Search PubMed⌕ Search

Biomedical subjects

H Kawashima

Publications and source records attributed to H Kawashima.

At least 127 records · Page 7Linked to original sources
PubMed1998

Cytokine profiles of aqueous humor and graft in orthotopic mouse corneal transplantation.

BACKGROUND: Cytokine profile is a key in understanding the mechanisms of allograft rejection. Cytokine expression in the aqueous humor and the correlation between the aqueous humor cells and corneal infiltrating cells are not fully understood in corneal transplantation. METHODS: Orthotopic mouse corneal transplantation was performed using BALB/c (H2d) mice as recipients, and C3H/He (H2k) and BALB/c mice as donors for allografts and isografts, respectively. Immunocytochemistry was performed on aqueous humor cells. Corneal graft was studied immunohistochemically. Cytokine gene expressions of the cells infiltrating the aqueous humor and corneal grafts were determined by the semiquantitative reverse transcription and polymerase chain reaction method. RESULTS: Interferon-gamma, interleukin (IL)-2, IL-4, and IL-10 were detected in the cells infiltrating the aqueous humor and corneal grafts at both the protein and gene expression levels. T helper 1 (Th1) cytokine expressions at the protein level, however, were consistently predominant in the rejected allografts compared to those of Th2 cytokines. The cytokine and surface marker profiles of the cells in the aqueous humor corresponded well to those of the cells infiltrating the corneal grafts. Cytokine protein and mRNA expression levels in the aqueous humor decreased rapidly. CONCLUSIONS: Allorejection in corneal transplantation is Th1 cytokine-predominant. Infiltrating cells do not express Th2 cytokine so much in allograft rejection, as compared with Th1 cytokine. The cell infiltration patterns of the aqueous humor were well correlated with those of the cornea.

Animals↗
PubMed1998

Isolation of partially purified P450 2D18 and characterization of activity toward the tricyclic antidepressants imipramine and desipramine.

Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine. Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His]4 tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparent KM values for P450 2D18 toward imipramine and desipramine were 374 and 314 microM, respectively. While apparent KM values were similar, P450 2D18 was shown to have a fivefold increased Vmax (2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of the CYP2D6 inhibitor quinidine, the CYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Usefulness of nested PCR and sequence analysis in a nosocomial outbreak of neonatal enterovirus infection.

BACKGROUND: Non-polio enterovirus infections are recognized in children during summer-fall seasons and they sometimes cause large outbreaks. We experienced a nosocomial infection in the neonatal nursery and echovirus type 7 was isolated from samples of four patients. OBJECTIVES: We diagnosed the horizontal infection of four neonates by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) and the nucleotide sequence. STUDY DESIGN: Total RNA was extracted from clinical isolates, serum samples and cerebrospinal fluid (CSF). We amplified enterovirus genome in the 5'-noncoding region by nested PCR and determined the nucleotide sequences. RESULTS: Enterovirus genome was detected in all isolates, in the acute-phase sera in all four patients and in the CSF in one patient by the first PCR. By using nested PCR, the genome was detected from convalescent-phase sera in two patients. All enterovirus genome obtained from the nursery outbreak showed the same sequences with 100% homology. CONCLUSION: We demonstrated the clinical advantages of RT-nested PCR from serum samples and the analysis of nucleotide sequencing gave the supportive evidence of identification of transmission pathway.

5' Untranslated Regions↗
PubMed1998

Cloning of PCPTP1-Ce encoding protein tyrosine phosphatase from the rat cerebellum and its restricted expression in Purkinje cells.

