Biomedical subjects

K Kodaira

Publications and source records attributed to K Kodaira.

At least 19 recordsLinked to original sources

PubMed2001

Endonuclease IV homolog from Dictyostelium discoideum: sequencing and functional expression in AP endonuclease-deficient Escherichia coli.

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).

Amino Acid Sequence↗

PubMed2001

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

Amino Acid Sequence↗

PubMed2000

Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli.

The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS-PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA.

Acetyl Coenzyme A↗

PubMed2000

Purification and DNA-binding properties of the cro-type regulatory repressor protein cng encoded by the Lactobacillus plantarum phage phi g1e.

The putative repressor protein Cng (10kDa on an SDS gel) for the lytic pathway of Lactobacillus plantarum phage φg1e was purified using the Escherichia coli Pt7 system, and its DNA-binding ability for the seven operator-like sequences, the GATAC-boxes (Gb1 to Gb7), was investigated in vitro. In gel-shift assays, Cng selectively bound to the DNA fragments containing the GATAC-box(es). In addition, DNase I footprinting analysis with supercoiled DNA demonstrated that Cng can specifically cover about a 25bp region centered around each of the GATAC-boxes, although two boxes, Gb4 and Gb6, were only partially protected. Moreover, protein crosslinking experiments using glutaraldehyde suggested that Cng most likely functions as a dimer. On the other hand, the binding ability of Cpg for the GATAC-boxes in supercoiled DNA was also examined under the same conditions as in Cng; unlike Cng, Cpg covered Gb4 and Gb6 completely sufficiently as well as the other five boxes. Thus, the present and previous [Kakikawa et al., Gene 215 (1998) 371-379; 242 (2000) 155-166] results indicate a possibility that the two proteins Cng and Cpg selectively bind to the GATAC-boxes that act as operators, and can decide between the lytic or lysogenic pathways through repression of the promoter activity of P(R) as well as P(L).

Bacteriophages↗

PubMed2000

Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA.

The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP-). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP-. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and beta-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and beta-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.

Animals↗

PubMed1999

Cloning and characterization of the gene encoding mouse osteoclast differentiation factor.

Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.

Animals↗

PubMed1999

High-level expressing YAC vector for transgenic animal bioreactors.

The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

Animals↗

PubMed1999

Analysis of control elements for position-independent expression of human alpha-lactalbumin YAC.

A major problem in the production of transgenic animal bioreactors using microinjections is the low production rate of high-expressing transgenic animals due to the position effect. We previously reported that transgenic rats carrying the 210 kb yeast artificial chromosome (YAC) including the human alpha-lactalbumin gene express the transgene in a position-independent manner. The 210 kb YAC was thought to have all the elements necessary for position-independent expression. In this paper, we constructed fragmented YAC clones and a cosmid clone, and produced transgenic rats to analyze these elements. Transgenic rats with both the 50 kb upstream and downstream regions of the alpha-lactalbumin gene had position-independent expression. Transgenic rats with the 20 kb upstream and downstream regions, however, had position-dependent expression. Therefore, all the elements necessary for position-independent expression are thought to be located in the 50 kb upstream to 50 kb downstream region of the alpha-lactalbumin gene. Furthermore, we replaced the human alpha-lactalbumin promoter with the bovine alphaS1-casein promoter in the 210 kb YAC and produced transgenic rats. Position-dependent expression was observed. The elements required for position-independent expression of the bovine alphaS1-casein gene are different from those required for the human alpha-lactalbumin gene, despite the fact that the two genes have the same tissue and developmental specificity.

Animals↗

PubMed1999

Terrestrial gamma ray dose rates of Brunei Darussalam.

GPS-indexed in-situ and car-borne survey of terrestrial gamma-ray dose rates were carried out in Brunei and adjacent areas using two portable NaI(Tl) counters. The mean and population weighted average dose rates for Brunei are 34 and 33 nGy/h, respectively. The car-borne data and the in-situ data when spectral analysed separately, were found to show fractal behaviour with D of 1.7 and 1.8, respectively. A contour map of the dose rates was also produced.

Air Pollutants, Radioactive↗

PubMed1999

Basic subsite theory assumptions may not be applicable to hydrolysis of cellooligosaccharides by almond beta-glucosidase.

