Search PubMed⌕ Search

Biomedical subjects

I Kitajima

Publications and source records attributed to I Kitajima.

At least 73 records · Page 4Linked to original sources
PubMed1995

Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation.

Type I receptors for bone morphogenetic proteins (BMPs), i.e., BMPR-IA and BMPR-IB, are transmembrane serine/threonine kinases, that bind osteogenic protein-1 (OP-1, also termed BMP-7) and BMP-4. Using antibodies specific to BMPR-IA and -IB, we have studied the expression of BMP type I receptors in the bone formation process during embryonic development and fracture healing. In the mouse embryo, both BMPR-IA and -IB were expressed in condensing mesenchymal cells at 13.5 days post coitum (p.c.). At 15.5 days p.c., expression of BMPR-IB, but not of BMPR-IA, was observed in the cells in perichondrium of developing cartilage. At 17.5 and 19.5 days p.c., expression of both receptors was observed in chondrocytes and in osteoblasts. In normal rat adult bone, expression of BMPR-IA, but not of BMPR-IB, was observed in osteoblasts in the periosteum. Three days after the femoral fracture, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum. On day 7, both receptors were found in fibroblast-like spindle cells and chondrocytes in the endochondral ossification sites, and osteoblasts in the newly formed trabecular bone. Expression of BMPR-IA was higher than that BMPR-IB in osteogenic layer on day 3 and in osteoblasts in the trabecular bone on day 7. On day 14, expression of BMP type I receptors was observed at similar sites, albeit with lower expression levels than were observed on day 7. The present data suggest that expression of BMP type I receptors is up-regulated during bone formation, and that they may play important roles in bone morphogenesis.

Animals↗
PubMed1995

Thrombin receptor-mediated synovial proliferation in patients with rheumatoid arthritis.

Synovial cell proliferation is one of the pathological bases of rheumatoid arthritis (RA). Several cytokines including IL-1 and IL-6 and growth factors have been shown to be involved in the synovial cell proliferation in RA. Thrombin is a multifunctional protease and acts as a mitogen for several cell types through its specific receptor. To assess whether thrombin is involved in overproliferation of rheumatoid synovial cells, we measured the concentration of thrombin-anti-thrombin III (ATIII) complex (TAT) in synovial fluid obtained from patients with RA or osteoarthritis (OA). We also examined the effect of thrombin or thrombin receptor agonist peptide (TRAP) on cell growth of synovial cell clones (SCCs) established from an RA patient. The concentrations of TAT in the synovial fluid from patients with RA were significantly higher than in those with OA. Moreover, both thrombin and TRAP enhanced proliferation of synovial cells in vitro. We also characterized the expression of thrombin receptor mRNA by reverse transcription-PCR. The expression of mRNA for thrombin receptor was up-regulated by thrombin or TRAP stimulation. Thrombin receptor antigen was also detected on both SCCs and synovial tissue from RA patients by immunostaining using a monoclonal antibody against thrombin receptor. These findings indicate that thrombin may act as a mitogen for synovial cells through thrombin receptor and may play some role in synovial overproliferation and remodeling in RA.

Adult↗
PubMed1995

p53 gene mutations in skin cancers with underlying disorders.

Mutations in p53, a tumor suppressor gene, are one of the most common genetic lesions of human cancers. The relationship between p53 gene mutation and ultraviolet (UV) light has been demonstrated in skin cancers of sun-exposed sites. In this study, genomic DNA from 12 skin cancers was screened for mutations in exons 5 to 9 of this gene using the polymerase chain reaction--single strange configuration polymorphism (PCR-SSCP) analysis followed by DNA sequencing. DNA samples were obtained from 8 basal cell carcinomas (BCCs): 1 from an organoid nevus, 1 from a patient with basal cell nevus syndrome, 1 from a patient with xeroderma pigmentosum, and 1 from a recurrent and 4 from primary sporadic lesions on actinic damaged skin, and from 4 squamous cell carcinomas (SCCs): 1 from a burn scar, 1 from a patient with epidermodysplasia verruciformis, and 2 from actinic keratosis. Mutation of the p53 gene was detected in only 1 case of SCC which had arisen from actinic keratosis. The mutation occurred at codon 159 in exon 5 with a GCC to CCC base-pair substitution resulting in an amino acid change of alanine to proline. This mutation does not correspond to results of UV mutagenesis studies reported in the literature. Our findings imply that, although p53 gene mutation and UV exposure play an important role in the carcinogenesis of some skin cancers, they are not crucial, especially in skin cancers that develop from underlying skin disorders.

