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Biomedical subjects

H Sakamoto

Publications and source records attributed to H Sakamoto.

At least 433 records · Page 24Linked to original sources
PubMed1995

Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.

The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.

GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

Adenylate Kinase↗
PubMed1995

Vessel formation by choroidal endothelial cells in vitro is modulated by retinal pigment epithelial cells.

OBJECTIVE: To elucidate the mechanism of bovine choroidal endothelial (BCE) cell angiogenesis and, in particular, the role of retinal pigment epithelial (RPE) cells by use of an in vitro coculture assay system. METHODS: The BCE cells were isolated from choroidal tissues and cultured. They were embedded in type I collagen gel and incubated. The gel-embedded BCE cells were then covered with a monolayer of RPE cells, pericytes, choroidal fibroblasts, or additional BCE cells on culture day 0 or day 14. The BCE cells in culture formed a meshwork of tubelike structures. The length of the tubelike structures (micrometers per field) was quantified by image analysis, as an indicator of angiogenesis. The effect of RPE cells on normal and growth-arrested BCE cell tube formation was evaluated in this assay system. The mechanism of RPE-induced angiogenesis was studied by parallel experiments with the use of neutralizing antibodies against specific growth factors (basic fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor beta). RESULTS: Ultrastructural analysis revealed that the tubelike structures had features typical of choroidal endothelial cells. Cocultures initiated on day 0 revealed that BCE cell angiogenesis was promoted by overlying RPE cells and, to a lesser extent, by pericytes, choroidal fibroblasts, and additional BCE cells when compared with BCE cells without covering cells. In cocultures initiated after BCE tube formation (day 14), there was inhibition of BCE angiogenesis by overlying RPE cells when compared with cultures without overlay or with an overlay of BCE cells. The RPE cells stimulated tube formation of growth-arrested BCE cells less effectively than did normal BCE cells. Neutralizing antibody for basic fibroblast growth factor and vascular endothelial growth factor, but not transforming growth factor beta, inhibited control and RPE-induced tube formation by BCE cells. CONCLUSIONS: Overlying RPE cells stimulate the formation of tubelike structures by choroidal endothelial cells more effectively than do fibroblasts or pericytes but inhibit BCE tube formation in older cultures. The effect involves endothelial proliferation and differentiation. The stimulatory effect of overlying RPE cells can be inhibited by neutralizing antibodies to vascular endothelial growth factor and basic fibroblast growth factor, which suggests that these growth factors play an important role in this phenomenon.

Animals↗
PubMed1995

Muscles of the pelvic outlet in the rhesus monkey (Macaca mulatta) with special reference to nerve supply.

BACKGROUND: The manner of innervation of the muscles of the inferior limb, pelvic outlet, and tail in rhesus monkey (Macaca mulatta) was investigated in detail in 11 pelvic halves of four males and two females. RESULTS: The origins of the pudendal nerve were widespread and overlapped the sacral plexus. After removal of the bone structures, detailed dissection revealed the origin of the pudendal nerve to ventrocaudally overlap the sacral plexus. This dorsoventral branching pattern between the sacral plexus and the pudendal nerve is similar to that in Urodela (Akita, 1992b), Lacertilia (Akita, 1992a) and Aves (Akita et al., 1992a), and is a basis for morphological understanding of the muscles of the pelvic outlet. CONCLUSIONS: The muscles of the pelvic outlet consist of the levator, sphincter, and tail muscle groups, based on the stratification of the supplying nerves the former two groups likely derive from the ventral muscles of the inferior limb, and the latter group from the ventral caudal (trunk) muscles.

Anatomy, Artistic↗
PubMed1995

Learning versus performance effects of cocaine on discriminative heart rate conditioning in rats.

The study examined the effects of cocaine on learning and performance of a classically conditioned heart rate (HR) discrimination in rats involving two auditory conditioned stimuli (CSs). In the discrimination protocol, one CS (CS+) was paired with the shock unconditioned stimulus (US) on a consistent basis and the other CS (CS-) was always presented alone. Four groups received an IP injection of 1, 3, 10, or 30 mg/kg cocaine and a fifth group received saline. Shortly after the injections, all groups were given six CS-alone trials, followed by 24 randomly sequenced discrimination conditioning trials (12 CS+ and 12 CS-). Approximately 72 h later, all groups were given six test trials with each CS in the absence of cocaine to evaluate the presence or absence of discrimination learning. All cocaine groups showed impaired discrimination performance on the discrimination conditioning trials, reductions in early pretest CS-alone responses, and reductions in resting HR. However, on the non-drug test trials discrimination performance was normal in all cocaine groups. The results established that in spite of major changes in HR dynamics, learning of the HR discrimination was not affected by cocaine but that cocaine did interfere with the performance of the discrimination. Except for the highest 30 mg group, the performance decrement appeared to be related to a cocaine-produced reduction in the capacity to inhibit bradycardia responding to the safe CS-. It was suggested that this loss of inhibitory control may have been due to cocaine changes in a corticothalamic pathway that controls inhibition of bradycardia to a safe CS-.

