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Biomedical subjects

H Fujisawa

Publications and source records attributed to H Fujisawa.

At least 145 records · Page 8Linked to original sources
PubMed1997

Molecular cloning of Ca2+/calmodulin-dependent protein kinase kinase beta.

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous and immune systems, is markedly activated upon phosphorylation through the action of CaM-kinase kinase. Our previous immunotitration analysis suggested the existence of an isoform different from CaM-kinase kinase alpha, the beta isoform, in rat brain [Okuno, S., Kitani, T., and Fujisawa, H. (1996) J. Biochem. 119, 1176-1181]. In the present study, cDNA for CaM-kinase kinase beta was cloned from a rat cerebellar cDNA library. The coded protein consisted of 587 amino acids with a molecular weight of 64,445. Western blot analysis revealed that CaM-kinase kinase beta significantly existed only in the brain. The enzyme was not significantly detected in the retina where CaM-kinase kinase alpha exists.

Amino Acid Sequence↗
PubMed1997

Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha.

Ca2+/calmodulin-dependent protein kinase (CaM-kinase) kinase a, which is known to activate CaM-kinases IV and I by phosphorylation of Thr196 and Thr177, respectively, can only phosphorylate Thr196 among many phosphorylation sites of CaM-kinase IV [Kitani, T., Okuno, S., and Fujisawa, H. (1997) J. Biochem. 121, 804-810], indicating its high degree of substrate specificity. In the present study, the substrate specificity of CaM-kinase kinase a was examined using various proteins and synthetic peptides as substrates as a means to address its physiological function. Among a number of proteins and synthetic peptides, including several known as good substrates for various protein kinases, only CaM-kinases IV and I and peptides containing the sequence surrounding Thr196 of CaM-kinase IV or Thr177 of CaM-kinase I were significantly phosphorylated by CaM-kinase kinase alpha, while the heat-denatured (at 60 degrees C for 5 min) CaM-kinases IV and I were not phosphorylated. Peptides containing the phosphorylation site of CaM-kinase IV or I were far less active as substrates for CaM-kinase kinase a than were native CaM-kinase IV or I. Thus, CaM-kinase kinase a showed a high degree of substrate specificity, recognizing not only specific amino acid sequences but also the native conformation of CaM-kinases IV and I.

Adenosine Triphosphate↗
PubMed1997

Perinuclear membrane localization of alphaKAP, a protein produced from a gene within the gene of calmodulin-dependent protein kinase IIalpha.

AlphaKAP is a protein produced from a gene within the gene of the a isoform of calmodulin-dependent protein kinase II (CaM-kinase IIa). It consists of the association domain of CaM-kinase IIa and a highly hydrophobic amino-terminal stretch consisting of 25 amino acids which is absent from CaM-kinase IIalpha. We previously demonstrated that alphaKAP is an integral membrane protein by subcellular fractionation analysis [Sugai, R., Takeuchi, M., Okuno, S., and Fujisawa, H. (1996) J. Biochem. 120, 773-779], but the exact subcellular localization of alphaKAP was not well understood. Here we demonstrate that alphaKAP is localized on the nuclear membrane of COS-7 cells transiently expressing alphaKAP. The nuclear membrane and perinuclear small vesicles were immunostained with an antibody against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids of alphaKAP. In contrast to the intact alphaKAP, the mutant alphaKAP, from which the hydrophobic amino-terminal segment had been deleted, accumulated within nuclei. Thus, alphaKAP may function as an anchoring protein for CaM-kinase II and/or other proteins in the perinuclear membrane.

Animals↗
PubMed1997

Regulation of Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) by changing its susceptibility to phosphorylation by CaM-kinase kinases.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is markedly activated on the phosphorylation of Thr196 by an upstream protein kinase, CaM-kinase kinase. The phosphorylation of CaM-kinase IV by CaM-kinase kinase is strongly suppressed on incubation with calmodulin in the presence of Ca2+, which results in a decrease in the enzyme activity, and completely restored on subsequent incubation with Mg2+ in the absence of Ca2+, which results in an increase in the activity. That a downstream protein kinase regulates its activity through reversible changes in its susceptibility to phosphorylation by an upstream protein kinase is a new regulatory mechanism.

Animals↗
PubMed1997

Mutations in the SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism.

