Search PubMed⌕ Search

Biomedical subjects

K Morikawa

Publications and source records attributed to K Morikawa.

At least 73 records · Page 4Linked to original sources
PubMed1999

Functional interactions of a homolog of proliferating cell nuclear antigen with DNA polymerases in Archaea.

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (alpha-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.

Amino Acid Sequence↗
PubMed1999

The effect of tulobuterol tape on histamine-induced bronchoconstriction in conscious guinea pigs: long duration of action.

The objective of the present study was to determine the action duration of tulobuterol tape within a 24-hr period in conscious guinea pigs. The bronchoconstriction induced by histamine-inhalation was significantly inhibited by tulobuterol tape in comparison with its placebo tape 8 and 12 hr after binding, and the inhibitory rate was 50+/-11% and 35+/-13%, respectively. Twenty-four hours after binding, the inhibitory effect of tulobuterol tape gradually diminished, but the inhibitory rate was maintained at 30+/-14%. These results suggest that tulobuterol tape has a long lasting bronchodilatory action.

Administration, Cutaneous↗
PubMed1999

Symmetry and elongation of objects influence perceived direction of translational motion.

Five experiments were conducted to examine how perceived direction of motion is influenced by aspects of shape of a moving object such as symmetry and elongation. Random polygons moving obliquely were presented on a computer screen and perceived direction of motion was measured. Experiments 1 and 2 showed that a symmetric object moving off the axis of symmetry caused motion to be perceived as more aligned with the axis than it actually was. However, Experiment 3 showed that motion did not influence perceived orientation of symmetry axis. Experiment 4 revealed that symmetric shapes resulted in faster judgments on direction of motion than asymmetric shapes only when the motion is along the axis. Experiment 5 showed that elongation causes a bias in perceived direction of motion similar to effects of symmetry. Existence of such biases is consistent with the hypothesis that in the course of evolution, the visual system has been adapted to regularities of motion in the animate world.

Adult↗
PubMed1999

Phase-contrast magnetic resonance imaging study on cord motion in patients with spinal dysraphism: comparison with healthy subjects.

Cord motion at the cervical cord (C-3) level was measured with phase-contrast magnetic resonance imaging (MRI) in 39 healthy young subjects and 34 age-matched patients with spinal dysraphism. Cord velocity curves (composed of waves I, II, and III) were made in both sagittal images and transverse images and classified into 3 types. The measurement in the sagittal images were more reproducible than that in the transverse images. In healthy subjects, the mean cord velocity of wave I was 0.49 +/- 0.11 cm/s in the sagittal image. In the subgroup of patients with spinal dysraphism showing stable clinical courses, cord velocity was comparable to that in the healthy subjects. Patients of the subgroup with symptomatic aggravation showed a significant decrease in cord velocity in wave I, and a characteristic pattern in the cord velocity curve: the velocity change (wave I) was small. This decrease in cord velocity was thought to be due mainly to cord tethering. The measurement of cord motion with phase-contrast MRI could give objective and quantitative information about cord motion and tethering associated with spinal dysraphism.

Adolescent↗
PubMed1998

A structural basis for transition-state stabilization in antibody-catalyzed hydrolysis: crystal structures of an abzyme at 1. 8 A resolution.

The three-dimensional structure of a catalytic antibody, 6D9, has been solved as a complex with a transition state analog. The structure was determined from two different crystal forms, and was refined at a resolution of 1.8 A. The antibody 6D9, which was induced by immunization with the phosphonate transition state analog 3, hydrolyzes a prodrug of chloramphenicol monoester 1 to generate the parent drug 2. The kinetic studies have shown that the antibody is catalytic by virtue of the theoretical relationship between the affinity for the transition state and the catalytic efficiency (kcat/kuncat=KS/KTSA). The crystal structure makes it possible to visualize the theoretical relationship. A side-chain (Nepsilon) of HisL27D is placed in a key position to make a hydrogen bond to the phosphonate oxygen of the transition state analog with a distance of 2.72 A, suggesting a hydrogen bond to the oxyanion developing in the transition state of the hydrolysis. There are no catalytic residues, other than the histidine, around the phosphonate moiety. In addition, in the antibody-hapten complex, the hapten bears a folded conformation and the two stacked aromatic rings are buried deep in the antigen-combining site through aromatic-aromatic interaction with TrpH100I and TyrH58. The conformation of the bound hapten suggests that the antibody binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable E-form, thereby making it easy for the substrate to reach the transition-state during catalysis. These observations reveal that the catalytic mechanism is explained purely on the basis of the stabilization of the transition state. The refined high resolution structures reported here are envisaged to have an impact on the understanding of other hydrolytic antibodies, since their haptens share some unique features with the hapten used in this study.