Animals↗
PubMed1995

Secondary hyperparathyroidism shortens the action of vecuronium in patients with chronic renal failure.

The authors studied the duration of action of vecuronium in 15 patients with normal renal function and 40 patients with chronic renal failure to evaluate the effect of secondary hyperparathyroidism on the action of vecuronium. The patients were divided into four groups: 15 patients with normal renal function (Group A); nine patients with chronic renal failure who did not need haemodialysis (Group B); 15 anephric patients who did not require parathyroidectomy (Group C); and 16 anephric patients who underwent parathyroidectomy because of severe secondary hyperparathyroidism (Group D). The ratio of the height of the first twitch (T1) to the baseline value before vecuronium administration was measured by an electromyogram. Baseline T1 was obtained after anaesthesia induction with thiamylal iv. The time to 10% recovery of the first twitch (REC 10) after administration of vecuronium 0.12 mg.kg-1 iv was measured in each group. Anaesthesia was maintained with isoflurane and nitrous oxide in oxygen, and supplemented with fentanyl iv. Patients in Group D showed shorter REC 10 (51 +/- 4 min) than those in Groups B (71 +/- 6 min) and C (80 +/- 10 min) (P < 0.05), but similar REC 10 to patients in Group A (37 +/- 4 min). These results suggest that the duration of action of vecuronium in anephric patients with secondary hyperparathyroidism is shorter than in those without secondary hyperparathyroidism.

Anesthesia, Inhalation↗
PubMed1995

Interaction between substance P-immunoreactive central terminals and gamma-aminobutyric acid-immunoreactive elements in synaptic glomeruli in the lamina II of the chicken spinal cord.

We investigated the interaction between gamma-aminobutyric acid (GABA)-immunoreactive (IR) elements and substance P (SP)-IR central terminals in synaptic glomeruli in lamina II of the chicken spinal cord in order to ascertain how pain information is modulated in the spinal dorsal horn. We combined the peroxidase-antiperoxidase (PAP) technique and the protein A-gold (PAG) technique to observe the synaptic relationship between these two components. At the light microscopic level, we observed both GABA-IR and SP-IR elements in the lamina II. GABA-IR elements were also observed in the lamina III. At the electron microscopic level, the following three GABA-IR elements formed synapses with the SP-IR central terminals in synaptic glomeruli: (1) elements which appeared to be axon terminals containing tightly-packed pleomorphic clear vesicles; (2) elements which appeared to be vesicle-containing dendrites with loosely-packed clear and dense-cored vesicles (DCVs); and (3) dendrites without synaptic vesicles. The first type of element was always presynaptic to the SP-IR central terminal. The second type was postsynaptic, presynaptic or in some cases reciprocal to the SP-IR central terminals. The third type was postsynaptic to the SP-IR central terminal. These results suggest that the SP-containing primary afferents activate GABA-containing dendrites and that the SP-containing primary afferents are inhibited presynaptically by GABA-containing neurons through axo-axonic and dendro-axonic synapses.

Animals↗
PubMed1995

Atypicality of several antipsychotics on the basis of in vivo dopamine-D2 and serotonin-5HT2 receptor occupancy.

An in vivo receptor binding technique was used to evaluate the binding profiles of typical and atypical antipsychotic drugs to striatal dopamine-D2 and frontal serotonin-5-HT2 receptors in a rat brain using more specific ligands than those previously employed. [3H]-YM-09151-2 or [3H]-ketanserin was injected into the tail vein 10 minutes after administration of test drugs. One hour after the ligand injection, radioactivities in the striatum, frontal cortex, and cerebellum were counted to obtain receptor occupancies by the test drugs. Higher ratios of potency in occupying 5-HT2 versus D2 receptors were found for clozapine, RMI-81512, and tiospirone compared to haloperiodol and pimozide. Zotepine, mosapramine, and clocapramine produced ratios that fall between these two groups. Chlorpromazine was exceptional as a typical antispychotic by these criteria. Relatively strong antagonism of 5-HT2 receptors by atypical antipsychotics was confirmed by this in vivo measure of receptor binding using more selective ligands than those used in previous studies.

Animals↗
PubMed1995

Hst-1 (FGF-4) antisense oligonucleotides block murine limb development.