Recently, cDNAs encoding brain-specific transmembrane-type protein tyrosine phosphatases (PTPs) with single catalytic domain have been cloned. These include PC12-PTP, PCPTP1, PTPBR7, and PTP-SL, whose cytoplasmic domains had high similarity to STEP, a brain-specific nontransmembrane-type PTP. Based on the high similarity and expression pattern, PCPTP1 seems to be identical with PC12-PTP1 and to be the rat homologue of murine PTPBR7. Here, we report the molecular cloning and expression profile of PCPTP1-Ce, a variant of PCPTP1. Both PCPTP1 mRNA and PCPTP1-Ce mRNA seem to be derived from a single common region gene. Nucleotide and deduced amino acid sequence comparison between PCPTP1-Ce and PCPTP1 revealed that the predicted protein product of PCPTP1-Ce is identical with that translated from the third initiation methionine of the longest ORF of PCPTP1, and that these two clones differ in the 5'-untranslated sequences. Northern blot analyses with specific probes for PCPTP1 and PCPTP1-Ce confirmed our previous observation that PCPTP1-Ce mRNA was almost exclusively expressed in the cerebellum, whereas PCPTP1 was widely expressed in various brain regions dissected including cerebellum. In situ hybridization study demonstrated that PCPTP1-Ce mRNA was exclusively expressed in Purkinje cells of the cerebellum. In contrast, PCPTP1 mRNA was predominantly expressed in granule cells and less in Purkinje cells. Moreover, immunohistochemical analysis using an affinity-purified polyclonal antibody raised against the cytoplasmic region of PCPTP1/PCPTP1-Ce demonstrated that Purkinje cells were strongly immunostained, whereas granule cells were stained only faintly in the cerebellum. These observations clearly demonstrated that PCPTP1-Ce mRNA and its protein products are expressed in Purkinje cells and suggest that PCPTP1-Ce may play an important role in Purkinje cell function in the rat cerebellum.

Animals↗
PubMed1998

A novel isoform of rat estrogen receptor beta with 18 amino acid insertion in the ligand binding domain as a putative dominant negative regular of estrogen action.

A novel isoform of rat estrogen receptor (ER) beta, ER beta 2, which is a putative alternative splicing product of the reported ER beta (ER beta 1) has been identified. Rat ER beta 2 cDNA contains an additional, in-frame 54 base pair insertion in the ligand binding domain of ER beta 1, which generates an 18 amino acid residue insertion. Northern blot and RT-PCR analyses revealed that ER beta 2 coexists with ER alpha and ER beta 1 in all tissues examined including brain, lung, liver, kidney, fat, bone, uterus, prostate, and ovary. The insertion caused loss of ligand binding activity of ER beta 2, whereas the ability to bind the palindromic estrogen response element (ERE) was retained. In an ERE-containing luciferase reporter gene assay using COS-1 cells, ER beta 2 failed to activate estrogen-dependent transcription. Furthermore, ER beta 2 dose dependently suppressed the ER alpha- and ER beta 1-mediated transcriptional activation. These results suggest that rat ER beta 2 functions as a negative regulator of estrogen action.

Alternative Splicing↗
PubMed1998

Direct antigen presentation through binding of donor intercellular adhesion molecule-1 to recipient lymphocyte function-associated antigen-1 molecules in xenograft rejection.

Cellular interactions that lead to graft rejection were examined in a rat-to-mouse xenogeneic combination using species-specific monoclonal antibodies (mAbs) against donor and recipient intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) molecules, respectively. Although both mAbs displayed moderate blocking activity in an in vitro mixed lymphocyte response assay, strong suppression was observed when anti-donor (rat) ICAM-1 mAb was combined with anti-recipient (mouse) LFA-1 mAb. Likewise, significant prolongation of islet xenograft survival was observed with these mAbs. Thus, 0.05 mg of anti-mouse LFA-1 mAb and anti-rat ICAM-1 mAb given on days 0 and 1 produced significant prolongation of graft survival over the control (51+/-20 days vs. 10+/-3 days, P<0.0001), but not when anti-mouse ICAM-1 mAb was combined with anti-mouse LFA-1 mAb (13+/-3 days). In this species combination, mouse T cells were able to proliferate in the presence of rat antigen-presenting cells (APCs) in a cell number-dependent manner, but not in the presence of mouse APCs. The binding assay showed that LFA-1 molecules on mouse T cells can bind immobilized rat ICAM-1 molecules. These results suggest that rat ICAM-1 molecules on APCs can interact with mouse LFA-1 molecules on T cells across a species barrier and that this binding generates the consequent immune responses leading to rejection. mAb treatment against these adhesion molecules of recipient as well as donor is crucial for preventing rejection in a xenogeneic transplantation model.

Animals↗
PubMed1998

Calcium-sensing receptor in mature osteoclasts, which are bone resorbing cells.