Antibodies, Catalytic↗
PubMed1998

Crystal structure and mutational analysis of the Escherichia coli putrescine receptor. Structural basis for substrate specificity.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-A resolution. The PotF molecule has dimensions of 54 x 42 x 30 A and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.

Base Sequence↗
PubMed1998

Functional analyses of the domain structure in the Holliday junction binding protein RuvA.

BACKGROUND: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA-RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein. RESULTS: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction. CONCLUSIONS: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction-DNA binding.

Adenosine Triphosphate↗
PubMed1998

Crystallization and preliminary X-ray analysis of the periplasmic receptor (PotF) of the putrescine transport system in Escherichia coli.

The primary receptor (PotF) of the putrescine transport system in E. coli has been crystallized by the hanging-drop vapor-diffusion technique. The crystals belong to the space group P21212 with unit-cell dimensions a = 269.4, b = 82.33 and c = 93.74 A. The crystals diffract beyond 2.2 A with a rotating-anode X-ray source. A complete data set from the native crystals has been collected and processed at 2.3 A resolution. Two heavy-atom derivatives have been prepared from the same Pt compound at 293 and 277 K. The difference Patterson maps revealed completely different major heavy-atom sites between these two derivatives.

Biological Transport↗
PubMed1998

Cloning of the fatty acid synthetase beta subunit from fission yeast, coexpression with the alpha subunit, and purification of the intact multifunctional enzyme complex.

We have cloned and sequenced the fission yeast (Schizosaccharomyces pombe) fas1+ gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two expression vectors, containing the lsd1+/fas2+ gene and the fas1+ gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.

Amino Acid Sequence↗
PubMed1998

Effects of MK-801 on bladder overactivity in rats with cerebral infarction.

PURPOSE: Our objective was to evaluate the underlying mechanisms of neurogenic voiding dysfunction following cerebral infarction. MATERIALS AND METHODS: The left middle cerebral artery (MCA) was occluded using 4-0 monofilament nylon thread in male S-D rats. Cystometric examination was performed in unanesthetized and urethane-anesthetized rats through a catheter chronically implanted in the dome of the bladder. RESULTS: Bladder capacity of unanesthetized or urethane anesthetized rats was significantly reduced just after occlusion of the left MCA; 2 weeks after the occlusion, the capacity was less than half that in sham-operated rats. Intravenous administration of N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 to the unanesthetized sham-operated rats led to a marked dose-dependent decrease in bladder capacity. Its administration to unanesthetized rats with cerebral infarction resulted in a slight decrease in bladder capacity. In the urethane-anesthetized state, the bladder capacity of the rats with cerebral infarction was significantly increased by MK-801, 0.1 mg./kg., without inhibiting the contraction pressure or increasing the amount of residual urine. A high dose (1 mg./kg.) of MK-801 was required to increase the bladder capacity of sham-operated rats. This led to an inhibition of contraction pressure and an increase in residual urine. CONCLUSION: Results in urethane anesthetized rats indicate that NMDA glutamatergic transmission is important in the overactivity of the bladder following a cerebral infarction. This model is useful in studying the neurogenic voiding dysfunction observed in patients with cerebrovascular disease.

Anesthesia↗
PubMed1998

Change in bladder contractility associated with bladder overactivity in rats with cerebral infarction.

PURPOSE: To evaluate the contractile properties of overactive bladder from rats in the chronic stage of experimental cerebral infarction. MATERIALS AND METHODS: Cystometry was performed in conscious male S-D rats after inducing occlusion of the left middle cerebral artery. Bladder muscle strips were evaluated for force development in response to field stimulation, acetylcholine and KCl. By measuring the contractile response to field stimulation after adding atropine and alpha,beta-methylene-ATP, contributions of cholinergic and purinergic transmission were determined. RESULTS: Bladder capacity of cerebral-infarcted rats was <50% of the capacity of sham-operated rats and significantly less than that of sham-operated rats even 4 months after surgery. There was no significant difference in bladder weight between sham-operated rats and cerebral-infarcted rats. No differences in the contractile response of detrusor strips to field stimulation and acetylcholine, or in the relative contribution of cholinergic and purinergic transmission to the contractile response, were observed over time or between strips from sham-operated rats and cerebral-infarcted rats. KCl induced significantly less contraction in strips from 4 month infarcted rats than in strips from 4 month sham-operated rats, 2 week infarcted rats and 2 month infarcted rats. CONCLUSIONS: This animal model will be useful for chronic studies on the mechanism of detrusor hyperactivity (DH).

