human gastrin gene in normal and gastrinoma tissues.

The complete sequence of the human gastrin gene is reported here. This gene consists of three exons. Nine Alu family sequences are found within the gene and in the surrounding region. S1 mapping study showed that the transcription of gastrin gene starts at 60, 57, or 55 bp upstream from the 3' end of the first exon. The mechanism of mRNA synthesis in a gastrinoma tissue was studied to clarify the ectopic production of gastrin. It was found that mRNA synthesis starts from the same three transcriptional start points. Southern blotting profiles for normal gastric antrum and gastrinoma DNA were indistinguishable from each other within the 18-kb region containing the gastrin gene, showing that no genomic rearrangements are associated with the gastrinoma formation. Thus, the overproduction of gastrin in this tumor is likely to be due to an aberrant expression control system of the cell, rather than a change in the control region of the gastrin gene.

Amino Acid Sequence↗
PubMed1986

Structure of human cholecystokinin gene and its chromosomal location.

We have determined the entire structure of human cholecystokinin (CCK) gene, which is 7 kb in size and separated into three exons. S1 endonuclease analysis has shown two putative transcription initiation sites that are preceded by 'TATA' equivalent sequences located 39 bp and 35 bp upstream from these sites. The promoter region contains five 'G-C box'-like sequences, which are believed to be sp 1-binding sites. By chromosome sorting in combination with velocity sedimentation and Southern hybridization, the human cck gene was mapped on the short arm of human chromosome 3.

Amino Acid Sequence↗
PubMed1986

Detection of delta 9-THC in saliva by capillary GC/ECD after marihuana smoking.

A method is described for the determination of delta 9-tetrahydrocannabinol (delta 9-THC) in the saliva by the use of a combination of moving-precolumn injector and glass capillary gas chromatograph with electron capture detector (GC/ECD). There were no interfering peaks due to impurities around the peak of pentafluoropropyl derivative of delta 9-THC (delta 9-THC-PFP). This GC/ECD method was linear over the range of 5-200 ng/ml of delta 9-THC-PFP. The lower detection limit was approximately 1 ng/ml. delta 9-THC content in the saliva after experimental marihuana smoking was measured by this method. It was demonstrated that for at least 4 h after smoking the level of delta 9-THC was sufficient for detection.

Adult↗
PubMed1986

Purification and properties of the mini-F plasmid-encoded E protein needed for autonomous replication control of the plasmid.

Mini-F plasmid encodes a protein, E protein, that is indispensable for its autonomous replication. We have constructed a plasmid that overproduces the E protein and have purified the protein to apparent homogeneity. Using nitrocellulose filter binding and nuclease digestion assays, we demonstrated that the E protein binds to three unique regions of the mini-F DNA sequence: the replication origin (ori2) and an incompatibility locus (incB), another incompatibility locus (incC), and the promoter for the E gene. These binding sites have a common 8-base-pair sequence. These findings suggest the direct role of the E protein in initiation of mini-F replication and copy number control. They are also in line with the in vivo evidence that the incompatibility phenotype caused by incB and incC DNA is due to titration of a factor(s) indispensable for replication and that the production of the E initiator protein of the mini-F plasmid is under autoregulatory control.

Amino Acid Sequence↗
PubMed1986

Molecular cloning of an oncogene from a human hepatocellular carcinoma.

A transforming DNA, named lca (for liver cancer), was obtained from a primary human hepatocellular carcinoma (HCC) in transformation assays using NIH 3T3 cells and a calcium phosphate coprecipitation method. High molecular weight DNA obtained from the HCC tissue was employed for this purpose. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage for further studies. Restriction enzyme analyses showed that the minimal size of the lca transforming DNA is about 10 kilobase pairs and that its cleavage profiles are different from those of any one of the previously reported human transforming genes or retroviral oncogenes. No cross-hybridization was observed between these genes and the lca DNA. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC.

Animals↗
PubMed1986

Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons.

Two recombinant plasmids were constructed that allow expression of the hepatitis B core (HBc) antigen gene in the yeast Saccharomyces cerevisiae under the control of the repressible acid phosphatase promoter. One plasmid was designed to produce polypeptide I, which consists of 183 amino acids, and the other plasmid was designed to produce polypeptide II, which has an additional 29-amino-acid sequence at the amino terminus of polypeptide I. The viral genome may code for either one or both of these two polypeptides, depending upon the selection of initiation codons. Both polypeptides produced in yeast cells reacted with anti-HBc antibody and were assembled into spherical particles approximately 27 nm in diameter. Particles made of polypeptide I were stable, whereas those made of polypeptide II readily dissociated when exposed to high salt levels. The antigenicity of the HBc (as defined by its reactivity to anti-HBc antibody in the reversed passive hemagglutination assay) disappeared as the particle dissociated, leaving materials that sedimented slowly and that reacted to anti-hepatitis B e antibody. These observations strongly suggest that native viral cores are mostly (if not all) made of polypeptide I, because it is reasonably stable, and that the N-terminal portion of this polypeptide has some, but not a profound, influence on the assembly of polypeptides into particles.

Capsid↗
PubMed1986

Localization of a novel v-erbB-related gene, c-erbB-2, on human chromosome 17 and its amplification in a gastric cancer cell line.

The c-erbB-2 gene is a v-erbB-related proto-oncogene which is distinct from the gene encoding the epidermal growth factor receptor. By using two independent methods, hybridization of both sorted chromosomes and metaphase spreads with cloned c-erbB-2 DNA, we mapped the c-erbB-2 locus on human chromosome 17 at q21, a specific breakpoint observed in a translocation associated with acute promyelocytic leukemia. Furthermore, we observed amplification and elevated expression of the c-erbB-2 gene in the MKN-7 gastric cancer cell line. These data suggest possible involvement of the c-erbB-2 gene in human cancer.

