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Biomedical subjects

F Yoshikawa

Publications and source records attributed to F Yoshikawa.

At least 19 recordsLinked to original sources

Intravenous sedation and general anesthesia for a patient with Gilles de la Tourette's syndrome undergoing dental treatment.

Patients with Gilles de la Tourette's syndrome develop symptoms during childhood. Repetitive various motor tics or speech tics that are spontaneous, aimless, and involuntary are characteristic of the syndrome (1). Patients with the syndrome have been considered to have an aggressive, impulsive, and obsessive character (2). (3). Suicide is one of the mental symptoms of the syndrome. Routine dental treatment with this syndrome can be difficult.

Adolescent↗

Inositol 1,4,5-trisphosphate receptors are strongly expressed in the nervous system, pharynx, intestine, gonad and excretory cell of Caenorhabditis elegans and are encoded by a single gene (itr-1).

Inositol 1,4,5-trisphosphate (InsP3) activates receptors (InsP3Rs) that mediate intracellular Ca(2+ )release, thereby modulating intracellular calcium signals and regulating important aspects of cellular physiology and gene expression. To further our understanding of InsP3Rs we have characterised InsP3Rs and the InsP3R gene, itr-1, from the model organism Caenorhabditis elegans. cDNAs encoding InsP3Rs were cloned enabling us to: (a) identify three putative transcription start sites that result in alternative mRNA 5' ends: (b) detect alternative splicing at three sites and: (c) determine the full genomic organisation of the itr-1 gene. The InsP3R protein (ITR-1) is approximately 42 % identical with known InsP3Rs and possesses conserved structural features. When the putative InsP3 binding domain was expressed in Escherichia coli, specific binding of InsP3 was detected. Using antibodies against ITR-1 we detected a protein of 220 kDa in C. elegans membranes. These antibodies and itr-1::GFP (green fluorescent protein) reporter constructs were used to determine the expression pattern of itr-1 in C. elegans. Strong expression was observed in the intestine, pharynx, nerve ring, excretory cell and gonad. These results demonstrate the high degree of structural and functional conservation of InsP3Rs from nematodes to mammals and the utility of C. elegans as a system for studies on InsP3R mediated signalling.

Amino Acid Sequence↗

Real time analysis of interaction between inositol 1,4, 5-trisphosphate receptor type I and its ligand.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger that releases intracellular Ca(2+) by binding to its specific receptor, inositol 1,4,5-trisphosphate receptor (IP(3)R), in a wide range of cellular processes. We report here large-scale expression and purification of N-terminal 604 amino acids of IP(3)R type 1 (T604) expressed in E. coli, which contains the ligand binding domain. Surface plasmon resonance biosensor studies showed that purified T604 could bind to its ligands with binding specificity identical to that of full-length native IP(3)R type 1. Kinetic parameters of T604 for IP(3) consisted of a fast association rate constant (K(ass) = 1.2 x 10(6) M(-1) s(-1)) and a rapid dissociation rate constant (k(diss) = 1 s(-1)), and the equilibrium dissociation constant was determined to be 336 nM, at 150 mM NaCl and pH 7.4. However, association and dissociation patterns depended on the pH level and ionic strength. These results pave the way toward detail analysis of structure-function analysis of the ligand binding domain of IP(3)R type 1 for its ligands.

Amino Acid Sequence↗

High efficient expression of the functional ligand binding site of the inositol 1,4,5-triphosphate receptor in Escherichia coli.

Type 1 inositol 1,4,5-trisphosphate receptor (IP3R1), an inositol 1, 4,5-trisphosphate (IP3)-gated Ca2+ release channel, binds IP3 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IP3 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IP3 binding constructs expressed in E. coli significantly affect their production as soluble protein. Residues 1-604 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IP3 of T604 (Kd = 45 nM) is comparable to that of the native IP3R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IP3R1 for IP3.

Amino Acid Substitution↗

Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor. Structural and functional coupling of cleaved ligand binding and channel domains.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel (calculated molecular mass = approximately 313 kDa/subunit). We studied structural and functional coupling in this protein complex by limited (controlled) trypsinization of membrane fractions from mouse cerebellum, the predominant site for IP3R1. Mouse IP3R1 (mIP3R1) was trypsinized into five major fragments (I-V) that were positioned on the entire mIP3R1 sequence by immuno-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I-IV were derived from the N-terminal cytoplasmic region where the IP3-binding region extended over two fragments I (40/37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the membrane-spanning channel region. All five fragments were pelleted by centrifugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggested that the native mIP3R1-channel is an assembly of four subunits, each of which is constituted by non-covalent interactions of five major, well folded structural components I-V that are not susceptible to attack by mild trypsinolysis. Ca2+ release experiments further revealed that even the completely fragmented mIP3R1 retained significant IP3-induced Ca2+ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP3-induced Ca2+ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP3R1, that is neighboring coupling between components I and II for IP3 binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.

