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Biomedical subjects

K Fujisawa

Publications and source records attributed to K Fujisawa.

At least 127 records · Page 7Linked to original sources
PubMed1996

Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain.

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

Adaptor Proteins, Signal Transducing↗
PubMed1996

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

HLA class II alleles in Ainu living in Hidaka District, Hokkaido, northern Japan.

The Ainu people are considered to be the descendants of preagricultural native populations of northern Japan, while the majority of the population of contemporary Japan (Wajin) is descended mainly from postneolithic migrants. Polymorphisms of the HLA-DRB1, DRB3, and DQB1 alleles were investigated in DNA samples of 50 Ainu living in Hidaka district, Hokkaido. Unique features of the Ainu in this study were high incidences of DRB1*1401, DRB1*1406, and a newly described allele, DRB1*1106 (20%, 17%, and 5%, respectively). On the other hand, several common alleles in Wajin (DRB1*1502, 1302, 0803, and 1501) were found at relatively low frequencies (1-2%) in Ainu. Previously DRB1*1406 was described as a characteristic allele of some Native American or northeast Asian ethnic groups, and DRB1*1106 had been found in only two Singapore Chinese and one Korean. Principal component analysis of various populations based on HLA class II allele frequencies places the Ainu population midway between other east Asian populations, including Wajin, and Native Americans. These observations may support the hypothesis that the Ainu people are the descendants of some Upper Paleolithic populations of northeast Asia from which Native Americans are also descended.

Alleles↗
PubMed1996

Activation of transcription factor NF-kappa B in human synovial cells in response to tumor necrosis factor alpha.

OBJECTIVE: To examine whether nuclear factor kappaB (NF-kappaB) is activated in cultured synovial cells in response to tumor necrosis factor alpha (TNFalpha) and to investigate the correlation between NF-kappaB activation and synovial cell proliferation. METHODS: Activation of NF-kappaB was detected by electrophoretic mobility shift assay. The transcription of several NF-kappaB-dependent genes was evaluated by reverse transcriptase polymerase chain reaction and transient expression assay using human immunodeficiency virus-long terminal repeat chloramphenicol acetyltransferase. Proliferative activity was determined by measurement of 3H-thymidine incorporation. RESULTS: Stimulation of synovial cells with TNFalpha activated NF-kappaB and subsequent transcription of several genes. Treatment of synovial cells with N-acetyl-L-cysteine (NAC), an antioxidant agent, inhibited TNFalpha-induced NF-kappaB activation and transcription. Moreover, NAC also inhibited synovial cell proliferation induced by TNFalpha. CONCLUSION: Our results suggest that NF-kappaB plays a pivotal role in synovial cell activation by TNFalpha. Thus, suppression of NF-kappaB could be a potential therapeutic modality for synovitis such as that of rheumatoid arthritis.

Acetylcysteine↗
PubMed1996

Hepatic iron contents and response to interferon-alpha in patients with chronic hepatitis C. Relationship to genotypes of hepatitis C virus.

Recent reports have shown that response to interferon treatment is influenced by hepatic iron contents in patients with chronic hepatitis C. In those reports, however, hepatitis C virus (HCV) genotypes and serum HCV-RNA levels were not examined. The aim of the present study was to investigate whether hepatic iron contents influence the response to interferon in patients with chronic hepatitis C and whether HCV genotypes and serum HCV-RNA levels play a role in this relationship. Among 65 patients with chronic hepatitis C, hepatic iron contents were significantly high in patients with a history of excess drinking of alcohol (more than 80 g/day) compared to those without, and significantly low in female patients before menopause. Having excluded these patients, hepatic iron contents were significantly higher in patients with genotype 1b infection than those with genotype 2a and 2b infection. There was no significant correlation between hepatic iron contents and plasma HCV-RNA levels. Among the patients with genotype 1b infection, hepatic iron contents were significantly lower in the responders to interferon than those in the nonresponders (429 +/- 100 vs 875 +/- 110 micrograms/g liver, P < 0.05). From these results, it is concluded that response to interferon is mainly influenced by HCV genotypes, while hepatic iron contents may play an important role in response to interferon in patients with genotype 1b infection.

Adolescent↗
PubMed1996

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes.

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A1 (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *B101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.

ABO Blood-Group System↗
PubMed1996

Serum type IV collagen in various liver diseases in comparison with serum 7S collagen, laminin, and type III procollagen peptide.

The clinical significance of the immunoreactive triple helical domain of type IV collagen in serum was evaluated in 73 healthy controls and 161 patients with various biopsy-proven liver diseases. Although serum levels of type III procollagen peptide were increased in all liver diseases, those of type IV collagen, 7S collagen, and laminin were principally increased in chronic liver diseases associated with hepatic fibrogenesis/fibrosis. In both non-alcoholic and alcoholic liver diseases, 7S collagen was increased in serum, while type IV collagen and laminin in serum were particularly increased in alcoholic liver diseases and in hepatocellular carcinoma, in which latter the sensitivity was greater for type IV collagen than for laminin. Gel filtration analysis in Sephacryl S-400 revealed type IV collagen in serum to be a single molecular form with a molecular weight that correspond to type IV collagen, whereas 7S collagen was recognized as several heterogeneous macromolecules. These findings indicate that serum type IV collagen is derived from the type IV protocollagen pool, and is a sensitive marker for the fibrogenetic process in hepatic basement membranes.