The initiation of limb development depends on the site specific proliferation of the mesenchyme by the signals from the apical ectodermal ridge (AER) in embryonic mouse. We have previously reported that the local expression of Hst-1/Fgf-4 transcripts in AER of the mouse limb bud is developmentally regulated, expressed at 11 and 12 days post coitus (p.c.) embryo. In an effort to further understand the role of Hst-1/FGF-4 in mouse limb development, an antisense oligodeoxynucleotides (ODNs) study was performed. We first established a novel organ culture system to study mouse limb development in vitro. This system allows mouse limb bud at 9.5-10-d p.c. embryo, when placed on a sheet of extracellular matrix in a defined medium, to differentiate into a limb at 12.5-d p.c. embryo within 4.5 d. Using this organ culture system, we have shown that exposure of 9.5-10-d p.c. embryonal limb bud explants to antisense ODNs of Hst-1/FGF-4 blocks limb development. In contrast, sense and scrambled ODNs have no inhibitory effect on limb outgrowth, suggesting that Hst-1/FGF-4 may work as a potent inducing factor for mouse limb development.

Animals↗
PubMed1995

Development and characterization of a new model of hematogenous osteomyelitis in the rat.

Hematogenous osteomyelitis was produced in the tibia or the mandible of rats by drilling a hole into the bone, injecting sodium morrhuate, and inoculating Staphylococcus aureus Phillips into the femoral vein. Animals were sacrificed after 2 weeks and examined. The infection was characterized grossly and radiographically by bone deformation, histopathologically by a characteristic suppurative reaction, and microbiologically by the recovery of S. aureus Phillips from the infected tissue. These findings indicate that the model mimics human osteomyelitis with respect to its inflammatory bone changes. In contrast to earlier rat models in which bacteria were injected directly into the bone, this new experimental model allows study of the initiating events of osteomyelitis such as bacterial attachment and might assist as a model for both prophylactic and therapeutic trials.

Animals↗
PubMed1995

Fetal loss in the first trimester after demonstration of cardiac activity: relation of cytogenetic and ultrasound findings.

A retrospective comparison of cytogenetic and ultrasound findings in first trimester spontaneous fetal loss after demonstration of cardiac activity was made. The crown-rump length (CRL) was measured twice for each fetus resulting in spontaneous abortion: (i) CRL was measured in the viable state while demonstrating cardiac activity, and the growth deviation was expressed as the measured/expected CRL ratio (M/E CRL ratio); (ii) in the same fetus, CRL was measured after confirmation of fetal death, and designated as the post-mortem CRL. The chorionic tissues of these abortuses were karyotyped. The CRL of fetuses which resulted in normal deliveries were also measured as controls. As a result, 16 of 24 abortuses displayed an abnormal chromosomal analysis (67%). The mean M/E CRL ratio of still-viable fetuses was smaller than that of control fetuses (0.74 +/- 0.20 versus 0.98 +/- 0.13 respectively, P < 0.01). The differences in ratio between karyotypically normal and abnormal abortuses were not statistically significant. The post-mortem CRL of dead fetuses was > 20 mm in four of five monosomy X, two of three 21-trisomy, one of three triploidy and none of eight embryos with normal karyotype and five other trisomies. In conclusion, our study demonstrated that the M/E CRL ratio could be used as a predictor of spontaneous abortions, although it does not discriminate abnormal karyotypes from normal ones. The embryos with a post-mortem CRL more than 20 mm have a higher likelihood of suffering monosomy X or 21-trisomy. The ultrasonographic findings might offer a cytogenetic clue as to a possible cause to the developmental arrest.

Abortion, Spontaneous↗
PubMed1995

Management of meconium aspiration syndrome by tracheobronchial lavage and replacement of Surfactant-TA.

Meconium aspiration syndrome creates mechanical airway obstruction with air trapping and atelectasis. Tracheobronchial saline lavage to dislodge meconium may precipitate respiratory distress, a wet lung appearance and respiratory failure. Two case studies are reported in which meconium aspiration resulted in mechanical obstruction and displacement of surfactant and in whom tracheobronchial saline lavage and artificial surfactant replacement reversed respiratory failure.

Female↗
PubMed1995

Local continuity of myocardial blood flow studied by monochromatic synchrotron radiation-excited x-ray fluorescence spectrometry.