Amino Acid Sequence↗

Cooperative formation of the ligand-binding site of the inositol 1,4, 5-trisphosphate receptor by two separable domains.

Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.

Animals↗

Solitary fibrous tumor of the buccal mucosa: report of a case with immunohistochemical studies.

We describe a case of a solitary fibrous tumor of the buccal mucosa and report the results of immunohistochemical studies of the lesion. Solitary fibrous tumors are extremely rare in the intraoral region. These tumors are generally difficult to diagnose because of their broad range of morphologic characteristics. We regard the expression of CD34 within the appropriate clinical and morphologic setting, in the absence of reactivity for other specific markers of differentiation, as evidence supporting the diagnosis of solitary fibrous tumor.

Adult↗

[Transient global amnesia after general anesthesia].

A 61 year old male patient with left mandibular cyst, received marsupialization of the left mandible under general anesthesia. Four hours after the end of anesthesia, his memory for the past 4 months and short term memory plastisity were impaired. No neurological abnormalies were found at that time. On the 2nd postoperative day, he recovered his lost memory for the past 4 months. The memory of events between 6 hours before operation and next morning, however, remained lost. It is suggested that the memory disorder is the TGA due to various causes including transient hypertension, operative stress, postoperative pain and diazepam.

Adjuvants, Anesthesia↗

Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.

To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Amino Acid Sequence↗

Immunohistochemical analysis of the peritoneum adjacent to endometriotic lesions using antibodies for Ber-EP4 antigen, estrogen receptors, and progesterone receptors: implication of peritoneal metaplasia in the pathogenesis of endometriosis.

This study was designed to investigate whether or not the pelvic peritoneum exhibits a metaplastic process into müllerian-type epithelium using a marker for epithelial differentiation (Ber-EP4 antigen) and markers that indicate müllerian differentiation (estrogen receptors and progesterone receptors). The peritoneum and/or ovarian surface epithelium adjacent to endometriotic lesions were obtained from 24 patients with endometriosis at operation, and peritoneum and ovarian surface epithelium without any lesions were also obtained from control patients without endometriosis. The specimens were immunohistochemically analyzed using antibodies for epithelial antigen Ber-EP4, estrogen receptor (ER), and progesterone receptor (PR) on frozen sections. Normal peritoneal mesothelium showed negative staining for Ber-EP4, ER, and PR. The mesothelium of the peritoneum adjacent to the endometriotic lesions showed focal positivity for Ber-EP4, ER, and PR. Several cases of ovarian surface epithelium from normal control ovaries and ovaries adjacent to endometriotic lesions also showed focal positivity for Ber-EP4, ER, and PR. Stromal cells accompanying these foci were sporadically positive for ER and/or PR but negative for Ber-EP4. Focal expression of Ber-EP4, ER, and PR in the mesothelium of the peritoneum and the ovarian surface epithelium adjacent to endometriotic lesions suggests that mesothelium possibly acquires characteristics of epithelial as well as müllerian-type nature. These results support an existence of a metaplastic process of the peritoneal mesothelium in the pathogenesis of endometriosis. The more frequent Ber-EP4 positivity in normal ovarian surface epithelium compared to normal peritoneal mesothelium also suggests a fundamental difference in these tissues that may be related to the greater prevalence of epithelial neoplasms arising in ovarian tissue.

Adult↗

Autosomal codominant inheritance and Japanese incidence of deficiency of OKT4 epitope with lack of reactivity resulting from conformational change.