Adult↗
PubMed1996

HLA-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method.

We developed a system for discriminating HLA-B40, B18, B27, and B37 alleles using a two-step PCR method followed by SSCP analysis. Fragments (0.8 kb) including exon 2, intron 2, and exon 3 were amplified in the first PCR. We used two sets of primers, one specific for HLA-B60-related alleles and the other specific for HLA-B61-related, B18, B27, and B37 alleles. No amplifications of other class I genes or pseudogenes were observed. In the second PCR, exon 2 and exon 3 were amplified separately, using diluents of the first PCR products as templates. HLA-B61-related, B18, B27, B37, and B60-related alleles were clearly discriminated in the SSCP analysis of the second PCR products. In a population study in which B61 alleles were analyzed, B*4003 was detected in two Japanese individuals in addition to two B61 alleles previously reported to occur in Japanese, B*4002 and B*4006. The relative frequencies of B*4002, B*4006, and B*4003 in Japanese were 58, 35, and 6%, respectively. The individuals having B*4003 are the first non-South Americans in whom this allele has been detected. The SSCP banding patterns of 18 HLA-B60-positive Japanese population samples were identical to those of a B*40012 sample for both exon 2 and exon 3. We also demonstrated that the B37 allele occurring in some Japanese is B*3701.

Alleles↗
PubMed1996

Congenital triggering of the index finger at the A2 pulley.

A case of trigger index finger is reported. The diagnosis was made in the neonatal intensive care unit. The proximal interphalangeal joint (PIP) was locked in a flexed position. A nodular thickening of the flexor tendon was felt at the A2 pulley level. Surgery revealed thickening of both the A2 pulley and the radial slip of the flexor superficialis tendon. Division of the A2 pulley released the PIP joint locking.

Finger Joint↗
PubMed1996

Urinary uric acid excretion in term and premature infants.

OBJECTIVE: To clarify postnatal changes in urinary uric acid (UA) excretion in normal term infants and to examine the effects of prematurity or illness on the UA excretion. METHODOLOGY: Measurements of urinary UA were performed in term and premature infants at the ages of 1 and 7 days and at 1 and 4 months, as well as 7 months in term infants. RESULTS: Urinary UA levels were lowest on day 7 in term infants. The levels were highest on day 1 in premature infants and remained significantly higher compared to term babies during the first month of life. Respiratory failure requiring ventilation and oxygen supply resulted in further significant elevation of urinary UA in premature infants. CONCLUSIONS: With the reference values obtained in the study reported here, urinary UA can now be used for the diagnosis and monitoring of inherited disorders of purine metabolism and for the assessment of oxygen radical insult to sick infants.

Age Factors↗
PubMed1996

Testicular morphological changes in children with acute lymphoblastic leukemia following chemotherapy.

Morphological changes in the testis induced by chemotherapy given according to the Tokyo Children's Cancer Study Group (TCCSG) regimens were studied in children with acute lymphoblastic leukemia (ALL). After informed consent, testicular biopsies were performed 14 times in 12 patients at the end of treatment. The testicular morphology in all cases had sustained a degree of damage. The tubular fertility index (TFI), calculated as the percentage of seminiferous tubules containing identifiable spermatogonia, was from 0 to 42.8% (mean 33.4%) below the normal value. Infiltration of leukemic cells was the most significant factor contributing to the decrease in TFI. There were no differences in the TFI among the TCCSG protocols. Formation of sperm was recognized in six cases, whose ages were 7, 8, 9, 10, 15 and 19 years. In two children, testicular biopsy was performed twice. In the second biopsy, TFI was elevated and sperm formation with the maturation of Leydig cells was observed. A number of other pathological changes were observed: modification of spermatogonia, Sertoli cells and inclusion bodies in spermatogonia, abnormal maturation of Leydig cells, evidence of interstitial fibrosis and thickening of the basement membrane. These results suggest that recent strong chemotherapy for the treatment of ALL might cause severe but not fatal damage to children's testicular tissue. As chemotherapy escalates, more investigation of testicular function will be necessary.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Vertebral bone mineral density in postpartum women.

