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Biomedical subjects

A Kaji

Publications and source records attributed to A Kaji.

At least 91 records · Page 5Linked to original sources
PubMed1989

Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome.

The ribosome-releasing factor (RRF) gene was localized at a position between 2 and 6 min on the Escherichia coli chromosome by measuring the gene-dosage-dependent production of RRF in various E. coli F' merozygotes. This position was confirmed and refined by using a nucleotide probe corresponding to a 16-amino-acid sequence in RRF. It was found that the RRF gene was contained in pLC 6-32 of the Clark-Carbon Gene Bank. Restriction enzyme mapping of E. coli genomic DNA with the above probe led us to conclude that the RRF gene is situated in the 4-min region, somewhere downstream (clockwise) of the elongation factor Ts gene, tsf. A pLC 6-32-derived DNA fragment which carries the RRF gene was found to contain a partial sequence of tsf. The exact location of the translational initiation site of the RRF gene was determined to be 1.1 kilobases downstream from the translational termination site of tsf. The RRF gene is designated frr.

Amino Acid Sequence↗
PubMed1988

Separation of formyl-methionyl transfer RNA, methionyl transfer RNA, and transfer RNAfmet using mixed-mode high-performance liquid chromatography on C6-modified aminopropylsilyl-hypersil.

Preparative amounts of formyl-methionyl-tRNAfmet, methionyl-tRNAfmet, and tRNAfmet were separated from each other with baseline resolution in 30 min using mixed-mode HPLC on hexanoic anhydride-modified aminopropylsilyl-Hypersil 2. Pure tRNAfmet was aminoacylated with [35S]methionine in the presence or absence of a formyl donor and was immediately fractionated on the column. Two isoacceptors, tRNA1fmet and tRNA2fmet, as well as aminoacyl-tRNA synthetases were clearly separated from each other. The purified f[35S]-methionyl-tRNA was biologically active in that as much as 98% could be bound to ribosomes in response to AUGUAA in vitro. Formyl-methionine was released from this complex by the action of termination factor and greater than 92% of bound formyl-methionine was released by puromycin.

Caproates↗
PubMed1988

Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip.

The distribution and origin of perivascular acetylcholinesterase-active and vasoactive intestinal polypeptide-immunoreactive nerve fibers were studied in the rat lower lip by means of acetylcholinesterase histochemistry and vasoactive intestinal polypeptide immunohistochemistry. The perivascular nerve fibers stained intensely with both histochemical techniques and were widely distributed on small arteries and arterioles of the lower lip, especially in the transitional zone between the hairy skin and the mucous membrane. The distributions of the two types of fibers were very similar and most of them showed overlapping coloration, on consecutive staining for vasoactive intestinal polypeptide and acetylcholinesterase. Both acetylcholinesterase-positive and vasoactive intestinal polypeptide-immunoreactive fibers were completely lost on removal of the otic ganglion, while they were not affected by sympathetic ganglion removal or sensory nerve sectioning. In the otic ganglion, most cells exhibited acetylcholinesterase activity, and about 60% of the cells showed light to heavy vasoactive intestinal polypeptide immunoreactivity. These findings indicate that vessels in the rat lip are innervated by parasympathetic fibers originating from the otic ganglion and support the view that vasoactive intestinal polypeptide is present in cholinergic neurons. This may suggest the possible control by the parasympathetic nervous system of cutaneous blood vessels through vasoactive intestinal polypeptide-containing cholinergic neurons, in general or at least in the facial area.

Acetylcholinesterase↗
PubMed1988

Nucleotide sequence of an Rts1 fragment causing temperature-dependent instability.

Rts1 is a multiphenotypic drug resistance plasmid which is eliminated from host bacteria at 42 degrees C but not at 32 degrees C. This phenotype has been called temperature-dependent instability (Tdi). We determined the nucleotide sequence of the Rts1 DNA b' segment which causes this phenotype. Within this 786-base-pair segment, several open reading frames (ORFs) were found, including one which encodes a protein with a molecular weight of 16,000. A protein approximately corresponding to this protein is expressed in Escherichia coli minicells harboring plasmids containing the b' segment. In addition, we found the chi sequence at 112 bases proximal to this ORF. Temperature-dependent elimination due to this segment was not observed in the RecA strain of E. coli, but the RecB protein was not required for expression of this phenotype. We constructed various deletion derivatives and found that three portions, the region containing the chi (nucleotides 1 to 24), ORF (nucleotides 25 to 546), and tail (nucleotides 631 to 786) sequences are necessary for Tdi activity. Site-directed mutagenesis studies indicated that ORF I is required for Tdi expression.

Bacterial Proteins↗
PubMed1987

Identification of the Rts 1 DNA fragment responsible for temperature sensitive growth of host cells harboring a drug resistant factor Rts 1.