Bone metabolism consists of osteoblast-mediated bone formation coupled to osteoclastic resorption of bone. Osteoclastic bone resorption plays an important role in normal skeletal development and the maintenance of its integrity throughout life. Although osteoclastic activity is thought to be under the control of feedback regulation by extracellular cations, the molecular mechanism of detecting extracellular cations within the bone microenvironment remains to be clarified. In the present study we showed by reverse transcription-polymerase chain reaction and Northern blot analysis that cultured mature osteoclasts express the calcium-sensing receptor (CaSR) mRNA. The nucleotide sequence of rabbit osteoclast CaSR was approximately 90% identical to that of CaSR cDNA from human, bovine, and rat parathyroid glands. Moreover, the activity of osteoclastic bone resorption, as determined by pit formation, was regulated by extracellular calcium ion as well as its agonists that are known to act through the CaSR. We conclude that CaSR, homologous to that identified in parathyroid glands, is present in mature osteoclasts and calcium ion released from bone may directly regulate osteoclastic bone resorption.

Animals↗
PubMed1998

Rare case of small-cell carcinoma arising from the endometrium with paraneoplastic retinopathy.

We present the fourth known case of endometrial carcinoma, and the second case of endometrial small-cell carcinoma, to be associated with paraneoplastic retinopathy. Initial symptoms were decreased visual acuity and a narrowing of the visual field. Endometrial carcinoma was diagnosed several months later. An antibody to 34-kDa bovine retinal antigen was detected in the patient's serum. Thus, autoimmunity was suspected as the cause of the retinopathy. In patients with endometrial carcinoma with visual disturbance of unknown cause, paraneoplastic retinopathy should be suspected.

Carcinoma, Small Cell↗
PubMed1998

Pharmacokinetics of nimustine, methotrexate, and cytosine arabinoside during cerebrospinal fluid perfusion chemotherapy in patients with disseminated brain tumors.

OBJECTIVE: This study was conducted to evaluate the pharmacokinetics of anticancer drugs in cerebrospinal fluid (CSF) perfusion chemotherapy. METHODS: We administered CSF perfusion chemotherapy with nimustine (ACNU), methotrexate (MTX), and cytosine arabinoside (Ara-C) to three patients with disseminated malignant brain disease. The drugs were infused via Ommaya's reservoirs to the lateral ventricle and removed by drainage from the temporal lobe or lumbar spine. CSF and plasma concentrations of the anticancer drugs were determined by high-performance liquid chromatography and fluorescence polarization immunoassay. RESULTS: The concentrations of anticancer drugs in the discharged CSF peaked about 40 min after the start of a 1-h CSF perfusion. After the perfusion, the drug level in CSF decreased exponentially in a monophasic manner. ACNU and Ara-C were not detectable in the discharged CSF in the temporal lobe at 6 h and 48 h after perfusion, respectively, but MTX was detectable at 48 h. The maximum concentration ratio of anticancer drugs and the duration of perfusion were inversely correlated. The plasma concentrations of anticancer drugs were much lower than those in CSF. The half-life of ACNU was very short (0.2-1.1 h), whereas the half-lives of MTX and Ara-C were relatively long (2.81-13.5 h and 1.84 6.25 h, respectively). The half-lives of the anticancer drugs in CSF tended to decrease with repeated CSF perfusion chemotherapy. CONCLUSION: Results suggest that CSF perfusion chemotherapy enables a high concentration of anticancer drug to be administered for dissemination in the spinal cord within a short period of time, with minimal adverse effects.

Adult↗
PubMed1998

Complete nucleotide sequence of the genome organization of RNA2 of patchouli mild mosaic virus, a new favavirus.

The complete nucleotide sequence and the genome organization of the RNA 2 of a patchouli mild mosaic virus (PaMMV) was determined. The sequence consists of 3591 nucleotides and contains a single long open reading frame sufficient to code for 118 K protein. Three proteins of 52 K, 44 K and 22 K could be encoded by the PaMMV RNA 2 genome. Our analysis of the N-terminal sequences of two species of coat protein (CP) allowed precise location of the CP cistrons within the polyprotein. 44 K and 22 K proteins are the coat proteins. The positions of the cleavage sites are Gln/Ala between 44 K and 22 K coat proteins and Gln/Gly between 52 K and 44 K proteins. Comparison of PaMMV RNA 2 with comoviral and nepoviral RNA 2 showed no sequence similarity. These results as well as previous serological studies strongly suggest that PaMMV is a member in the genus Fabavirus.