We have developed a monochromatic synchrotron radiation-excited system for two-dimensional mapping of x-ray fluorescence evoked from heavy element-loaded microspheres, which can evaluate myocardial blood flow in small contiguous regions with a small methodological error: 10.8 +/- 2.4% of the average of difference of the dual flow for 7- to 10-mg myocardial tissue (4 dogs). The fractal D value obtained from the slope of the log relative dispersion-log mass plot was 1.21 +/- 0.08 for a voxel size of 7 to 1260 mg (5 dogs) and that for a voxel size of 2.5 to 40 mg (1.12 +/- 0.06) was smaller than that for a voxel size of 40 to 1280 mg (1.25 +/- 0.14, P < .05, ANOVA, 4 dogs). The distance-correlation coefficient relation for paired myocardial regions was attenuated (correlation analysis), and the correlation coefficients between the original grouping and the two aggregates of the adjacent regions were dissociated (extended correlation analysis) under reduction of coronary perfusion pressure (6 dogs). Suppression of myocardial contraction with lidocaine (3 dogs) and vasodilation with adenosine partly improved the distance-correlation coefficient relation under reduced coronary perfusion pressure. Thus, an x-ray fluorescence system designed for precise flow measurement shows that the fractal nature of local flow distribution can be extended into regions smaller than previously reported, that in these regions the flow becomes more homogeneous, and that the self similarity and continuity of local flow are attenuated by the reduction of coronary perfusion pressure and improved by contractile suppression and coronary vasodilation.

Animals↗
PubMed1995

Modulation of in vitro splicing of the upstream intron by modifying an intra-exon sequence which is deleted from the dystrophin gene in dystrophin Kobe.

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing 52-bp deletion was skipped during splicing, although the known consensus sequences at the 5' and 3' splice sites of exon 19 were maintained (Matsuo, M., T. Masumura, H. Nishio, T. Nakajima, Y. Kitoh, T. Takumi, J. Koga, and H. Nakamura. 1991. J. Clin. Invest. 87:2127-2131). These data suggest that the deleted sequence of exon 19 may function as a cis-acting element for exact splicing for the upstream and downstream introns. To investigate this potential role of exon 19, an in vitro splicing system using artificial dystrophin mRNA precursors (pre-mRNAs) was established. Pre-mRNA containing exon 18, truncated intron 18, and exon 19 was spliced precisely in vitro, whereas splicing of intron 18 was almost completely abolished when the wild-type exon 19 was replaced by the dystrophin Kobe exon 19. Splicing of intron 18 was not fully reactivated when dystrophin Kobe exon 19 was restored to nearly normal length by inserting other sequences into the deleted site. These results suggest that the presence of the exon 19 sequence which is lost in dystrophin Kobe is more critical for splicing of intron 18 than the length of the exon 19 sequence. Characteristically, the efficiency of splicing of this intron seemed to correlate with the presence of polypurine tracks within the downstream exon 19. Moreover, an antisense 31-mer 2'-O-methyl ribonucleotide complementary to the 5' half of the deleted sequence in dystrophin Kobe exon 19 inhibited splicing of wild-type pre-mRNA in a dose- and time-dependent manner. This first in vitro evidence that dystrophin pre-mRNA splicing can be modulated by an antisense oligonucleotide raises the possibility of a new therapeutic approach for Duchenne muscular dystrophy.

Base Sequence↗
PubMed1995

Effective prevention of thrombocytopenia in mice using adenovirus-mediated transfer of HST-1 (FGF-4) gene.

HST-1 (FGF-4) gene product is a member of the fibroblast growth factor family with a signal peptide and plays a crucial role in limb development. We showed previously that an intraperitoneal injection of replication-deficient adenovirus containing the HST-1 gene (Adex1HST-1) into normal mice caused a twofold increase in peripheral platelet count. To investigate whether Adex1HST-1 could effectively prevent experimentally induced thrombocytopenia in mice, we injected Adex1HST-1 intraperitoneally into thrombocytopenic mice induced by administration of a chemotherapeutic agent and/or by irradiation. A single Adex1HST-1 injection caused continuously increased levels of serum HST-1 protein for at least 30 d and increased the count of large megakaryocytes in bone marrow, which specifically recovered platelet counts and more efficiently diminished the extent and duration of thrombocytopenia than any other reported cytokine or any combination of cytokines so far. In the other peripheral hematological parameters, no discernible differences were detected. No other apparent side effects were observed. Therefore, this method could be useful for treatment and/or prevention of thrombocytopenia induced by chemotherapy and/or irradiation for cancer treatment.

Adenoviruses, Human↗
PubMed1995

Whole-body retention and fetal uptake of 65Zn in pregnant mice fed a Zn-deficient diet.

Whole-body retention and fetal uptake of 65Zn under a Zn-deficient diet were studied in pregnant mice in the late gestational stage after a single oral administration of 65Zn. Whole-body retentions were much greater in mice given a Zn-deficient diet than in those given a Zn-normal diet. Accordingly, the amount of 65Zn transmitted to the offspring in utero was greater in the Zn-deficient diet group. In another experiment, fetal uptake of 65Zn in dams on gestation day 17 was examined over a period of 24 hr after a single intravenous administration of 65Zn to the Zn-deficient and Zn-normal animals. There was no major difference in fetal uptake between the two groups, indicative that approximately a similar proportion of the 65Zn retained in the maternal body was transmitted to the in utero offspring in both groups.

Animals↗