Shoots of higher plants grow upward in response to gravity. To elucidate the molecular mechanism of this response, we have isolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana. In this report, we describe three novel mutants, sgr4-1, sgr5-1 and sgr6-1 whose inflorescence stems showed abnormal gravitropic responses as previously reported for sgr1, sgr2 and sgr3. These new sgr mutations were recessive and occurred at three independent genetic loci. The sgr4-1 mutant showed severe defect in gravitropism of both inflorescence stem and hypocotyl but were normal in root gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1 mutants showed reduced gravitropism only in inflorescence stems but normal in both hypocotyls and roots as sgr3. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs in A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that SGR4, SGR5 and SGR6 genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response.

Arabidopsis↗
PubMed1997

The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana.

In higher plants, shoots show negative gravitropism and roots show positive gravitropism. To elucidate the molecular mechanisms of root and hypocotyl gravitropism, we segregated the second mutation from the original phyB-1 mutant line which impaired both root and hypocotyl gravitropism and characterized this novel mutation named rhg (for root and hypocotyl gravitropism). The rhg is a single recessive nuclear mutation and it is mapped on the lower part of the chromosome 1. Analyses on the gravitropic responses of the rhg mutant indicate that root and hypocotyl gravitropism are severely impaired but inflorescence stem gravitropism is not affected by the rhg mutation. In the rhg mutant seedlings, amyloplasts (statoliths for gravity-perception) were present in the presumptive statocytes of roots and hypocotyls. Phototropism by roots and hypocotyls was not impaired in the rhg mutant. These results suggest that the RHG gene product probably acts on the gravity-perception and/or the gravity-signal transduction in root and hypocotyl gravitropism. This is the first report about the genetic locus specifically involved in both root and hypocotyl gravitropism but not inflorescence stem gravitropism, supporting our hypothesis that the mechanisms of gravitropism are genetically different between hypocotyls and inflorescence stems.

Arabidopsis↗
PubMed1997

Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant.

Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.

Amino Acid Sequence↗
PubMed1997

CD45 molecule in gammadelta T-cell generation: disruption of CD45 exon 6 does not affect Vgamma3 dendritic epidermal T-cell development.

There are two distinct lineages of T cells: T-cell receptor (TCR) alphabeta-bearing cells (alphabeta T cells) and TCR gammadelta-bearing cells (gammadelta T cells). All of the alphabeta T cells and most subsets of gammadelta T cells develop in the thymus. It has been demonstrated that the protein tyrosine phosphatase CD45 plays a pivotal role in the intrathymic development of alphabeta T cells. Thymocyte maturation is arrested at the transitional stage from immature CD4+ CD8+ double-positive to mature CD4+ or CD8+ single-positive cells after CD45 exon 6 gene disruption. In this study, we examined whether Vgamma3 dendritic epidermal T cells (DETC), a subset of thymus-dependent gammadelta T cells uniquely residing in the murine epidermis, are altered in the CD45 exon 6-deficient mice. In situ immunolabeling on epidermal sheets demonstrated that the CD45-deficient mice had a normal density and immunophenotype of Vgamma3 DETC compared with the wild-type control mice. Reverse transcriptase polymerase chain reaction revealed that similar levels of Vgamma3 TCR mRNA were present in the epidermis of CD45-deficient mice and wild-type controls. Flow cytometry demonstrated no significant difference in the proportion of Vgamma3 T cells in the epidermis between the genotypes. In addition, Vgamma2 T cells, another subset of gammadelta T cells, were also examined by flow cytometry. The frequency of Vgamma2 T cells in lymph nodes was normal in the CD45-deficient mice. Our results indicate that although CD45 is crucial for the development of alphabeta T cells, this molecule is not necessary for the thymic maturation of gammadelta T cells, including Vgamma3 DETC and Vgamma2 T cells.

Animals↗
PubMed1997

Conformability and irritancy of adhesive tapes on the skin.

The relationship between the conformability of adhesive tape, the mechanical property of the adhesive tape not to interfere with skin movement, and irritation of the skin was investigated. In order to assess the conformability of adhesive tape to the skin, a uniaxial method employing film strips connected to a strain gauge was used to measure the elastic property of the skin, with or without application of various elastic tapes. The tension loaded on the strain gauge was measured while the skin was extended by 15% of its original length in a direction across the humeral axis on the flexor side of the upper right arm. The most elastic adhesive tape showed the best conformity to the skin. The same adhesive tapes were applied on the flexor side of both upper arms so that the tape held a piece of sanitary cotton in place for 24 h. Dermal irritation was not so remarkable in the skin under the inner part of the tapes. On the other hand, the skin reaction was much more severe on the skin under the edge portion of the applied tape which showed poor conformability to the skin. These findings seemed to indicate that the skin reaction was caused by localized distortion of the skin under the edge portion of the applied tapes, during movement of the underlying muscle. Actually, the distortion of the skin surface was great in the areas immediately outside the edge of the applied tape. In conclusion, adhesive tape conformable to skin movement reduced localized distortion of the skin during application, resulting in low irritation at the edge of the applied tape.