Aged↗
PubMed1995

Molecular typing of the methicillin resistance determinant (mec) of clinical strains of Staphylococcus based on mec hypervariable region length polymorphisms.

We used a method for molecular typing the methicillin resistance determinant (mec) based on the size of the mec-associated hypervariable region amplified by the polymerase chain reaction (PCR) to examine 61 methicillin-resistant Staphylococcus aureus (MRSA), 15 methicillin-resistant (Mcr) S. epidermidis, and 11 Mcr S. haemolyticus clinical isolates. In the 61 MRSA isolates, five sizes of PCR products were observed. The MRSA isolates were grouped into five hypervariable region (HVR) genotypes on the basis of the size of the PCR product. Three different sizes were detected among 15 isolates of Mcr S. epidermidis and two sizes among 11 isolates of Mcr S. haemolyticus. The PCR products amplified from 14 of 15 Mcr S. epidermidis isolates were the same as products amplified from MRSA isolates, which was confirmed by the PCR-SSCP (single-strand conformation polymorphism) method. In methicillin-susceptible isolates, the target gene was not amplified. This method is thought to be useful in epidemiologic investigations of nosocomial infections caused by MRSA. This is the first typing method capable of comparing the mec determinants of MRSA isolates and Mcr coagulase-negative staphylococcal isolates to establish the origin of the mec determinant.

Base Sequence↗
PubMed1995

[Study of signal transduction through thrombin receptor and anti-thrombotic strategy using its controls].

Thrombin, a key enzyme in the hemostatic pathway, also has various effects on the function of human platelet, endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC). A thrombin receptor (TR) has been cloned and is thought to mediate a variety of thrombin-induced responses. The post-receptor signals are mediated by several protein kinases responsible for NF-kappa B activation, and most thrombin-inducible genes have the kappa B sequence in the regulatory elements. TR stimulation resulted in a biphasic activation of NF-kappa B and the late phase of which required new NF-kappa B synthesis. We showed that the antisense oligodeoxynucleotides (ODNs) of NF-kappa B have a marked inhibitory effect on thrombin-induced cellular responses. Furthermore, E5510, a compound with anti-platelet activity preferentially inhibited the thrombin-inducible NF-kappa B activation. Therapeutic potential of inhibition of TR-NF-kappa B activation signaling for treatment with thrombotic disease is also indicated.

Arteriosclerosis↗
PubMed1994

Involvement of NF-kappa B activation in thrombin-induced human vascular smooth muscle cell proliferation.

A thrombin receptor has been cloned and is thought to mediate a variety of thrombin-induced responses. However, the transcription factors important for postreceptor signaling have been little clarified. The post-receptor signals are mediated by several protein kinases responsible for NF-kappa B activation, and most thrombin-inducible genes have the kappa B sequence in the regulatory elements. The possibility that NF-kappa B may participate in thrombin signaling was therefore investigated in cultured human vascular smooth muscle cells (VSMCs). Thrombin receptor stimulation resulted in activation of NF-kappa B. Furthermore, treatment of cells with antisense p65 ODNs of NF-kappa B inhibited thrombin-stimulated growth of VSMC in vitro. Results indicate that the activation of NF-kappa B is involved in thrombin signaling and that this pathway causes the proliferation of VSMC induced by thrombin. Therapeutic potential of antisense NF-kappa B ODNs for the treatment with atherosclerosis and restenosis is also indicated.

Acid Phosphatase↗
PubMed1994

Nitric oxide-mediated apoptosis in murine mastocytoma.

To investigate how the number of mast cells is controlled, we studied a murine mastocytoma cell line. Based on electron microscopic observation of nuclear condensation and electrophoretic evidence with DNA fragmentation, these mastocytoma wells were shown to undergo apoptosis. This apoptosis was dependent on the concentrations of serum and L-arginine and was enhanced by TNF-alpha. We confirmed that apoptosis was mediated by nitric oxide (NO) synthase; inducible NO synthase (iNOS) mRNA was strongly expressed in apoptotic cells, while an inhibitor of NOS, NG-monomethyl-L-arginine, and dexamethasone prevented apoptosis in addition to inhibiting iNOS mRNA expression. Our results suggest that iNOS expression is very important in regulating the proliferation of mast cells under pathological conditions.

Amino Acid Oxidoreductases↗
PubMed1994

E5510 antagonizes thrombin receptor signals by inhibiting NF-kappa B activation.