Animals↗
PubMed1998

Solution structure of the DNA- and RPA-binding domain of the human repair factor XPA.

The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), was determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel alpha/beta structure with a positively charged superficial cleft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.

Amino Acid Sequence↗
PubMed1998

Deoxyspergualin preferentially inhibits the growth and maturation of anti-CD40-activated surface IgD+ B lymphocytes.

Deoxyspergualin (DSG), an analogue of spermidin, is a potent immunosuppressive drug with an action quite distinct from that of cyclosporin, rapamycin, or FK506. In this study we investigated the effect of DSG and methyldeoxyspergualin (MeDSG) on the proliferation and differentiation of human B cells stimulated with anti-CD40 MoAb. Highly purified B cells obtained from tonsillar samples were used as target cells. Both agents inhibited the proliferative response of anti-CD40-stimulated B cells in the absence and presence of IL-4, IL-2 or IL-10 in a dose-dependent manner. This inhibitory effect differed markedly among cell populations based on surface IgD expression: strong inhibition of sIgD+ B cells but little inhibition of sIgD- B cells. The drugs also suppressed the production of IgG, IgM and IgA by unfractionated B cells, which suggests that DSG acts against post-switch (sIgD-) B cells. Although the drugs suppressed immunoglobulin synthesis by both sIgD+ and sIgD- B cells, the effect was more marked in the sIgD+ B cells. Analysis of the subclass of IgG secreted by sIgD+ B cells revealed a decline in IgG1 and IgG3 in the presence of DSG. These results suggest that DSG preferentially inhibits the growth and maturation of sIgD+ naive B cells.

Antibodies, Monoclonal↗
PubMed1998

A new stereoscopic illusion: eyes popping out and sinking in.

A stereoscopic illusion is produced by manipulating the binocular disparity of the irises in stereo pairs of human faces. These stimuli elicit unnatural percepts of the irises popping out of the face or sinking in inside the head.

Depth Perception↗
PubMed1998

A new anti-metastatic drug, ND-2001, inhibits lung metastases in rat hepatoma cells by suppressing haptotaxis of tumor cells toward laminin.

We examined the effects of ex vivo treatment of tumor cells with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). The ex vivo treatment of rat hepatoma cKDH-8/11 cells with this new synthetic product of the antibiotic nojirimycin, ND-2001 (50 microg/ml), inhibited the experimentally induced lung metastases of the tumor cells significantly at an inhibition rate of 69.2% (one of 10 animals remained metastasis free). Also, it was elucidated in in vitro tumor cell invasion assays that ND-2001 (50 microg/ml) suppressed the invasion activities of cKDH-8/11 cells to Matrigel Matrix at an inhibition rate of 69.3%. However, phagokinetic track assays revealed that ND-2001 did not suppress the random motility of cKDH-8/11 cells. However, ND-2001 (50 microg/ml) suppressed the haptotaxis, another important role in tumor invasion, of cKDH-8/11 cells toward laminin (inhibition rate of 77.0%). These results suggest that ND-2001 suppressed the haptotaxis of tumor cells toward laminin directly at the step of invading the basement membrane and brought about the inhibition of lung metastases.

Animals↗
PubMed1998

ND-2001 suppresses lung metastasis of human renal cancer cells in athymic mice.

Explants of highly metastatic human renal cell carcinoma SN12Cpm6 cells in athymic mice were treated with sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam; ND-2001). ND-2001 (50 micrograms/ml) caused 78% inhibition of lung metastasis of SN12Cpm6 cells (two of five animals remaining metastasis free). The in vitro tumor cell invasion assay showed that ND-2001 (100 micrograms/ml) suppressed the invasive activity of SN12Cpm6 cells to Matrigel matrix at an inhibition rate of 72%. These results suggest that ND-2001 may be a new anti-metastatic drug against human cancer cells.

Animals↗