Biomedical subjects

A Nakata

Publications and source records attributed to A Nakata.

At least 73 records · Page 4Linked to original sources

PubMed1995

Analysis of the tyrosinase gene of the Japanese pond frog, Rana nigromaculata: cloning and nucleotide sequence of the genomic DNA containing the tyrosinase gene and its flanking regions.

Three genomic DNA fragments containing the tyrosinase-encoding gene (TYR) of the Japanese pond frog, Rana nigromaculata, were cloned. The first, clone I, was isolated from a genomic library of sperm DNA using the mouse TYR cDNA as the probe and contained a DNA segment similar to exon 4 of the mouse TYR gene. Subsequently, the TYR cDNA was isolated by screening a frog embryo cDNA library using clone I as the probe. Two clones that contain genomic DNA of the TYR gene were isolated also from a blood cell DNA library using the frog TYR cDNA as the probe. Comparison of the nucleotide (nt) sequences of the genomic clone II DNA and the cDNA revealed that clone II contained a 3,140-bp DNA fragment consisting of the 5'-flanking region, the first exon, and a part of the first intron. The region upstream of the coding region contained the characteristic sequences for regulatory elements, including TATA- and CAAT-motifs, and also a pigment cell-specific promoter element, which is shared by the promoter regions of the vertebrate TYR genes. A 764-bp segment containing an upstream 748-bp non-coding region and 16-bp coding region was functional for expression of the promoter-less cat gene on a plasmid in the transiently transformed albino frog melanophore. The genomic clone III contained the 3'-untranslated region of the mRNA and its 3'-flanking region. Thus, the cDNA plus genomic DNA fragments isolated here cover the entire TYR gene and its flanking regions.

Animals↗

PubMed1995

I-123 metaiodobenzylguanidine cardiac scintigraphy in patients with an implanted permanent pacemaker.

T1 scintigraphic abnormalities have been reported in patients with an implanted permanent pacemaker, but little is known about the MIBG scintigraphic findings in such patients. This study was performed to assess the MIBG scintigraphic findings in patients with an implanted permanent pacemaker, and to test the hypothesis that imaging characteristics of MIBG scintigraphy differ according to its mode. Twelve patients (4 men and 8 women, mean age: 72.4 +/- 9.5 years), who had undergone the implantation of a permanent pacemaker for bradyarrhythmias, underwent MIBG scintigraphy. The patients were divided into VVI pacemaker and DDD pacemaker groups. The tomograms were divided into nine segments and the MIBG defect in each segment scored on a scale ranging from 0 (normal uptake) to 3 (no uptake). Total MIBG defect scores were generated by summing the scores for the nine segments in each patient. MIBG scintigraphic abnormalities were found in ten of the twelve patients. The six patients with the VVI pacemaker manifested MIBG scintigraphic abnormalities. These MIBG scintigraphic abnormalities were observed in all segments, particularly in the posterior segments. The mean total defect score of the VVI group was higher than that of the DDD group (14.8 +/- 9.8 vs 3.0 +/- 3.5, respectively p < 0.05). Therefore, we conclude that despite several limitations of the study, MIBG scintigraphic abnormalities occur in patients with implanted permanent pacemakers, and that such abnormalities are more prominent with the VVI than DDD pacemaker.

3-Iodobenzylguanidine↗

PubMed1994

Conjugation of recombinant reverse transcriptase of HIV-1 to beta-D-galactosidase from Escherichia coli for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG.

Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to beta-D-galactosidase from Escherichia coli in three different ways. Maleimide groups were introduced into beta-D-galactosidase molecules using N,N'-o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into beta-D-galactosidase molecules, which had been treated with excess of 4,4'-dithiodipyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III). Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl-S-acetylmercaptoacetate. The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-beta-D-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N epsilon-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads with (anti-human IgG gamma chain) IgG. The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.

Animals↗

PubMed1994

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection.

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-beta-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. This assay was at least 3,000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine.

Adolescent↗

PubMed1994

Facilitatory but nonessential role of the muscarinic cholinergic system in the generation of long-term potentiation of population spikes in the dentate gyrus in vivo.