We have recently demonstrated that NF-kappa B is involved in a thrombin-signaling and that the antisense oligodeoxynucleotides (ODNs) of NF-kappa B has a marked inhibitory effect on thrombin-induced cellular responses. In this study, we demonstrate that E5510 (4-cyano-5,5-bis(methoxyphenyl)-4-pentenoic acid), a compound with anti-platelet activity preferentially inhibits the thrombin-inducible NF-kappa B activation and then antagonizes the following thrombin-induced cellular responses, proliferation and cytokines production in vascular smooth muscle cell, and the adherency of differentiated HL-60 cells. These data suggest that E5510 is an anti-atherosclerotic or anti-restenotic drug induced by thrombin.

Amino Acid Sequence↗
PubMed1994

Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region.

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.

Animals↗
PubMed1994

[Development and application of non-radio isotropic PCR-SSCP analysis for genetic diagnosis].

We applied a new and powerful technique for genetic diagnosis, so called reverse transcript-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) without using radio labeled materials (non-RI). The principle of this method is mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits. In this study, this method was used to determine the mutations of the LDL receptor or insulin receptor. We found new mutations in Exon 14 of the LDL receptor, and a patient had a new missense mutation in which Asn461 was substituted for Thr461 in the alpha-subunit of the insulin receptor. Our findings indicate that this analysis can be successfully used to not only rapidly and easily but also safely screen mutation containing exons in large genes, compared to the conventional Southern blotting analysis. Moreover, this analysis has the advantage over the RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in the DNA fragments. This "non-RI" RT-PCR-SSCP analysis is expected to be useful as a routine examination for genetic diagnosis in the ordinary laboratory.

Adult↗
PubMed1994

[Molecular typing of the methicillin resistance determinant (mec) of clinical strains of Staphylococcus based on mec hypervariable region length polymorphisms].

We used a method for molecular typing of the methicillin resistance determinant (mec) based on the size of the mec-associated hypervariable region amplified by the polymerase chain reaction (PCR) to examine 106 methicillin-resistant Staphylococcus aureus (MRSA), 9 methicillin-resistant (Mcr) S. epidermidis and 5 Mcr S. haemolyticus clinical isolates. In the 106 MRSA isolates, 5 sizes of PCR products were observed. The MRSA isolates were grouped into five hypervariable region (HVR) genotypes on the basis of the size of the PCR product. The PCR products amplified from 8 of 9 Mcr S. epidermidis isolates were the same as products amplified from MRSA isolates, which was confirmed by the PCR-SSCP (single-strand conformation polymorphism) method. In 32 methicillin-susceptible isolates, the target gene was not amplified. This method is thought to be useful in epidemiological investigations of nosocomial infections caused by MRSA. This is the first typing method capable of comparing the mec determinants of MRSA isolates and Mcr CNS isolates to establish the origin of the mec determinant.

Base Sequence↗
PubMed1994

[The diagnosis of a mutation of the insulin receptor by non-radioisotropical RT-PCR-SSCP analysis].

We evaluated a 24 years old diabetes woman with type A insulin resistant (patient "Yakushima") who had some typical symptoms as acanthosis nigricans, hirsutism and virilization. Insulin binding to the patient's erythrocytes was significantly decreased to about 30% of the normal control. In order to determine the mutation of the insulin receptor, we used for reverse transcript-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis without using radiolabeled materials. We also analysed the nucleotide sequence with non-isotropical probe. Our results suggested that the mutation was heterozygous, and the patient had a new missense mutation substituted Asn461 for Thr461 in the alpha-subunit.

Adult↗
PubMed1994

Olfactory bulbectomy leads to learning/memory impairments in 3-panel runway and 3-lever operant tasks.

The effects of olfactory bulbectomy on the learning and memory of rats were examined, using several memory tasks. In reference memory and working memory tasks using a 3-panel runway apparatus, acquisition was delayed markedly by bilateral ablation of the olfactory bulb (OB). OB lesion performed after acquisition markedly impaired both reference and working memories. Even when the tasks were repeated for several sessions, the impairment of memory in OB-lesioned rats did not recover to the control level of the sham operation group. The delayed matching-to-lever location (DMLL) performance, which was examined using a 3-lever operant apparatus, was markedly impaired by OB lesions. This impairment was mild immediately after surgery, but tended to increase with time. Rats with OB-olfactory tubercule lesions show more severe impairment of memory in the DMLL performance. Reversal learning, using a 2-lever operant apparatus, was markedly impaired by OB lesions. The impairment of working and reference memories in OB lesioned rats, which was assessed using a 3-panel-runway apparatus, was reduced by cholinesterase inhibitor physostigmine and NIK-247. These findings suggest that the OB plays a very important role in the learning and memory processes necessary for both a working memory task and a reference memory task and that, at least in part, the memory impairment in OB lesioned rats is mediated by lowering of cholinergic function.