We have placed a kanamycin resistance SalI fragment (3.65 Kb) from the drug resistance factor Rts1 into pUC19 and pBR322. These chimeric plasmids containing the kanamycin resistance fragment from Rts1 cause temperature sensitive growth in E coli. The orientation of the kanamycin resistance fragment in the vector plasmids does not influence this phenotype.

DNA Restriction Enzymes↗
PubMed1986

Demonstration of sensory cutaneous nerve fibers in guinea-pig lip using endogenous oxidase histochemistry.

We developed a new histochemical method based on endogenous oxidase activity for the detection of cutaneous nerves in guinea-pig lips. After the application of a reaction mixture containing 0.14 mM 3,3'-diaminobenzidine(DAB) and 15 mM nickel ammonium sulfate in Tris-HCl buffer (pH 7.6), many blue-black sensory fibers were observed, i.e., dermal nerve fiber bundles, perifollicular plexuses, Merkel-cell endings, and Meissner-like nerve endings. The autonomic fibers of the skin were not stained. Electron microscopy revealed that the reaction product was mainly localized on mitochondrial cristae. The addition of 0.01 M sodium azide completely inhibited the reaction. This technique is simple and preferentially stains sensory nerves.

3,3'-Diaminobenzidine↗
PubMed1985

Reduced cleavage by sodium hydroxide of methyladenine in DNA sequencing.

When the Maxam and Gilbert DNA sequencing method which is modified by Bencini et al. (Biotechniques Jan/Feb pp4-5, 1984) is applied to DNA containing methylated adenine in a GATC sequence, the cleavage reaction by sodium hydroxide is found to be greatly reduced in comparison to that of non-methylated adenine. Thus, a faint band in A greater than C reaction suggests a methyl adenine and can be used for its detection. That the faint band corresponds to a methyladenine was confirmed by Sanger sequencing of the same fragment and further by Maxam and Gilbert sequencing of the complementary strand of DNA, which was replicated in an E. coli strain either having or lacking methylation enzymes.

Adenine↗
PubMed1985

Two modes of amber codon read-through in vitro.

Read-through translation of bacteriophage R17 amB2 coat cistron carrying an amber mutation at the seventh codon was studied in vitro using the crude cell extract (S30) derived from an Escherichia coli nonsuppressor strain. Despite the presence of termination factors as well as ribosome-releasing factor (RRF) which prevent the read-through translation [M. Ryoji, J. W. Karpen, and A. Kaji (1981) J. Biol. Chem. 256, 5798-5801], synthesis of coat-like protein still persists at a low level in this system. Characterization of this protein by peptide fingerprinting and amino acid sequencing was performed to reexamine the generally accepted notion that it is produced by amino acid misinsertion to the amber mutation codon. The results indicated, however, that the major population of this coat-like protein is produced as a result of reinitiation of translation from the eighth codon. Read-through by amino acid misinsertion in this system becomes predominant only when the Mg2+ concentration is higher than 16 mM.

Cell-Free System↗
PubMed1985

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids.

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.

Ampicillin↗
PubMed1984

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin.

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.

Amino Acid Sequence↗
PubMed1983

Mode of action of bottromycin A2: effect of bottromycin A2 on polysomes.

When bottromycin A2 was added to an in vitro protein synthesis system carried out by naturally occurring polysomes, it inhibited protein synthesis effectively. Examination of the 3 steps of peptide chain elongation revealed that the binding of aminoacyl-tRNA to the polyribosomes was inhibited by bottromycin A2. In contrast, we concluded that the peptide bond formation and the translocation steps in this system were not inhibited by bottromycin A2 on the basis of the following observations: (1) The break-down of polysomes, which is dependent on EFG, puromycin and RR (ribosome releasing) factor, was insensitive to bottromycin A2; (2) The puromycin dependent release of polypeptide from polysomes, with or without EFG, was not inhibited by bottromycin A2. Thus bottromycin specifically interferes with proper functioning of the A sites of polysomes. This is consistent with the results obtained using the model system with synthetic polynucleotides.

Anti-Bacterial Agents↗
PubMed1983

Inhibition of precartilaginous chick somites by oncogenic virus.

Infection of embryonic chicken notochord-somite explants with Rous sarcoma virus inhibited the in vitro differentiation of somites into cartilage. Visual inspection of the explants revealed that viral infection reduced the size of cartilage nodule formation. Formation of the complex of sulfated proteoglycans with hyaluronic acid was inhibited by RSV infection, and sedimentation analysis of the sulfated proteoglycans showed that very little fast sedimenting proteoglycans were synthesized by RSV-infected explants. The infected explants primarily synthesize a slowly sedimenting sulfated proteoglycan which was chondroitinase resistant. These slow-sedimenting sulfated proteoglycans lack the ability to associate with hyaluronic acid and appear to be noncartilaginous. These effects of RSV are apparently due to the src gene of this virus since the mutant td108, which lacks part of the src gene, has no detectable influence on the chondrogenic differentiation of somite explants. Similarly, infection with RAV-2 as well as with uv-irradiated virus had no detectable effect. The inhibition of synthesis of fast sedimenting proteoglycans was observed at 41 degrees C with explants infected with tsNY68, suggesting that residual activity of transforming gene of this virus at the non-permissive temperature is sufficient for this inhibition in the explants.