Steady-state kinetic parameters for the hydrolysis of cellooligosaccharides by almond beta-glucosidase were evaluated at pH 5.0 and 25 degrees C in relation to the subsite theory (K. Hiromi, Biochem. Biophys. Res. Commun., 40, 1-6, 1970). The value of k0/Km decreased monotonously with increasing degree of polymerization (DP) of the substrates (DP = 2-6). Also, the Km and k0 values for cellotriose were smaller than those for cellobiose. These DP dependencies differ from those of most amylases and glucosidases studied so far, to which the subsite theory has been successfully applied. The subsite parameters could not be consistently obtained, which suggests that one or both of the two basic assumptions of the subsite theory might not be applicable to the hydrolysis of cellooligosaccharides by the enzyme. That is, the intrinsic rate of the hydrolysis may depend on the DP and/or there may be interaction between subsites for binding the glucose residues of a substrate.

Journal Article↗

PubMed1999

Analysis of the disruption mutant of the oscillin homolog gene of Dictyostelium discoideum.

A homolog of oscillin, the Ca2+ oscillation-inducing factor of the hamster, was identified from the cellular slime mold Dictyostelium discoideum and designated Dd-oscillin. In the developmental stages of D. discoideum, the gene is expressed at the prestalk region which contains a higher concentration of cytosolic Ca2+ than the prespore region. The Dd-oscillin null strain aggregated but did not develop further when the cells were plated on non-nutrient agar at a density of 1.5x10(6) cells/cm2, showing that the Dd-oscillin gene is important for development. Since the null cells carrying the hamster oscillin gene formed fruiting body, the hamster oscillin was the homolog of Dd-oscillin as far as function is concerned. In addition, the null cells formed fruiting body in the presence of 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ: a specific inhibitor of Ca2+-ATPase activity in the endoplasmic reticulum). These results suggest that Dd-oscillin will increase cytosolic Ca2+ in the cells and promote further development.

Animals↗

PubMed1997

Functional and structural features of the holin HOL protein of the Lactobacillus plantarum phage phi gle: analysis in Escherichia coli system.

Lactobacillus plantarum phage phi gle has two consecutive cell lysis genes hol-lys (Oki et al., 1996b). In the present study, functional and structural properties of the hol protein (Hol) were characterized in Escherichia coli. Electron microscopic examinations showed that hol under plac in E. coli XL1-Blue injured the inner membrane to yield empty ghost cells with the bulk of the cell wall undisturbed. Northern blot analysis indicated that hol-lys genes under plac were co-transcribed, although the amount of hol transcript was larger than that of lys, ceasing via an apparently rho-independent terminator just downstream of hol. However, deletion and/or fusion experiments suggested that: (1) the N-terminal half of phi gle Hol composed of three putative transmembrane domains may be responsible for interaction with membrane; (2) the N-terminal end (five amino acids) seems nonessential; and (3) the C-terminal half containing charged amino acids appears to be involved in proper hol function. These results suggest that phi gle Hol is a member of the lambdoid holin family, but divergent in several properties from lambda holin.

Amino Acid Sequence↗

PubMed1996

Cloning and nucleotide sequence of the major capsid proteins of Lactobacillus bacteriophage phi gle.

Bacteriophage phi gle was induced from a lysogenic Lactobacillus strain Gle. phi gle genome is double-stranded DNA of approximately 42.5 kilo-base (kb) pairs. SDS poly-acrylamide gel electrophoresis demonstrated that the phage particles contain 4 major structural (capsid) proteins, gpB, gpG, gpO, and gpP, whose molecular weights (MW) are estimated to be 64, 43, 29 and 26 kilodaltons (kDa), respectively. More than 16 minor proteins ranging from 113 to 9.6 kDa were also detected. The genes for the major capsid proteins were cloned and each DNA sequence was determined. N-terminal amino acid alignments determined by protein sequencing completely coincided with those deduced from the nucleotide sequences.

Amino Acid Sequence↗

PubMed1996

Transgene expression in mammary glands of newborn rats.