The role of the muscarinic cholinergic system in the generation of LTP in the medial perforant path-dentate granule cell synapses in vivo was investigated using anesthetized rats. Cholinergic denervation with AF64A, a cholinergic toxin, did not significantly affect LTP induced by a strong tetanus (100 pulses at 100 Hz), but attenuated the LTP induced by a weak tetanus (30 pulses at 60 Hz). The i.c.v. injection of scopolamine (1.5-50 nmol) did not significantly affect the LTP induced by the strong tetanus but attenuated the magnitude of LTP produced by the weak tetanus in a concentration-dependent manner. These results suggest that the cholinergic system is not essential for induction of LTP by strong stimuli but plays a role in facilitating the generation of LTP by weak stimuli. Furthermore, the induction of LTP by a weak tetanus was blocked by pirenzepine but affected by neither AF-DX116 nor 4-diphenylacetoxy-N-methylpiperidine. The LTP-facilitatory action of the cholinergic system is probably mediated by muscarinic M1 receptors.

Animals↗

PubMed1994

Detection of anti-human immunodeficiency virus type 1 (HIV-1) immunoglobulin G in urine by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) with recombinant reverse transcriptase as an antigen.

Anti-human immunodeficiency virus type 1 immunoglobulin G in urine was detected by an immunoassay with reverse transcriptase as the antigen and beta-D-galactosidase as the label; this immunoassay was 30-fold more sensitive than the previous immunoassay with peroxidase as the label. The sensitivity and specificity were both 100%. The lowest signal for asymptomatic carriers was 20-fold higher than the highest signal for seronegative subjects.

Adolescent↗

PubMed1994

Anatomical structure and action of the tail muscles in the cat.

Anatomical structure and movements of tail muscles were studied in 12 adult cats. Tail muscles mainly consisted of the following 6 muscles on both sides, M. sacrocaudalis dorsalis medialis (SDM), M. sacrocaudalis dorsalis lateralis (SDL), M. coccygeus lateralis (CL), M. coccygeus medialis (CM), M. sacrocaudalis ventralis lateralis (SVL) and M. sacrocaudalis ventralis medialis (SVM). Experiments by electrical stimulation of muscles demonstrated that the tail is bent to the dorsal direction by actions of SDM and SDL, curved laterally by those of SDL, CL, CM and SVL, bent to the ventral direction by those of SVL and SVM, and rolled by those of CL and CM on both sides. Contraction of SDL and SVL produced rapid and strong tail movements by actions of their long tendons.

Animals↗

PubMed1994

Crossed and uncrossed segmental synaptic effects on obliquus externus abdominis motoneurons from cutaneous and cutaneous-muscle nerve in spinal cats and alpha-chloralose-anesthetized cats.

Segmental synaptic responses in the obliquus externus abdominis (OEA) for stimulation of the dorsal cutaneous and cutaneous-muscle (CD) nerve and the ventral cutaneous and cutaneous-muscle (CV) nerve were investigated in 49 spinal cats and 28 cats under alpha-chloralose anesthesia (chloralose cats) with an intracellular recording method. The result that there was no monosynaptic PSP in OEA motoneurons for stimulation of the CD or CV nerves (containing the cutaneous-muscle nerve) indicates that the cutaneous muscle of the dorsal or ventral area does not connect monosynaptically to the OEA motoneuron. The OEA motoneurons in chloralose cats received few effects from descending pathways coming from the upper spinal cord and the brain. Almost all OEA neurons had polysynaptic EPSP responses for stimulation of the ipsilateral or contralateral CD or CV nerves at 5-10T stimulus intensity in spinal or chloralose cats, which indicates that the OEA muscle can contract bilaterally when strong impulses of superficial afferents from the left or right skin or cutaneous muscle enter in the spinal cord.

Anesthesia, General↗

PubMed1994

Crossed and uncrossed segmental synaptic effects on rectus abdominis motoneurons from cutaneous and cutaneous-muscle nerve in spinal cats and alpha-chloralose-anesthetized cats.