Capsid↗
PubMed1998

Construction and expression of a mouse-human chimeric antibody against human tumor necrosis factor-alpha.

A mouse anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (MoAb), designated as 3B10, has previously been produced and characterized by our laboratory. We report here the construction and the expression of mouse-human chimeric antibody derived from the MoAb. cDNAs encoding variable regions of heavy and light chains were prepared from 3B10 cells by polymerase chain reaction, and introduced to mammalian expression vectors containing cDNA for human gamma1 and kappa constant regions, respectively. Cotransfection of the vectors into CHO cells resulted in production of antibody reacting with human TNF-alpha. In SDS-PAGE analysis, the chimeric antibody, c3B10, migrated at 170 kDa under a nonreducing condition, whereas two bands with 58 and 28 kDa appeared following treatment with 2-mercaptoethanol. Both c3B10 and mouse 3B10 neutralized the cytotoxic activity of human TNF-alpha to the same level, indicating that c3B10 holds the binding activity of its original MoAb. These findings suggest that the introduced genes for chimeric heavy and light chains are transcribed and translated to produce the chimeric heavy and light chain peptides, and that the peptides are assembled to form native IgG molecule. The chimeric anti-TNF-alpha antibody described in this study is expected to be less immunogenic and thus more suitable for possible clinical use.

Animals↗
PubMed1998

Development of step-specific PET tracers for studying fatty acid beta-oxidation: biodistribution of [1-(11)C] octanoate analogs in rats and a cat.

To evaluate the potential of [1-(11)C]-3-(R,S)-methyloctanoate (BMOA), [1-(11)C]-2-octynoate, and [1-(11)C]-2-decynoate as PET tracers for studying particular steps in fatty acid beta-oxidation, we examined the pharmacokinetics of these compounds in rats and a cat. In rats given these compounds, high levels of radioactivity accumulated in the heart, liver, and kidneys, suggesting their potential as tracers for studying beta-oxidation in these tissues. These organs were clearly visible with PET in a cat given BMOA, indicating the utility of BMOA for imaging these organs.

Animals↗
PubMed1998

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

BACKGROUND: Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS: We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION: From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general.

Animals↗
PubMed1998

A functional epitope on P-selectin that supports binding of P-selectin to P-selectin glycoprotein ligand-1 but not to sialyl Lewis X oligosaccharides.

P-selectin mediates the adhesion of leukocytes to activated platelets and endothelial cells. To characterize the functional domains of P-selectin for ligand recognition, we established nine hybridoma cell lines secreting anti-rat P-selectin mAb. Among them, the mAb C215 bound both rat and human P-selectins, and inhibited binding of rat and human P-selectins to P-selectin glycoprotein ligand-1 (PSGL-1) from HL-60 cells. In contrast, mAb C215 failed to inhibit the binding of rat and human P-selectin-IgG to sialyl Lewis X (sLe(x)) oligosaccharides. Epitope mapping of mAb C215 using synthetic decapeptides revealed that mAb C215 binds specifically to an eight-residue epitope that spans amino acids 76-83 of rat P-selectin, a region completely conserved by human P-selectin. Synthetic peptides containing the mAb C215 epitope inhibited binding of P-selectin to PSGL-1, but not to sLe(x) oligosaccharides, suggesting that the C215 epitope on P-selectin may directly interact with a particular site on the PSGL-1 core protein essential for interaction with P-selectin, such as sulfated tyrosine residues. Our results suggest the presence of two ligand recognition sites on P-selectin necessary for binding to PSGL-1--one recognizes sLe(x), while the other recognises the PSGL-1 core protein.

Amino Acid Sequence↗
PubMed1998

Isolation of estrogen-responsive genes with a CpG island library.