Transgene expression in the mammary glands of newborn rats was studied to establish an early selection system for transgenic animals producing exogenous proteins in their milk during lactation. A fusion gene composed of the bovine alpha S1 casein gene promoter and the human growth hormone gene was microinjected into rat embryos. Transgenic lines that produced human growth hormone in their milk were established and used in this study. Immediately after birth, and without any hormone treatment, human growth hormone was found in the extracts of mammary glands from both male and female rats derived from the line secreting human growth hormone in their milk. The expression of the transgene in mammary glands of newborn rats was also detected by the presence of human growth hormone mRNA. Nontransgenic newborn rats did not express the human growth hormone gene in their mammary glands, while the mRNA for rat alpha casein, an endogenous milk protein, was found in all mammary glands from both transgenic and nontransgenic neonates. These results show that analyzing the expression of transgenes in the mammary glands of neonates is a valuable tool to select the desired transgenic animals and to shorten the selection schedules establishing the transgenic animals.

Animals↗

PubMed1996

Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes.

Transgenic rats expressing the rat c-myc gene under the control of the human metallothionein II A promoter were produced. We found that the female transgenic rats were fertile, but that the male transgenic rats were sterile. Atrophy of the seminiferous tubules and depletion of sperm were observed in the sterile male testes. The expression of differential stage-specific mRNAs, including those of the c-kit receptor proto-oncogene, meiotic heat-shock protein 70 gene, acrosin gene, and transition protein 1 gene, was analyzed by the reverse transcriptase-polymerase chain reaction during spermatogenesis. The results suggested that spermatogenesis in these sterile rats were arrested at the prophase of meiosis in the primary spermatocytes. We found that apoptotic DNA fragmentation occurred in primary spermatocytes of the sterile transgenic rats. These results suggest that overexpression of the c-myc gene induces apoptosis at the prophase meiosis of the primary spermatocytes thereby causing male sterility in the c-myc transgenic rats.

Animals↗

PubMed1996

The virion proteins encoded by bacteriophage phi K and its host-range mutant phi KhT: host-range determination and DNA binding properties.

The microvirid phage phi K, specific for Escherichia coli K12, contains a circular single-stranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core). phi KhT, a host-range mutant of phi K, can grow on E. coli C and B, besides K12, and is more thermosensitive than the parental phage phi K. Sequencing analysis revealed that the genome of phi K and phi KhT consists of 6,089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of alpha 3, phi X174, and G4 infective to strain C. In phi KhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala. Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to phi K SS DNA more tightly than and preferentially over the host E. coli SS-DNA-binding protein (SSB). These results indicate that the two spike proteins G and H are involved in the determination of phi K host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle.

Amino Acid Sequence↗

PubMed1996

Common carotid artery wall properties in Takayasu's arteritis.

The vessel wall properties of the common carotid artery have been noninvasively and quantitatively assessed with an ultrasonic instrument. Stiffness parameter beta, which represents the mechanical properties of the vessel, was calculated from the relationship between blood pressure and the diameter of the artery. There are no reports that quantitatively assess wall properties in Takayasu's arteritis. The authors compared, in vivo, the vessel wall properties of the common carotid artery in 14 patients with Takayasu's arteritis versus those in 60 normal subjects (controls). They measured changes in the inner diameter of the artery between systole and diastole with an ultrasonic, phase-locked, echo-tracking system. Beta was significantly higher in Takayasu's arteritis than in normal subjects (better than a 99% confidence interval). The findings for each decade were as follows: 3rd decade (20s): 35.7 +/- 28.9 vs 5.01-6.46, P=0.0001; 4th decade (30s): 19.5 +/- 9.71 vs 6.09-7.80, P=0.02; 5th decade (40s): 26.2 +/- 11.3 vs 7.26-9.28, P = 0.0001; 6th decade (50s): 19.1 +/- 4.27 vs 8.66-11.25, P = 0.0001. Takayasu's arteritis significantly impaired the mechanical performance of the common carotid artery. Thus, beta shows promise as a useful diagnostic indicator of Takayasu's arteritis.

Adult↗

PubMed1995

Determination of the single strand origin of Shigella sonnei plasmid pKYM.

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a -ori defective M13 vector phage, M13 delta lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RSA) which functions in plasmid sitespecific recombination and/or transfer.

Base Sequence↗