A large Japanese family in which some members were homozygous or heterozygous for OKT4 epitope deficiency was studied. Homozygotes, heterozygotes, and normal individuals were identified by differences in the number of OKT4 epitopes on the surfaces of lymphocytes. This deficiency was transmitted as an autosomal codominant trait. The internalization of CD4 molecules and the production of IL-2 by lymphocytes of these subjects were examined. The OKT4 epitope was not needed for internalization of CD4 molecules, and IL-2 was produced in the same amounts by these different kinds of subjects. DNA from four clones lacking OKT4 established from four individuals of this family was sequenced. As reported elsewhere for different subjects, a single nucleotide substitution (CGG-->TGG) was found in all four cell lines. The mutation results in arginine being replaced by tryptophan. Analysis showed different hydrophobicity at positions 239 and 240 from the control, probably giving rise to a conformational change in CD4 accounting for lack of reactivity with the OKT4 monoclonal antibody. The incidence of homozygotes in the Japanese population was found to be 0.47% by examination of 1478 random samples, and on the basis of this value, the incidence of heterozygotes was estimated to be 12.8%.

Amino Acid Sequence↗

Pedunculated cavernous hemangioma. A case report.

A rare case of pedunculated hemangioma of the oral mucosa has been reported. Clinically, it was diagnosed as a fibroma induced by irritation, but histologic examination followed to excisional biopsy demonstrated that it was a cavernous hemangioma.

Aged↗

Solitary eosinophilic granuloma of bone occurring in the buccal mucosa: report of a case.

Eosinophilic granuloma of bone is not uncommon in the craniofacial region (Anderson and kissane, 1977), and it sometimes occurs in organs such as the lung, stomach and spleen. However there are only few reports on solitary eosinophilic granuloma of bone occurring in oral soft tissue. In this report, we describe a case of eosinophilic granuloma of bone occurring in the soft tissue of a 40-year-old woman.

Adult↗

Pharmacological analysis of vasoconstrictor responses of isolated and perfused human umbilical arteries.

Using the cannula inserting method, we studied vascular responses of isolated human umbilical arteries to several vasoactive substances. ACh did not produce a vasodilation in non-constricted preparations but induced only a vasoconstriction. Histamine and 5HT produced strong vasoconstrictions in a dose-dependent manner. Epinephrine and norepinephrine in large doses induced only a slight vasoconstriction. The ACh-induced vasoconstriction was markedly suppressed by atropine and slightly, but significantly, suppressed by methylsergide. The vascular responses to ACh were not influenced by removal of the endothelium by an intraluminal bolus injection of saponin. These results suggest that the endothelium has no muscarinic receptors in the umbilical arteries, although cholinergic vasoconstrictor mechanisms may be partially involved in the regulation of umbilical circulation, and that human umbilical arteries exhibit different pharmacological responses from those of vessels of other organs.

Acetylcholine↗

The role of antibodies in the regulation of delayed-type hypersensitivity.

The contribution of B cells and antibodies to the regulation of delayed-type hypersensitivity (DTH) was investigated in mice rendered B-cell-deficient by treatment with anti-mu antibodies. In normal rabbit immunoglobulin (Ig)-treated mice as well as normal mice, the intravenous injection of a large amount of keyhole limpet haemocyanin (KLH) suppressed DTH, and serum titres of the anti-KLH antibody were significantly elevated. However, in anti-mu-treated mice, the intravenous injection of a large amount of KLH could not induce either suppression of DTH or the elevation of anti-KLH antibody titres. The transfer of anti-KLH antibodies suppressed DTH in a H-2 non-restricted, probably Igh-restricted, way in anti-mu-treated mice. In addition, the transfer of anti-KLH antibodies induced effector-phase suppressor T cells whose phenotype was L3T4-, Lyt-2+. We concluded that antibodies play a significant role in the regulation of DTH.

Animals↗

Production of self-migration stimulating factor by metastatic human malignant melanoma cells established in culture.

Establishment of human malignant melanoma of hard palate cell line derived from metastatic foci in lymph node is reported. We name it HM162. The shape of HM162 was almost spindle. HM162 cells secreted a factor into culture medium which stimulates motility of HM162 cell itself, and the mode of stimulated migration of HM162 cells were activated random migration, that is chemokinesis.

Cell Movement↗

Bilateral gonadoblastoma producing steroid hormones in a patient with 45,X/46,XY gonadal dysgenesis.

A case of bilateral gonadoblastoma in a phenotypic female with 45,X/46,XY gonadal dysgenesis is presented. Hormonal investigations revealed that serum testosterone, estradiol and beta-human chorionic gonadotropin decreased following excision of the tumors, but follicle-stimulating hormone and luteinizing homrone levels increased further. Immunohistochemical staining for testosterone and estradiol was positive in both Leydig and lutein-like cells in the tumor. It is suggested that gonadoblastoma is capable of producing testosterone and estradiol, and Leydig or lutein-like cells may be the actual source of these steroid hormones.

Adolescent↗