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Biomedical subjects

K Kodaira

Publications and source records attributed to K Kodaira.

At least 37 records · Page 2Linked to original sources
PubMed1995

Comparative studies on the minus origin mutants of Escherichia coli spherical single-stranded DNA phages.

The minus origins for complementary strand DNA synthesis (-ori) of Escherichia coli spherical single-stranded DNA (microvirid) phages G4, phi K, alpha 3, and St-1 closely resemble each other in DNA structure and contain two potential secondary hairpin loops (I and II) that have been implicated as direct recognition sites for host E. coli dnaG protein (primase). We introduced mutations (deletion or insertion) within the -ori regions of phi K and G4 by the nuclease digestion method. Mutants thus constructed produced minute plaques, showed thermosensitivity, and they remarkably reduced the phage yield and rate of viral DNA synthesis. Deletions in the phi K mutants (dTa) were ranging from 1 nucleotide (nt) to 102 nt centered at the hairpin II; a dTa8 mutant was entirely lacking in the two hairpins besides the starting point for primer RNA synthesis. On the other hand, the G4 mutants (dSa) had deletions centered at hairpin I; two mutants dSa35 and dXN completely lost the hairpin I and the primer RNA starting point. In addition, progeny phage populations of several phi K and G4 mutants contained revertant-like phages. DNA sequencing analysis revealed that these secondary phages had been generated by spontaneous DNA rearrangement with additional insertion or deletion near the parental mutation sites, via an unknown recA-independent pathway.

Base Sequence↗
PubMed1994

An acrosomal protein, sp32, in mammalian sperm is a binding protein specific for two proacrosins and an acrosin intermediate.

An acrosomal protein, sp32, was completely purified from acid extracts of ejaculated porcine sperm. Purified sp32 gave a single 32-kDa protein band on SDS-polyacrylamide gel electrophoresis and was characterized as a binding protein specific for 55-, 53-, and 49-kDa forms of (pro)acrosin. This protein was not capable of binding a 43-kDa acrosin intermediate and 35-kDa mature acrosin. sp32 significantly accelerated autoactivation of proacrosin at a basic pH in vitro and affected the maturation pathway of proacrosin. In the presence of sp32, the 49-kDa acrosin intermediate from the 55- and 53-kDa proacrosins was accumulated, instead of the 43-kDa acrosin intermediate. These results suggest that sp32 interacts with both the amino- and carboxyl-terminal sequences of the 53-kDa proacrosin. The cDNA clones coding for porcine and guinea pig sp32 have been identified from testis cDNA libraries in lambda gt11. The deduced amino acid sequence indicates that sp32 is initially synthesized as a 61-kDa precursor protein with a putative signal peptide at the amino terminus. The carboxyl-terminal half of the precursor molecule corresponds to the mature sp32. Thus, sp32 is produced by post-translational modification of the precursor. The binding of sp32 to proacrosin may be involved in packaging the acrosin zymogen into the acrosomal matrix.

Acrosin↗
PubMed1994

Correlation of ultrasound-measured common carotid artery stiffness with pathological findings.

To quantitatively and noninvasively evaluate common carotid atherosclerosis in a series of patients, we measured the stiffness parameter beta, which represents the mechanical properties of the vessel. beta was calculated from the relationship between blood pressure and the diameter of the artery as measured by an ultrasonic, phase-locked, echotracking system. Increases in the severity grade of atherosclerosis as subsequently determined at autopsy were correlated with increased beta values in 60 common carotid arteries (r = .68). Patients with beta values greater than 13 had a pathological diagnosis of atherosclerosis in the common carotid artery. The sensitivity of this discrimination ratio was 80%, and the specificity was 80% as well. Thus, beta shows promise as a useful diagnostic indicator for detecting asymptomatic common carotid atherosclerosis.

Adult↗
PubMed1993

The C. elegans unc-18 gene encodes a protein expressed in motor neurons.

The C. elegans unc-18 gene is required to maintain normal acetylcholine levels. We determined the complete structure of an unc-18 cDNA that encodes a protein of 591 highly charged and hydrophilic amino acids. The protein shows sequence similarity with elements of the secretory pathway in the yeast S. cerevisiae. Antibodies raised against a portion of the unc-18-encoded protein (UNC-18) detected a 68 kd soluble antigen on immunoblots and intensely stained all vertical cord motor neurons in situ. These findings suggest that UNC-18 participates in the axonal transport system and influences the acetylcholine flow in motor neurons.

Amino Acid Sequence↗
PubMed1993

A study on cerebral nicotine receptor distribution, blood flow, oxygen consumption, and other metabolic activities--a study on the effects of smoking on carotid and cerebral artery blood flow.

