Biomedical subjects

A Nakata

Publications and source records attributed to A Nakata.

At least 19 recordsLinked to original sources

PubMed1995

Autophosphorylation and activation of transcriptional activator PhoB of Escherichia coli by acetyl phosphate in vitro.

The PhoB protein, the transcriptional activator for the genes belonging to the phosphate regulon in Escherichia coli, was autophosphorylated in the presence of acetyl phosphate (acP) in vitro. The properties of phospho-PhoB, such as stability upon acid or alkali treatment and activating pstS transcription by RNA polymerase holoenzyme, were the same as those of phospho-PhoB produced from phospho-PhoR or phospho-PhoM. These results indicate that PhoB is an enzyme that catalyzes its own phosphorylation using acP, a low-molecular-weight metabolic intermediate.

Bacterial Proteins↗

PubMed1995

Mutational analysis of the role of the first helix of region 4.2 of the sigma 70 subunit of Escherichia coli RNA polymerase in transcriptional activation by activator protein PhoB.

Transcription of the genes belonging to the phosphate (pho) regulon in Escherichia coli requires the specific activator protein PhoB, in addition to RNA polymerase containing the major sigma factor, sigma 70, which is encoded by rpoD. We previously isolated two mutant sigma 70s (D570G and E575K) that were specifically defective in transcribing the pho genes. The mutated sites were located near and within the first helix of the helix-turn-helix (HTH) motif or region 4.2 of sigma 70. To study further the role of the first helix of the HTH motif of sigma 70 in transcriptional activation by PhoB, we made a series of rpoD mutations that alter the motif and purified the mutant sigma 70 proteins. RNA polymerases containing the mutant sigma 70s Y571A, T572L, V576T, K578E and F580V showed reduced in vitro transcription from the pstS promoter, a representative pho promoter, in the presence of PhoB, whereas RNA polymerase containing another mutant sigma 70 (E574K) showed enhanced transcription from the promoter. Transcription from the activator-independent tac promoter and the pBR-P4 promoter, which is independent of PhoB and requires cAMP-CRP (cAMP receptor protein) for transcription, was affected at most only marginally by these sigma 70 mutations. These results provide further evidence that the first helix plays an important role in the specific interaction between RNA polymerase and PhoB protein bound to the pho promoters in transcriptional activation.

Amino Acid Sequence↗

PubMed1995

Overproduction, purification, and diagnostic use of the recombinant HIV-1 Gag proteins, the precursor protein p55 and the processed products p17, p24, and p15.

HIV-1 Gag protein precursor p55, and its processed products, p17, p24, and p15 were overproduced in Escherichia coli and purified to near homogeneity. To study the antigenic properties and the potentiality as the diagnostic and prognostic reagents, varying amounts of the purified Gag proteins were dotted onto the polyvinylidene difluoride membrane and reacted with 40 sera of HIV-1-infected individuals (35 AC, 1 ARC, and 4 AIDS patients) and 10 sera of normal healthy donors. p55 reacted with 40 (100%) sera of HIV-1 carriers, while p17, p24, and p15 reacted with 37 (92.5%), 35 (87.5%) and 34 (85%) of the 40 sera of HIV-1 carriers, respectively. On the whole, the reaction of p55 was especially strong and that of p15 was the weakest. p55 showed the strongest reaction among the four Gag proteins with all specimens, and it showed a positive reaction with a carrier serum with which none of the processed Gag proteins showed a positive reaction. Therefore, p55 is the most useful antigen among the four Gag proteins for detection of the Gag antibodies and may even be one of the most useful antigens for the diagnosis of HIV-1 infection.

Chromatography, Gel↗

PubMed1995

Three antigenic regions in p17 of human immunodeficiency virus type 1 (HIV-1) revealed by mouse monoclonal antibodies and human antibodies in HIV-1 carrier sera.

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 x 10(5)-1.4 x 10(8) M-1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 x 10(5)-1.8 x 10(7) M-1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

Amino Acid Sequence↗

PubMed1995

Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects.

To clarify the physiological roles of CD36 as an oxidized low density lipoprotein (OxLDL) receptor, we analyzed the monocyte-derived macrophages from normal and two CD36-deficient subjects, since we identified the molecular abnormalities (Kashiwagi, H., Y. Tomiyama, Y. Kosugi, M. Shiraga, R. H. Lipsky, Y. Kanayama, Y. Kurata, and Y. Matsuzawa 1994. Blood. 83:3545-3552; and Kashiwagi, H., Y. Tomiyama, S. Honda, S. Kosugi, M. Shiraga, N. Nagao, S. Sekiguchi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1995. J. Clin. Invest. 95:1040-1046). Scatchard analysis of 125I-OxLDL binding showed a linear plot and the maximum binding was lower by approximately 40% in the macrophages from subjects with CD36 deficiency than those from normal controls. Competition studies showed that the uptake of 125I-OxLDL was suppressed by OKM5, an antibody against CD36, by 53% in normal control macrophages, but not in the CD36-deficient macrophages. After incubation with OxLDL for 24 h, cholesteryl ester mass accumulation was reduced by approximately 40% in the macrophages from CD36-deficient subjects than those from normal controls. These results suggest that CD36 is one of the physiological receptors for OxLDL. Since specific binding of OxLDL was only reduced by approximately 40% in spite of the complete deficiency of CD36, several other receptors also may have some role in OxLDL uptake. Further studies will be needed to assess the quantitative role of CD36 in foam cell formation in vivo.

Adult↗

PubMed1995

Distal vasoconstriction following coronary angioplasty--comparison of emergent and elective PTCA.

To examine the occurrence of distal vasoconstriction following primary percutaneous transluminal coronary angioplasty (PTCA) for acute myocardial infarction, quantitative coronary angiography was performed during and after acute myocardial infarction. An assessment of coronary artery vasoconstriction was made in patients undergoing primary and elective PTCA. The primary PTCA group was subdivided into two groups based on initial antegrade flow characteristics: group 1 (TIMI grade flow 0/1) and group 2 (TIMI grade flow 2/3). All balloon angioplasties were performed during continuous administration of nitrates. There was no significant difference with relation to lesion diameter, lesion stenosis, or diameter of the left circumflex coronary artery between the acute and chronic phase in both groups. The distal diameter, however, was smaller during the acute phase than in the chronic phase in both groups (1.7 +/- 0.5 vs 2.0 +/- 0.4 mm, p < 0.001; 2.0 +/- 0.4 vs 2.2 +/- 0.3 mm, p < 0.05, respectively). The percent vasoconstrictive response of the distal segment was greater in the primary PTCA group than in the elective PTCA group (11.2 +/- 18.1 vs -1.9 +/- 7.1%, p < 0.0001, respectively). In conclusion, nitrate-resistant coronary artery vasoconstriction following primary PTCA occurs distal to the site of balloon dilation.

Aged↗

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↗