The purpose of this study was to measure the bone mineral density of the lumbar spine (L2-L4) in healthy postpartum women following term delivery, and to evaluate factors associated with bone mineral density. Using dual energy X-ray absorptiometry, the bone mineral density of the lumbar spine was determined during 1 year in 123 healthy postpartum Japanese women following term delivery. The mean bone mineral density of the lumbar spine was 1.007 +/- 0.108 g/cm2 (range 0.731-1.252 g/cm2). Thus, there were wide inter-individual differences. Also, this result was in agreement with the peak bone mass previously reported for other Japanese women. Multiple regression analysis and linear regression analysis showed that weight and body mass index were each significantly correlated with bone mineral density of the lumbar spine. Age, parity, height, and baby's birth weight were not contributory.

Absorptiometry, Photon↗
PubMed1996

Multiple endocrine neoplasia type 1 associated with spinal ependymoma.

A 51-year-old man was hospitalized with a gait disturbance and hypoesthesia below the level of his chest. These symptoms were due to a spinal tumor which was surgically resected and identified as an ependymoma. Additionally, the patient had hypercalcemia and a family history of insulinoma. An endocrine evaluation revealed parathyroid hyperplasia and a pancreatic islet cell tumor. Magnetic resonance imaging disclosed a pituitary microadenoma. He was diagnosed with spinal ependymoma and multiple endocrine neoplasia type 1 (MEN 1). A review of the literature revealed that chromosome 11q13 abnormalities have been reported in both ependymoma and MEN 1. We discuss the pathogenesis of these diseases.

Chromosome Aberrations↗
PubMed1996

Glucose-dependent interleukin 6 and tumor necrosis factor production by human peripheral blood monocytes in vitro.

To clarify the mechanisms that cause elevation of plasma fibrinogen levels in diabetes, we first examined the effect of hyperglycemia on the production of interleukin 6 (IL-6) and tumor necrosis factor (TNF) by cultured human peripheral blood monocytes. Monocyte-enriched fractions isolated from 20 healthy volunteers were incubated with 11 mmol/l glucose, 33 mmol/l glucose, or mannitol as an osmolar control for 6 or 24 h. After 6 h of incubation, IL-6 and TNF-alpha mRNA levels were analyzed by reverse transcription and polymerase chain reaction. In addition, after 24 h of incubation, IL-6 and TNF-alpha immunoreactivity in the culture medium was measured by enzyme-linked immunoassay. Both IL-6 and TNF-alpha mRNA levels and immunoreactivity were significantly increased by treatment with 33 mmol/l glucose compared with treatment with 11 mmol/l glucose or 11 mmol/l glucose with 22 mmol/l mannitol. In addition, preincubation of the cells with an anti-TNF monoclonal antibody (mAb) blocked the stimulatory effect of 33 mmol/l glucose on IL-6 synthesis and secretion. Second, we examined the ability of conditioned media from human peripheral blood monocytes to stimulate beta-fibrinogen mRNA synthesis in HepG2 cells. The conditioned medium from monocytes treated with 33 mmol/l glucose increased beta-fibrinogen mRNA levels. The results of this study demonstrate that hyperglycemia stimulated IL-6 and TNF synthesis and secretion by human peripheral monocytes in vitro and that the IL-6 response to hyperglycemia may be mediated by TNF. Furthermore, hyperglycemia may increase fibrinogen levels through stimulation of peripheral monocytes. These results suggest that hyperglycemia may cause hyperfibrinogenemia in diabetic patients through an IL-6-dependent and TNF-dependent mechanism.

Antibodies, Monoclonal↗
PubMed1996

Distribution and characterization of proteoglycans associated with exfoliation material.

PURPOSE: To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials. METHODS: The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy. RESULTS: Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue. CONCLUSIONS: Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.

Aged↗
PubMed1996

Visual acuity disturbance in subjects over 50 years of age in a population-based cataract survey.

Visual acuity is still an essential examination item in cataract epidemiological studies, even though this parameter lacks objective reliability. A population-based epidemiological study was conducted in a rural area of Japan to find out the relationship between visual acuity levels and cataracts, the types of gradings of which were evaluated by an objectively reliable method through lens images, and to serve as a sample for researchers who perform epidemiological studies on cataract but lack the latest methodology. 863 participants above 50 years of age were examined and those previously diagnosed with ocular diseases which affect visual acuity were excluded from the analysis as much as possible. The mean visual acuity (LogMAR) in subjects in their 50s, 60s, 70s and over 80 years of age was 1.1, 1.0, 0.7 and 0.4, respectively. Both the mean and the distribution of visual acuity showed a statistically significant relationship to ageing (p < 0.01, p < 0.01). Both age and cataract grading showed a statistically significant relationship to visual acuity (p < 0.01), but there was no significant interaction effect between these two variables upon visual acuity. Visual acuity worsened remarkably with ageing in the eyes with grade III, whereas that of eyes with grades I and II remained fairly stable at a level of 0.7-1.0, except for those above 80 years of age. A fairly good visual acuity of 1.0, 0.8 and 0.7 remained in the eyes with pure cortical cataract of grades I, II and III, respectively, whereas those with mixed type cataract were 0.9, 0.6 and 0.3, respectively.

Aged↗