Segmental synaptic responses evoked by stimulation of the dorsal cutaneous and cutaneous-muscle (CD) nerve or the ventral cutaneous and cutaneous-muscle (CV) nerve were investigated with intracellular recording from rectus abdominis (RA) motoneurons in 49 spinal cats and 28 alpha-chloralose-anesthetized cats. No monosynaptic and few disynaptic connections existed in the circuit from the ipsilateral or contralateral CD or CV nerves to RA motoneurons in spinal cats. Polysynaptic EPSP responses equal to or over trisynaptic connections were dominant. In alpha-chloralose-anesthetized cats, RA motoneurons exhibited significantly more polysynaptic EPSP responses at lower stimulus intensities (1.5T and 2T) of ipsilateral CV nerve stimulation than at those of contralateral CV nerve stimulation. The result that there was no monosynaptic or disynaptic PSP in RA motoneurons produced by low-intensity stimulation of the ipsilateral CD or CV nerve containing the cutaneous-muscle nerves indicated that Ia afferents from the cutaneous-muscle did not connect mono- or di-synaptically to the RA motoneuron. RA motoneurons exhibited more polysynaptic IPSP responses in alpha-chloralose-anesthetized cats than in spinal cats. Segmental pathways from the CD or CV nerves to RA motoneurons might receive inhibitory descending inputs from the upper spinal cord or the brain, in contrast to those from the same nerves to obliquus externus abdominis motoneurons previously reported.

Anesthesia, General↗

PubMed1993

Detection of antibody IgG to HIV-1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV-1 infection.

For diagnosis of HIV-1 infection, attempts were made to detect anti-HIV-1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT) and p17 as antigens. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli beta-D-galactosidase for p17. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV-1 infection possible.

Adolescent↗

PubMed1993

Escherichia coli RuvA and RuvB proteins involved in recombination repair: physical properties and interactions with DNA.

Escherichia coli RuvA and RuvB proteins are encoded by an SOS-regulated operon, which is involved in DNA repair and recombination. RuvB has weak ATPase activity, which is enhanced by the addition of RuvA and DNA, and RuvA and RuvB in the presence of ATP promote branch migration at Holliday junctions. In this work, the physical states of RuvA and RuvB and their interactions with DNA were studied by sedimentation analysis and gel filtration chromatography. RuvA formed a stable tetramer in solution, which resisted dissociation by SDS at room temperature. RuvB formed a dimer in solution. When RuvA and RuvB were mixed, an oligomer complex was formed consisting of a tetrameric form of RuvA and a dimeric form of RuvB, and this complex bound to DNA. The maximal enhancement of the RuvB ATPase activity by RuvA was achieved at this stoichiometry in the presence of excess DNA.

Adenosine Triphosphatases↗

PubMed1993

Use of the recombinant chimera proteins, LacZ-Env and Gag-Env, for immunological studies on HIV-1 infection.

To use Env proteins as antigens for detection of the human immunodeficiency virus type-1 (HIV-1) antibodies, we attempted to overexpress the Env proteins in Escherichia coli. To study the epitopes in the Env proteins recognized by the sera of HIV carriers, various regions of the proviral DNA encoding the Env region were fused to the 3' end of the lacZ gene. The immunoblotting analysis of the LacZ-Env(512-611) and LacZ-Env(721-826) proteins with the 41 positive sera revealed that the former and the latter immunologically reacted with 100 and 78% of the sera, respectively. To avoid rare false-positive reactions due to the LacZ moiety of the fusion protein, we attempted to express the Env(512-611) alone or Gag-Env(512-611) under the control of bacteriophage T7 promoter. Although we could express only a low level of the Env(512-611) peptide in E. coli, we succeeded in producing large amounts of the Gag(121-406)-Env(512-611) and Gag(308-406)-Env(512-611) proteins as chimeric proteins. Both of these chimera proteins strongly reacted with the 41 positive sera. We purified these proteins and analyzed the immunological reactivity by dot blot with the 60 positive sera and the 84 normal sera. As little as 20 ng of the dotted proteins was enough for the reaction with the positive sera, whereas as much as 320 ng of them did not show false-positive reactions with the normal sera. We obtained highly purified Gag-Env proteins with highly specific seroreactivity, which should be useful for diagnosis and prognosis.

AIDS-Related Complex↗

PubMed1993

Role of the sigma 70 subunit of RNA polymerase in transcriptional activation by activator protein PhoB in Escherichia coli.

