Biomedical subjects
Publications and source records attributed to H Hirokawa.
The effect of tripeptide, Arg-Gly-Asp (RGD), on the transfection activity of Bacillus phage M2 DNA was examined. The transfection activity decreased when M2 DNA was preincubated with RGD before it was added to competent cells.
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Sensitized photo-induced changes of vitreous structure were investigated using both in vivo and in vitro model systems. In the former, rabbit eyes were injected with the photosensitizer riboflavin, and in the latter, calf vitreous samples were treated with riboflavin or Methylene Blue prior to irradiation with white light. The active species of oxygen, i.e. singlet oxygen, superoxide anion, hydroxyl radical and hydrogen peroxide, generated by the photodynamic action of the sensitizer, caused significant liquefaction of the calf vitreous in vitro. There was little liquefaction of the rabbit vitreous in vivo, suggesting the presence of a protective mechanism in vivo. hyaluronidase induced significantly greater liquefaction in vitro than either Methylene Blue or riboflavin. This study suggests that loss of gel vitreous structure can result from extensive depolymerization of hyaluronidase by hyaluronidase and less drastic conformation and molecular weight changes in the photosensitized reactions. Although light-induced liquefaction was less marked than enzyme-induced liquefaction, the mechanism of the former is more pertinent to age-related vitreous synchysis.
The vitreoretinal relationship in 59 eyes with asteroid bodies was studied by biomicroscopy with the El Bayadi-Kajiura lens. Complete posterior vitreous detachment (PVD) occurred less often in eyes with asteroid bodies than in control eyes (P less than .05), and partial PVD occurred more often than in control eyes (P less than .01). In eyes with asteroid bodies after the age of 70 years, the prevalence of PVD, either complete or partial, was lower than in age-matched control eyes (P less than .01), and the prevalence of liquefaction (19%) was lower than has been reported in controls. Of eight eyes with proliferative diabetic retinopathy and asteroid bodies, only one showed partial PVD. Patients who were diabetic and who had asteroid bodies were significantly younger than patients without diabetes with asteroid hyalosis (P less than .01). Our data suggest that the presence of asteroid bodies may arrest the process of vitreous collapse or contraction and that diabetes might influence the development of asteroid hyalosis.
Aphidicolin-resistant mutants (Aphr) of Bacillus subtilis bacteriophage phi 29 were isolated after mutagenesis with hydroxylamine. Efficiency of plating (e.o.p.) of the resistant mutants was not reduced at 500 microM aphidicolin, although e.o.p. of wild type phi 29 was less than 10(-5) at the same concentration of aphidicolin. By recombination and complementation analyses, both sites of the mutations, aph-71 and aph-101, of Aphr71 and Aphr101, respectively, were mapped in gene 2 which encodes phi 29 DNA polymerase. The activity of wild type phi 29 DNA polymerase, in a partially purified fraction, was inhibited by aphidicolin. DNA polymerases from Aphr71 and Aphr101, prepared in the same manner as that of wild type, were resistant to the drug. These results indicate that the acquisition of the aphidicolin resistance of Aphr71 and Aphr101 of bacteriophage phi 29 results from a structural alteration of phi 29 DNA polymerase which reduces sensitivity to aphidicolin.
It has been suggested that the heterologous population of Bacillus phage M2 is derived from an original clone, which is identical with phage Nf, by the deletion on a particular region of the genome. We have determined the nucleotide sequence of this region of M2 subclones and Nf genomes. The results clearly indicate that the homologous recombination through the short direct repeated sequence is the main cause of the varied deletions found in the genomes of M2 subclones.
Of 250 eyes with idiopathic preretinal macular fibrosis, 56 had no posterior vitreous detachment (Group 1), ten had partial posterior vitreous detachments without vitreous traction to the macula (Group 2), 29 had partial posterior vitreous detachments with vitreous traction to the macula (Group 3), and 155 had complete posterior vitreous detachments (Group 4). There were significantly more eyes with visual acuities of 20/60 or worse, cystoid macular changes, or macular fluorescein leakage in Group 3 than in Group 1 or Group 4. Thus, the presence of vitreous traction to the macula was associated with worse anatomic and functional findings.
The nucleotide sequence of Bacillus phage Nf gene F has been determined. The deduced amino acid sequence of gpF is very similar to that of gp4, the transcriptional activator of phage phi 29. Both proteins contain the consensus structure that is conserved for the DNA-protein interacting domain of DNA-binding proteins such as the repressor, Cro and CII proteins of phage lambda.