Adhesiveness↗
PubMed1997

Function of a cell adhesion molecule, plexin, in neuron network formation.

Plexin is a type I membrane protein which was identified in Xenopus nervous system by hybridoma technique. Molecular cloning studies demonstrated that the extracellular segment of the plexin protein possesses three internal repeats of cysteine cluster which are homologous to the cysteine-rich domain of the c-met proto-oncogene protein product. A cell aggregation test revealed that the plexin protein mediated cell adhesion via a homophilic binding mechanism, in the presence of calcium ions. Plexin was expressed in the neuronal elements composing particular neuron circuits in Xenopus CNS and PNS. These findings indicate that plexin is a new member of the Ca(2+)-dependent cell adhesion molecules, and suggest that the molecule plays an important role in neuronal cell contact and neuron network formation.

Amino Acid Sequence↗
PubMed1997

[Antigenicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a novel drug for urinary frequency and incontinence].

An antigenicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a new drug for the treatment of urinary frequency and incontinence, was conducted in Hartley guinea pigs and BALB/cAnN mice. The following results were obtained. No active systemic anaphylaxis reactions were found in guinea pigs immunized by subcutaneous injection of NS-21 alone or in combination with Freund's complete adjuvant (FCA). No 24-hr heterologous passive cutaneous anaphylaxis reactions were elicited in rats by sera from mice immunized by intraperitoneal injection of NS-21 alone or in combination with 3% aluminum hydroxide gel. No passive hemagglutination reactions were elicited by sera from mice immunized by subcutaneous injection of NS-21 in combination with FCA. These results show that NS-21 has no antigenicity under the present experimental conditions.

Animals↗
PubMed1997

Effects of glycemic control on plasma 3-deoxyglucosone levels in NIDDM patients.

OBJECTIVE: To clarify the effects of glycemic control on the level of 3-deoxyglucosone (3-DG), a reactive dicarbonyl compound, in plasma from diabetic patients. RESEARCH DESIGN AND METHODS: Fasting plasma samples were collected from 15 healthy volunteers and 27 patients with NIDDM. Samples were collected from six poorly controlled patients before and after improved glycemic control for at least 2 months. Plasma 3-DG was determined by high-performance liquid chromatography (HPLC) as a 2,3-diaminonaphthalene derivative. We observed the relationship of 3-DG levels with plasma glucose or HbA1c levels and examined changes in 3-DG levels after glycemic control in the six patients. RESULTS: Plasma 3-DG was significantly more increased in diabetic patients than in nondiabetic control subjects (31.8 +/- 11.3 vs. 12.8 +/- 5.2 ng/ml, means +/- SD, P < 0.001), but there was an approximately threefold difference in 3-DG levels among diabetic patients. 3-DG levels were well correlated with plasma glucose (r = 0.56, P < 0.005) and HbA1c levels (r = 0.74, P < 0.001) in diabetic patients. The improvement of hyperglycemia in six patients resulted in a significant decrease in 3-DG (35.2 +/- 13.2 vs. 21.3 +/- 3.4 ng/ml, P < 0.05). CONCLUSIONS: The results indicate that the plasma glucose level is a predominant determinant of the plasma 3-DG level in diabetic patients and good glycemic control would be important to reduce this reactive metabolite.

Adult↗
PubMed1997

[Study on the evaluation of total and regional liver function using 99mTc-GSA dynamic SPECT].

To evaluate regional and total liver function, 99mTc-GSA dynamic SPECT was studied in 58 patients with various liver disease and 5 normal volunteers. Using dual-head gamma camera, 60 projection data (90 seconds/rotation) were acquired to obtain SPECT imaging. Forty continuous SPECT were obtained for 60 minutes. Time activity curve (TAC) of each voxel was created. The TAC was assumed to be equally the function of C(t) = Cmax (l-e-kt). The liver uptake rate (K value) was calculated using the least squares method. The product of K value and functional volume of each voxel was defined as liver functional index and the sum of the indices was defined as total liver functional index. The total liver functional index had good correlation with various liver function test, HH15, LHL15, and the index showed significant difference between each group of Child-Pugh's liver dysfunction stage. Because of unnecessariness of setting up the ROI, this method is simple and the result is no difference among operators. Three-dimensional liver functional index map exactly shows regional liver function. So that, this method seems to be useful for predicting the residual liver function after hepatectomy.

Adult↗