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli, which are induced by phosphate starvation, requires the specific activator protein PhoB in addition to the RNA polymerase holoenzyme containing the major sigma-factor sigma 70. To study the mechanism of transcriptional activation and identify the subunit of RNA polymerase involved in specific interaction with PhoB, we attempted to isolate rpoA and rpoD mutants that are specifically defective in the expression of the pho genes. We isolated two rpoD mutants with such properties, but no rpoA mutant with similar properties. The rpoD mutations altered amino acids within and near the first helix of the putative helix-turn-helix (HTH) motif in the carboxy-terminal region of sigma 70. Activities of the pho promoters in vivo were severely reduced in these mutants, whereas those of the PhoB-independent promoters were affected only marginally at most. The reconstituted mutant RNA polymerase holoenzymes were severely defective in transcribing the pstS gene, one of the pho genes, whereas they were efficient in transcribing the PhoB-independent promoters. Phosphorylated PhoB, which binds to the pho promoters with high affinity, mediated the specific binding of the wild-type holoenzyme to the pstS promoter, but it did not mediate the binding of the mutant holoenzymes. These results suggest that PhoB promotes specific interaction between RNA polymerase and the pho promoters for transcriptional activation, and the first helix of the putative HTH motif plays an essential role in the interaction, probably by making direct contact with PhoB.

Alleles↗

PubMed1993

Molecular analysis of the phoH gene, belonging to the phosphate regulon in Escherichia coli.

By making operon fusions with lambda placMu53, we identified, cloned, and analyzed the phoH gene belonging to the phosphate (pho) regulon. We mapped the phoH gene at 23.6 min in the Escherichia coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Its nucleotide sequence revealed an open reading frame of 354 amino acids which contains sequences for nucleotide-binding motifs. From comparison of the DNA sequences, phoH was found to be identical to psiH, which had been identified as a phosphate starvation-inducible gene (W.W. Metcalf, P.M. Steed, and B.L. Wanner, J. Bacteriol. 172:3191-3200, 1990). The PhoH protein was overproduced by the T7 promoter system, identified as a protein of about 39 kDa, and purified. The amino-terminal amino acid sequence of the PhoH protein agreed with the one deduced from the DNA sequence. We demonstrated that PhoH has an ATP-binding activity by a photoaffinity labeling experiment. Two transcriptional initiation sites (P1 and P2) were identified by S1 nuclease mapping. The upstream P1 promoter contains a pho box, the conserved sequence shared by the pho regulon genes. The region containing the pho box was bound by PhoB protein, the transcriptional activator of the pho regulon, as revealed by footprinting. Regulation of phoH expression in vivo was studied by constructing plasmids containing transcriptional fusions of the phoH promoters with a promoterless gene for chloramphenicol acetyltransferase. Transcription from the P1 promoter required the phoB function and was induced by phosphate limitation, while transcription from the P2 promoter was independent of phoB and constitutive under tested conditions.

Affinity Labels↗

PubMed1993

Adrenocortical carcinoma with a giant pericardial mass.

A patient with a history of adrenocortical carcinoma presented with massive pericardial effusion and a giant pericardial mass. Death resulted from cardiac tamponade. At autopsy a large necrotic mass was histologically similar to the original adrenocortical carcinoma. We present this case and review the literature on metastatic pericardial disease.

Adrenal Cortex Neoplasms↗

PubMed1992

Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.

We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.

Amino Acid Sequence↗

PubMed1992

Systematic sequencing of the Escherichia coli genome: analysis of the 0-2.4 min region.

A contiguous 111,402-nucleotide sequence corresponding to the 0 to 2.4 min region of the E. coli chromosome was determined as a first step to complete structural analysis of the genome. The resulting sequence was used to predict open reading frames and to search for sequence similarity against the PIR protein database. A number of novel genes were found whose predicted protein sequences showed significant homology with known proteins from various organisms, including several clusters of genes similar to those involved in fatty acid metabolism in bacteria (e.g., betT, baiF) and higher organisms, iron transport (sfuA, B, C) in Serratia marcescens, and symbiotic nitrogen fixation or electron transport (fixA, B, C, X) in Azorhizobium caulinodans. In addition, several genes and IS elements that had been mapped but not sequenced (e.g., leuA, B, C, D) were identified. We estimate that about 90 genes are represented in this region of the chromosome with little spacer.

Bacterial Proteins↗

PubMed1992

Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration.

The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holiday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (> 20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (> 4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.

Bacterial Proteins↗