The vitreous in 76 eyes with peripheral uveitis was examined and compared with the vitreous in 77 eyes of age- and sex-matched controls. The vitreous findings in peripheral uveitis were inflammatory and cotton ball-like opacities, gel shrinkage, and posterior vitreous detachment. The prevalence of partial and complete vitreous detachment was significantly higher in the peripheral uveitis group than in the control group. The prevalence of partial vitreous detachment was significantly higher in eyes with macular change than in eyes without macular change, whereas the prevalence of complete vitreous detachment was significantly higher in eyes without macular change than in eyes with macular change. We also found a significantly higher prevalence of complete vitreous detachment in eyes with visual acuity of 20/50 or better than in those with an acuity of 20/60 or worse. The prevalence of partial vitreous detachment was significantly higher in eyes with a visual acuity of 20/60 or worse than in those with an acuity of 20/50 or better.
The genome structures of a temperate Lactobacillus phage, phi FSW, and its virulent mutants, phi FSVs, were examined by restriction, heteroduplex and nucleotide-sequence analyses. The results showed that two out of three phi FSVs had the same 1.3 kbp insertion (designated as ISL1) at different positions in the phi FSW sequence. ISL1 was 1,256 bp long and contained at least two long open reading frames of 279 and 822 bases on one strand. Inverted repeats were found at the termini of the ISL1 which was bracketed by 3 bp direct repeats of the phi FSW sequence. From this evidence, we concluded that ISL1 was a transposable element in Lactobacillus casei.
Vitreous examinations using the three-mirror Goldmann and the preset El Bayadi-Kajiura lenses were performed on 222 eyes. The presence of posterior vitreous detachment, vitreous traction, liquefaction, lacunae, and cells was noted. Clinical findings using the two lenses differed in 10% of the variables studied. The accuracy of the El Bayadi-Kajiura technique was confirmed by clinical and histological examination of 20 animal eyes with experimentally altered vitreous. The Goldmann lens afforded examination of peripheral vitreous and better identification of cells in the vitreous, whereas the El Bayadi-Kajiura lens permitted better visualization of complex vitreoretinal relationships.
From phi 29-infected Bacillus subtilis cells, we have isolated a protein fraction which promotes in vitro replication of phi 29 DNA. This fraction catalyses both initiation and elongation, indicating that it contains the product of gene 3 (tp: terminal protein) and the product of gene 2 (gp2: probably a DNA polymerase), since initiation requires the two products (Blanco et al. 1983; Matsumoto et al. 1983). The fractions isolated from cells infected with temperature-sensitive (ts) mutants of gene 2 and gene 3 were thermolabile in both the initiation and elongation assays. When the pre-initiated material from the ts fractions of each mutant was heat-inactivated and mixed no complementation, restoring the elongation activity, was found. These results indicate: (i) tp and gp2 participate not only in the initiation but also in the elongation of phi 29 DNA replication, (ii) they probably function in tight physical association with each other.
A multicopy plasmid, 2.1 kb in size, was isolated from Shigella sonnei and named pKYM. This plasmid is cryptic and isolated along with pKY-1, a ColE1-like plasmid. In this paper, we report the physical map of pKYM and some characters required for its multiplication. The replication of the plasmid DNA does not require DNA polymerase I but depends on protein(s) produced by itself. The plasmid is poorly mobilized by the F factor.
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Cell-free extracts prepared from phi 29 and M2-infected Bacillus subtilis cells catalyse the formation of complexes between terminal protein and [alpha-32P]-dAMP in the presence of [alpha-32P]-dATP, MgCl2, ATP, and phage DNA with terminal protein covalently linked at both the 5'ends. The complex formation does not take place when proteinase K-treated DNA is added or when uninfected extract is used. The phi 29 complex thus formed is smaller than the M2 complex, primarily due to the different molecular weights of the respective terminal proteins. Extracts prepared from cells infected with suppressor-sensitive mutants of genes 2 or 3 of phi 29 or genes G or E of M2 do not support complex formation. When the pair of extracts of phi 29 or M2-infected cells are mixed, however, formation of the complex takes place as a result of in vitro complementation. These results indicate that the complex formation observed in vitro reflects in vivo initiation of phage DNA replication. The product of gene 2 of phi 29 may be the enzyme that catalyses formation of the complex.