Animals↗
PubMed1993

Vesnarinone inhibits production of HIV-1 in cultured cells.

Vesnarinone, a synthetic oral cardiotonic agent that has been used for treatment of patients with congestive heart failure, was found to inhibit replication of HIV-1 in a peripheral blood lymphocytes model and in chronically infected macrophages at clinically achieved concentrations. Vesnarinone has no direct inhibitory activity against the reverse transcriptase of HIV-1, syncytium formation in short term assays, or retroviral protease. In addition, vesnarinone inhibits production of TNF-alpha and IL-6 by human peripheral blood mononucleated cells stimulated with LPS. These observations suggest that vesnarinone may be therapeutically useful in patients infected with HIV-1.

Antiviral Agents↗
PubMed1993

Ameliorative effects of the centrally active cholinesterase inhibitor, NIK-247, on impairment of working memory in rats.

Using a three-panel runway task, the effects of NIK-247 on impairment of working memory produced by scopolamine, hippocampal lesions, and cerebral ischemia were investigated in rats; these effects were compared with those of the well-known cholinesterase inhibitors, tetrahydroaminoacridine (THA) and physostigmine. Intraperitoneal injection of scopolamine (0.56 mg/kg) significantly increased the number of errors (pushes made on the two incorrect panels of the three-panel gates located at four choice points). NIK-247 (3.2-18 mg/kg PO), THA (1-10 mg/kg PO), and physostigmine (0.1 and 0.32 mg/kg IP) dose-dependently reduced the increase in errors induced by scopolamine. NIK-247 (32 mg/kg) was also effective in reducing the increase in errors produced by lesions of the dorsal hippocampus. A 5-min period of cerebral ischemia markedly increased the number of errors. NIK-247 (3.2 and 10 mg/kg), given immediately after blood flow recirculation and again 20 min before the runway test carried out 24 h after ischemia, significantly reduced the increase in errors expected to occur after ischemia. Tetrahydroaminoacridine (3.2 mg/kg) and physostigmine (0.1 mg/kg) similarly reversed the increased errors in ischemic rats. These results suggest that NIK-247 alleviates the impairment of working memory produced by scopolamine, hippocampal lesions, and cerebral ischemia, possibly through activation of the central cholinergic system.

Acetylcholinesterase↗
PubMed1992

Human T-cell leukemia virus type I tax transformation is associated with increased uptake of oligodeoxynucleotides in vitro and in vivo.

We have utilized antisense oligodeoxynucleotides (ODNs) to modulate transcriptional activation by the human T-cell leukemia virus type I (HTLV-I) tax gene, the major transcriptional regulator of this virus. 3'-Terminal phosphorothioate-modified antisense ODNs were shown to efficiently inhibit Tax protein expression both in vitro and in vivo. Terminal substitution did not affect the affinity of ODNs for their target sequence but conferred a 9-fold increase in tax inhibition in vitro. When delivered into mice by intraperitoneal injection, ODNs inhibited tax expression in established tumors by 90%. Unmanipulated tax-transformed mouse fibroblasts, or HTLV-I-transformed human lymphocytes, showed at least 5-fold higher ODN binding and uptake over control cells. Balb/3T3 cell binding was induced to similar levels by cellular activators. This suggests that constitutive activation by tax transformation may increase susceptibility of HTLV-I-transformed cells to antisense therapy, providing a rationale for the use of antisense ODN therapeutics in HTLV-I-associated diseases.

3T3 Cells↗
PubMed1992

Ablation of transplanted HTLV-I Tax-transformed tumors in mice by antisense inhibition of NF-kappa B.

Mice transgenic for the human T cell leukemia virus (HTLV-I) Tax gene develop fibroblastic tumors that express NF-kappa B-inducible early genes. In vitro inhibition of NF-kappa B expression by antisense oligodeoxynucleotides (ODNs) inhibited growth of these culture-adapted Tax-transformed fibroblasts as well as an HTLV-I-transformed human lymphocyte line. In contrast, antisense inhibition of Tax itself had no apparent effect on cell growth. Mice treated with antisense to NF-kappa B ODNs showed rapid regression of transplanted fibrosarcomas. This suggests that NF-kappa B expression may be necessary for the maintenance of the malignant phenotype and provides a therapeutic approach for HTLV-I-associated disease.

3T3 Cells↗