Animals↗
PubMed1983

Association of pp36, a phosphorylated form of the presumed target protein for the src protein of Rous sarcoma virus, with the membrane of chicken cells transformed by Rous sarcoma virus.

A cellular protein with a molecular mass of approximately 36 kilodaltons is the presumed target protein of the src protein [the transforming protein encoded by Rous sarcoma virus (RSV)]. The cellular location of the phosphorylated 36-kilo-dalton protein (pp36) in chicken embryo fibroblasts transformed by the Schmidt-Ruppin strain of RSV has been investigated. In these studies, two-dimensional electrophoresis was used for detection of the phosphoproteins in total cell extracts and also in fractionated subcellular components. We conclude that pp36 is localized in the plasma membrane, on the basis of the following observations. (i) Fractionation of 32P-labeled cell extracts showed that pp36 is almost exclusively localized in the crude membrane fraction and no appreciable amount was found in nuclear or cytoplasmic fractions. (ii) On further fractionation of the crude membrane fraction, pp36 was localized mostly in the plasma membrane rather than in other membranous fractions. (iii) Isolated plasma membrane by itself phosphorylated the 36-kilodalton protein on incubation with [gamma-32P]ATP.

Animals↗
PubMed1983

Transformation-defective Rous sarcoma virus mutants with altered p19 of the gag gene and their inhibitory effect on host cell growth.

Mutants (PH2010, PH2011, PH2012) of Rous sarcoma virus which have a growth-inhibitory effect on chicken embryo fibroblasts were isolated from a temperature-sensitive mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (tsNY68). The growth rate of fibroblasts infected with these viruses was about 50 to 60% of that of uninfected fibroblasts. A morphological difference between mutant-infected and uninfected fibroblasts was observed at logarithmic phase but not at stationary phase. Neither the protein p60src nor its associated protein kinase activity was significantly detected by an immunoprecipitation assay in the cells infected with these mutants. Analysis of the unintegrated DNA of the mutant PH2010 showed that a sequence of about 1.4 kilobase pairs at the src gene region is deleted. Further examination of the viral structural proteins in infected cells as well as in virions by immunoprecipitation and peptide mapping revealed that the molecular size of the Pr76 gag protein of the mutant RSV is smaller than that of the mutant tsNY68 because of partial deletion at the p19 gag gene. The peptide maps suggest that the deleted region of the altered p19 of the mutant is near the carboxy terminal of p19. The amount of Prgp92env synthesized in the mutant-infected cells was about fivefold more than that in tsNY68-infected cells.

Animals↗
PubMed1982

Sequence analysis of the heat-labile enterotoxin subunit B gene originating in human enterotoxigenic Escherichia coli.

In this study, we determined the amino-terminal coding sequence, covering the signal peptide and the amino-terminus of the mature peptide, of the heat-labile enterotoxin subunit B (LT-B) gene originating in human enterotoxigenic Escherichia coli. Neither the signal sequence nor the amino-terminal sequence of the mature LT-B was identical to those sequences from porcine enterotoxigenic E. coli, but there was an extensive homology.

Amino Acid Sequence↗
PubMed1981

Effects of viral transformation on synthesis and secretion of collagen and fibronectin-like molecules by embryonic chick chondrocytes in culture.

Monolayer cultures of chondrocytes isolated from 11-day-old chick embryo vertebral cartilage were transformed by Rous sarcoma virus, and the effects of transformation on synthesis and secretion of extracellular proteins by these cells were studied. Transformation resulted in decreased synthesis of type II collagen which did not appear to be due to underhydroxylation of collagenous protein but to a decrease in the total amount synthesized. Carboxymethyl-cellulose chromatography and polyacrylamide disc gel electrophoresis failed to demonstrate any alpha 2 chains as a result of the transformation, suggesting that conversion of type II to type I collagen did not occur. In contrast to the decrease in collagen synthesis, synthesis of a molecule with biochemical characteristics similar to fibronectin increased markedly in virally transformed cultures. Although there were no significant differences in the amount of fibronectin-like molecules in the cell layers of normal and transformed chondrocytes, a marked increase of these molecules in the culture media of the transformed cells was demonstrated. These findings were confirmed by experiments with temperature-sensitive mutants of the virus.

Animals↗