Cell Line↗
PubMed1986

Saccharomyces cerevisiae PHO5 promoter region: location and function of the upstream activation site.

Saccharomyces cerevisiae repressible acid phosphatase (PHO5) is induced when inorganic phosphate in the culture medium is depleted. To study the mechanism of this regulation, we constructed various deletions in the 5'-flanking region of the PHO5 gene. Two elements were revealed by this analysis: an upstream activation site (UAS) and a downstream element, both playing parts in the expression of this gene. The UAS is located between -384 and -292 upstream of the initiation codon and activates expression of the gene when inorganic phosphate is depleted. It consists of two homologous regions (UAS I and UAS II) that contain CTGCACAAATG and an adenine-plus-thymine-rich sequence, either one of which suffices for the effect. The downstream element includes a putative TATA box at -100 from the ATG codon and is necessary for efficient transcription and expression of the normal-sized PHO5 transcript. The distance between the UAS and the downstream element can be altered without causing loss of expression efficiency, and the action of the UAS is not affected by its orientation. These results are consistent with a model wherein UAS acts as a site of activation for transcription by interaction with a protein factor(s) that becomes active when inorganic phosphate is depleted from the culture medium.

Base Sequence↗
PubMed1986

beta-Endorphin involvement in the antidopaminergic effect of caerulein.

Caerulein has been shown to possess a long-lasting antagonistic effect on amphetamine hyperactivity in rats when given in combination with haloperidol. We found that this effect of caerulein involved beta-endorphin. Naloxone pretreatment and hypophysectomy abolished the caerulein effect, while intracerebroventricular or intra-nucleus accumbens injection of beta-endorphin together with haloperidol administration produced an effect similar to that of caerulein. The results suggest that the long-term antagonism of the amphetamine effect of caerulein is mediated by the endogenous opioid beta-endorphin.

Amphetamine↗
PubMed1986

A novel oncogene from a human hepatocellular carcinoma.

Primary human hepatocellular carcinomas (HCC) were surveyed for oncogenes by transformation assays using NIH 3T3 cells and calcium phosphate coprecipitation method. One new transforming DNA, called lca (for liver cancer) was obtained, and its properties were studied in detail. The lca DNA, localized in a 10.5 kb DNA fragment, was assigned to human chromosome 2. It showed identical restriction enzyme cleavage profiles as its counterpart DNA obtained from normal tissue, indicating that there is no extensive DNA rearrangement associated with activation process of the lca DNA. The normal counterpart DNA shows no transforming activity. Recombinant genes of the laca and its normal counterpart made in vitro have allowed localization of the site of "activation" to a limited region of the 10.5 kb fragment. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC.

Animals↗
PubMed1986

The microvascular architecture of the vestibule and the endolymphatic duct and sac of the rat in vascular corrosion casts.

The blood vessels of the vestibule and the endolymphatic duct and sac (ES) of the rat were reproduced with methacrylate casting medium and observed under a scanning electron microscope. Dense capillary networks of the macula utriculi and the macula sacculi were observed. The collecting venules from the vestibule emptied into the vein of the vestibular aqueduct (VVAQ). The plexus of the vessels in the ES was triangular in shape and had anastomoses with vessels of the bone and dura and drained into the VVAQ. The posterior meningeal artery (PMA) gave off two branches to the ES. These findings supported the similarity of the vascularization of the vestibule and the ES between the human and the rat.

Animals↗
PubMed1986

Tetrahydro-beta-carbolines in human urine and rat brain--no evidence of formation by alcohol drinking.

1-Methyl-1,2,3,4-tetrahydro-beta-carboline (1-MeTHBC) and 6-methoxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline (6-M-1-MeTHBC) were identified and quantified in the urine of 131 healthy volunteers by gas chromatography/mass spectrometry (GC/MS) with a chemically-bonded glass-capillary column. 1-MeTHBC was present naturally in the human urine, whereas 6-M-1-MeTHBC was observed in only 20% of all subjects at a much lower level. No significant relationship was observed between these substances and alcohol consumption in the subjects. Various foods and alcoholic beverages were found to contain 1-MeTHBC but not 6-M-1-MeTHBC; thus, the urinary 1-MeTHBC detected could be of dietary origin. Salsolinol, on the other hand, was significantly elevated in the urine without increase of THBCs by long-term moderate drinking. 1-MeTHBC and 6-M-1-MeTHBC were also analysed in the brains of rats following inhalation of ethanol and acetaldehyde for four days. No evidence of formation of these compounds was observed in any regions of the brain.

Acetaldehyde↗
PubMed1985

Molecular cloning and nucleotide sequence of human pancreatic secretory trypsin inhibitor (PSTI) cDNA.

We have isolated and sequenced a cDNA clone coding for the human pancreatic secretory trypsin inhibitor (PSTI) from a human pancreatic cDNA library. The predicted product consists of 79 amino acids, and contains no apparent functional polypeptide other than PSTI. Southern blot analysis suggests that there is one copy of PSTI gene per haploid genome. This gene seems to be expressed not only in pancreas, but also in gastric mucosa, since a Northern blot analysis demonstrated the presence of a poly(A) RNA of the same size as in the pancreas. A comparison of sequences between human PSTI mRNA and mouse epidermal growth factor (EGF) mRNA revealed a high homology, suggesting that they share a common ancestral DNA sequence.

Animals↗