We investigated middle cerebral artery flow velocity (MCA-FV) by a noninvasive method to determine whether or not smoking causes an increase in cerebral blood flow (CBF). Furthermore we determined sequentially the changes in CBF caused by smoking in order to evaluate changes in responses at different times in daily activities and the effect from meals. The subjects were 25 healthy individuals ranging in age from 20 to 36 yr. MCA-FV was measured by a transcranial Doppler system. They smoked a filtered cigarette for 5 min at 1 P.M., 3 P.M., 6 P.M., 8 P.M., 10 P.M., 8 A.M., and 11 A.M. Results (1) Smoking caused increases in both common carotid artery flow volume and MCA-FV, and the percentage increase of these parameters showed a good correlation (r = 0.809). (2) MCA-FV increased significantly during the first (by 6.6%) and second halves (by 5.4%) of the smoking period. (3) The change in MCA-FV after meals was slight. (4) Smoking tended to increase MCA-FV during each smoking session but the changes were not significant. The pulsatility index reduced significantly during almost every smoking session. These results lead to the conclusion that smoking reduces vascular resistance in cerebral arteries and increases CBF.

Adult↗
PubMed1992

Nucleotide sequence of the genome of the bacteriophage alpha 3: interrelationship of the genome structure and the gene products with those of the phages, phi X174, G4 and phi K.

The complete nucleotide sequence of the genome of the circular single-stranded DNA (isometric) phage alpha 3 has been determined and compared with that of the related phages phi X174 and G4. The alpha 3 genome consists of 6087 nucleotides, which is 701 nucleotides longer than the nucleotide sequence of the phi X174 genome and 510 nucleotides more than that of the G4 genome. The results demonstrated that the three phage species have 11 homologous genes (A, A*, B, C, K, D, E, J, F, G and H), the order of which is fundamentally identical, suggesting that they have evolved from a common ancestor. The sequence of some genes and untranslated intergenic regions, however, differs significantly from phage to phage: for example, the degree of amino acid sequence homology of the gene product is averaged at 47.7% between alpha 3 and phi X174 and 46.9% between alpha 3 and G4, and alpha 3 has a remarkable longer intergenic region composed of 758 nucleotides between the genes H and A compared with the counterparts of phi X174 and G4. Meanwhile, in vivo experiments of genetic complementation showed that alpha 3 can use none of the gene products of phi X174 and G4, whereas the related phage phi K can rescue alpha 3 nonsense mutants of the genes B, C, D and J. These sequencing and in vivo rescue results indicated that alpha 3 is closely related to phi K, but distantly remote from phi X174 or G4, and supported an evolutional hypothesis which has been so far proposed that the isometric phages are classified into three main groups: the generic representatives are phi X174, G4 and alpha 3.

Amino Acid Sequence↗
PubMed1991

Correlation of common carotid flow volume measured by ultrasonic quantitative flowmeter with pathological findings.

To evaluate the possibility of quantitatively diagnosing carotid and cerebral atherosclerosis noninvasively, we measured common carotid flow volume in 60 sides (30 patients), using an ultrasonic quantitative flowmeter, and then compared these findings to the severity score of carotid and cerebral atherosclerosis as determined at autopsy. Stenosis decreased common carotid flow volume in the carotid and cerebral arteries. Increases in the severity score varied inversely with reduced flow volume, which was high in inverse correlation (r = -0.696). Patients with flow volumes of 8.5 ml/sec or greater did not have stenosis greater than or equal to 75%, whereas all patients with flow volumes of 6.4 ml/sec or less had stenosis greater than or equal to 50%, with 45% of these having stenosis greater than or equal to 75%. These pathological findings confirm that the common carotid flow volume reflects the degree of carotid and cerebral atherosclerosis present and that the lower limit of common carotid flow volume in healthy subjects is 6.5 ml/sec.

Arteriosclerosis↗
PubMed1990

Acrosin biosynthesis in meiotic and postmeiotic spermatogenic cells.

It has been widely accepted that mammalian sperm acrosin is first synthesized only in the postmeiotic stages of spermatogenic cells. In this study, we carried out Northern blot analysis of RNAs prepared from purified populations of mouse spermatogenic cells. The acrosin mRNA was obviously found in meiotic pachytene spermatocytes, and the mRNA content markedly increased in postmeiotic round spermatids. Also, the acrosin mRNA in pachytene spermatocytes was functionally associated with polysomes. These results provide evidence that acrosin biosynthesis is already started in meiotic cells and continues through the early stages of spermiogenesis.

Acrosin↗
PubMed1990

Properties of the bacteriophage alpha 3 mutants with deletion and/or insertion in the complementary strand origin.

Bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated in direct recognition sites for host Escherichia coli dnaG protein. We have introduced nucleotide deletion or insertion within the -ori region, by nuclease digestion and polymerase treatment of alpha 3 replicative form DNA. Deletion mutants (delo) showing the following in vivo properties were isolated: minute plaque size, longer latent period, and reduction both in phage yield and viral DNA synthesis as compared with the wild-type phage. In addition, several strains among the delo mutants did not grow on host E. coli dnaB cells at 42 degrees C. These results suggest that certain type of deletion in the -ori region converts simple priming of the complementary strand synthesis to a more complicated phi X174-type initiation that depends on the primosome, rather than on dnaG protein alone.

Bacteriophage phi X 174↗
PubMed1990

Novel replication mutant of microvirid phage alpha 3 deleted in the complementary strand origin.

The bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated as direct recognition sites for host Escherichia coli DnaG protein. To elucidate to what extent such structures are essential, we introduced a nucleotide deletion within the -ori region, by nuclease digestion of alpha 3 replicative form DNA. A mutant, delB, thus constructed had a 121 nucleotide deletion within the -ori region and was completely lacking in the two putative hairpin loops, I and II. The delB mutant formed smaller plaques on the host E. coli C and had a longer latent period, but the mean burst size at 37 degrees C was almost the same (400 phages) as that of the wild type. In contrast to the parental phage, growth of the mutant depends on host dnaB and dnaC functions. These results indicate that the prototype secondary structures in the alpha 3 origin of complementary strand synthesis are dispensable for delB and that the alpha 3 mutant has an additional replication origin whose function is dependent on DnaB and DnaC proteins, rather than on DnaG protein alone.

Bacteriophages↗
PubMed1989

Possible finger structure in gene A protein of Microviridae.

Microvirid phages alpha 3 and phi K encode for A protein which functions in initiation of the viral DNA synthesis. By nucleotide sequencing analysis, we have found that each gene A protein has 'finger motif structure' which conserves two cysteine and histidine residues similar to the consensus sequence deduced from more than thirty finger motifs reported from many eukaryotic regulatory proteins. In closely related phages, phi X174 and G4, we have detected the same structures in their gene A proteins also.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of the bacteriophage alpha 3 origin of complementary DNA synthesis: in vivo properties of mutants.

Bacteriophage alpha 3 origin of complementary strand DNA synthesis contains two potential secondary loop structures, I and II, which have been implicated in direct recognition sites for host Escherichia coli dnaG protein. To elucidate the function of the hairpin loops, we have introduced point mutations within the stem of the hairpin II so as to disturb its base-pairings. A mutant, oriAA, which had two point mutations in the region, formed minute plaques on E. coli host cells and its mean burst size at 37 degrees C was about 50, whereas that of wild-type was 250. In addition, the growth of oriAA at 42 degrees C was thermosensitive and the burst size was reduced to 5. From the oriAA, a revertant-like phage oriGA occurred spontaneously with a high-frequency of about 2.10(-2). It retained one point mutation and the plaque size and phage yield were nearly same as those of wild-type. These results are discussed with respect to the role of secondary structure as well as specific nucleotide sequence in the recognition site for the dnaG protein.

Base Sequence↗
PubMed1985

Function and structure of microvirid phage alpha 3 genome. DNA sequence of H gene and properties of missense H mutant.

The nucleotide sequence of wild-type alpha 3 H gene and its surrounding region was determined and compared with those of phi X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Bacteriophage phi X 174↗
PubMed1984

Function and structure of microvirid phage alpha 3 genome. II. Isolation and properties of various mutants of alpha 3.

Various mutants were isolated from a microvirid (isometric single-stranded DNA) phage alpha 3, by mutagenesis with hydroxylamine or nitrous acid. They were divided into eight complementation groups, and mainly by genetic crosses the gene alignment was determined as -A-B-C'-D-J'-F-G-H-. Except for groups C' and J', each defective gene product was clearly discerned in electropherograms of proteins extracted from the phage-infected suppressor-negative (Su-) Escherichia coli. Only gene A mutants abolished synthesis of the progeny replicative-form DNA (RF), whereas mutants belonging to groups B, C', D, E, F and J' affected RF replication at late stage, as well as synthesis of the single-stranded DNA (SS). Additional properties of several mutants are also discussed.

Bacteriophage phi X 174↗
PubMed1984

Isolation of human erythrocyte membranes in glucose solution.

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.

Cell Fractionation↗
PubMed1984

Isolation and some properties of bacteriophage alpha3 gene J mutant.

To elucidate the in vivo function of the J gene of microvirid (isometric) phages, we isolated several strains carrying a double mutation in J and H genes from phage alpha3 and then constructed single mutants each having an amber codon in the J gene. The J mutants could not multiply in suppressor-less hosts and were deficient in single-stranded progeny DNA synthesis. Nucleotide sequences of the wild-type and mutant alpha# J genes were analyzed to determine the mutation sites. The amino acid sequence of the J gene was also deduced from the nucleotide sequence and compared with those of phiX174 and G4.

Amino Acid Sequence↗