In order to isolate novel estrogen-responsive genes, we utilized a CpG island library in which the regulatory regions of genes are enriched. CpG islands were screened for the ability to bind to a recombinant estrogen receptor protein with a genomic binding site (GBS) cloning method. Six CpG islands were selected, and they contained perfect, imperfect, and/or multiple half-palindromic estrogen-responsive elements (EREs). Northern blot analysis of various human cells showed that all these genomic fragments hybridized to specific mRNAs, suggesting that the genes associated with these EREs might be transcribed in human cells. Then cDNAs associated with two of them, EB1 and EB9, were isolated from libraries of human placenta and MCF-7 cells derived from a human breast cancer, respectively. Both transcripts were increased by estrogen in MCF-7 cells. The increase is inhibited by actinomycin D but not by cycloheximide, indicating that no protein synthesis is required for the up-regulation. The cDNA associated with EB1 encodes a 114-amino-acid protein similar to the cytochrome c oxidase subunit VIIa, named COX7RP (cytochrome c oxidase subunit VII-related protein). The cDNA associated with EB9 is homologous only to an express sequence tag and was named EBAG9 (estrogen receptor-binding fragment-associated gene 9). The palindromic ERE of EB1 is located in an intron of COX7RP, and that of EB9 is in the 5' upstream region of the cDNA. Both EREs had significant estrogen-dependent enhancer activities in a chloramphenicol acetyltransferase assay, when they were inserted into the 5' upstream region of the chicken beta-globin promoter. We therefore propose that the CpG-GBS method described here for isolation of the DNA binding site from the CpG island library would be useful for identification of novel target genes of certain transcription factors.

Amino Acid Sequence↗
PubMed1998

[Protective effects of teprenone and gefarnate against taurocholate/hydrochloric acid-induced acute gastric mucosal lesions in rats].

To evaluate the effects of teprenone on acute gastritis, its inhibitory effects on gastric mucosal damage were compared to that of gefarnate in taurocholate/hydrochloric acid-induced acute gastric mucosal lesions in rats. After oral administration of 160 mM taurocholic acid and 250 mM hydrochloric acid, hemorrhage and erosion were macroscopically observed in the gastric mucosal surface layer. Edema in the submucosal tissue and decreased PAS staining in the mucosa were histomorphologically observed. Concerning macroscopic findings, pretreatment with teprenone at a dose of 50 mg/kg or more significantly reduced pathological changes in the mucosa of the fundic glandular area. However, gefarnate slightly inhibited these changes in at a dose of 50 mg/kg and significantly inhibited them at a dose of 200 mg/kg. With regards to histomorphological findings in the fundic glandular area, teprenone slightly inhibited erosion at a dose of 50 mg/kg, and it significantly and slightly inhibited the decrease in PAS staining in this area at doses of 50 and 200 mg/kg, respectively. Gefarnate at doses of 50 and 200 mg/kg showed significant inhibition of decreased PAS staining in the fundic glandular area. In the pyloric mucosa, decreased PAS staining was slightly inhibited by teprenone at both doses but not by gefarnate at either dose. The differences between teprenone and gefarnate observed in this model appear to be due to their differences in mucus production ability. These results suggest that teprenone was more effective than gefarnate for the treatment of gastritis.

Acute Disease↗
PubMed1998

Pharmacokinetics of anticancer drugs in cerebrospinal fluid.

OBJECTIVE: To examine the pharmacokinetics of anticancer drugs in the cerebrospinal fluid (CSF) during chemotherapy by the lumbar-ventricular (LV) and ventricular-lumbar (VL) routes. CASE SUMMARY: A 69-year-old Japanese woman with disseminated glioblastoma received two LV and four VL courses of CSF perfusion chemotherapy with methotrexate, nimustine, and cytarabine hydrochloride. Samples of CSF from the ventricles and lumbar spinal canal were obtained via Ommaya reservoirs during one LV and one VL course. Drug concentrations in the CSF were measured by fluorescence polarization immunoassay or HPLC. RESULTS: During LV CSF perfusion, the highest CSF drug concentrations in both the ventricles and the lumbar spinal canal were observed at the end of perfusion. During treatment, the concentrations of all three drugs in the lumbar spinal canal were higher than those in the ventricles. The CSF AUC of methotrexate in the ventricles was 16.1% of that in the lumbar spinal canal. During VL CSF perfusion, the highest drug concentrations were also observed at the end of perfusion. The drug concentrations in the lumbar spinal canal were initially lower than those in the ventricles. However, the concentrations of methotrexate and cytarabine in the lumbar spinal canal exceeded those in the ventricles 3 hours after perfusion. The AUC of methotrexate in the lumbar spinal canal was 174.9% of that in the ventricles. CONCLUSIONS: The pharmacokinetics of anticancer drugs in ventricular CSF differ from those in lumbar CSF during LV and VL perfusion chemotherapy.

Aged↗