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Biomedical subjects

H Fujisawa

Publications and source records attributed to H Fujisawa.

At least 181 records · Page 10Linked to original sources
PubMed1996

Patterns of increased glucose use following extracellular infusion of glutamate: an autoradiographic study.

An apparent transient increase in local glucose utilization has been demonstrated in certain brain areas after global and focal ischemia in several models. A coincident transient increase in extracellular glutamate has been shown in the same brain regions in many of these models. To test the hypothesis that an increase in metabolism is an important component of the excitotoxic effect of glutamate, we perfused glutamate at different concentrations (0.01, 0.1, 0.5, 1 M) into the extracellular space, and performed 2-deoxyglucose autoradiography after 90 min of infusion. Furthermore, we infused 14C-labeled glutamate to investigate its diffusion characteristics within the brain using autoradiographic methods. Glutamate at 0.5 and 1 M concentration caused large consistent areas of brain damage with all the histological features of acute infarction, although ischemia does not occur in this model. Glucose utilization was significantly increased (115 +/- 20 vs. 56 +/- 13 mumol/100 g/min in controls p < 0.01) in a sharply demarcated concentric zone, at the boundary between histologically damaged and normal brain, suggesting that viable cells not yet destroyed by glutamate respond by increased glucose metabolism. [14C] Glutamate diffused into the brain in a dose-dependent manner, and the pattern of its diffusion corresponded closely to that of the histological lesion and the zone of increased glucose uptake. We speculate that the increase in glucose use, which is not caused by ischemia in this model, is due to a metabolic response to glutamate and may be due to attempts to restore ionic homeostasis or repair cell damage.

Animals↗
PubMed1996

Evidence for the existence of Ca2+/calmodulin-dependent protein kinase IV kinase isoforms in rat brain.

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous system and immune system, is markedly activated upon phosphorylation by the action of CaM-kinase IV kinase. Northern and Western blot analyses of CaM-kinase IV kinase showed relatively weak reactions in the rat cerebellum, where the activity of CaM-kinase IV kinase has been demonstrated to exist, indicating that CaM-kinase IV kinase isoforms distinct from the enzyme cloned from the cerebral cortex may exist in the cerebellum. When the crude extracts of rat cerebral cortex, brain stem, and cerebellum were immunotitrated with antibody against the cloned enzyme, only approximately 46, 56, and 25% of the enzyme activity of the respective extracts were immunoprecipitated. Thus, at least two distinct isoforms of CaM-kinase IV kinase appear to exist in the brain.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a novel protein containing the association domain of calmodulin-dependent protein kinase II.

The cDNA of a novel protein, which contains the association domain of alpha isoform of calmodulin-dependent protein kinase II (CaM-kinase II alpha), was cloned from rat skeletal muscle. This protein, called alpha KAP, consisted of 200 amino acid residues with a molecular weight of 22,583. alpha KAP has a highly hydrophobic amino-terminal stretch of 25 amino acids which is absent from CaM-kinase II alpha, suggesting that this protein is either a secretory protein or an integral membrane protein. Northern blot analysis with a probe specific for alpha KAP detected three distinct mRNA species of 4.0, 2.4, and 1.5 kb in rat skeletal muscle. The 4.0- and 2.4-kb RNAs were also detected in heart, and at much lower levels in lung, kidney, and testis. Western blot analysis, using antibody raised against a synthetic peptide corresponding to the carboxyl-terminal 15 amino acids, revealed a single band corresponding in mobility to a molecular weight of 21,000 in crude extracts of both rat skeletal muscle and bacteria transformed with the cDNA, suggesting that no significant post-translational modification, such as excision of the amino-terminal hydrophobic segment, occurred. This, together with the fact that alpha KAP was recovered in the high-speed pellet in skeletal muscle, indicated that this protein may be an integral membrane protein.

Amino Acid Sequence↗
PubMed1996

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.

Arabidopsis↗
PubMed1996

SGR1, SGR2, SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana.

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1-1, sgr2-1, and sgr3-1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1-1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2-1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3-1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.

Arabidopsis↗
PubMed1996

Hyporesponsiveness in contact hypersensitivity and irritant contact dermatitis in CD4 gene targeted mouse.

To determine the role of CD4 molecules in the generation and regulation of contact hypersensitivity (CHS), we treated mice lacking the CD4 gene as a result of targeted disruption with dinitrofluorobenzene to induce CHS. The mutant mice lacking CD4 (CD4(-) mice) showed marked hyporesponsiveness in CHS compared with normal syngeneic C57BL/6 mice (38.3 +/-9.0% of normal at 24 h after the challenge assessed by net ear swelling; p < 0.025). CD4(-) mice had a larger CD4-8- double negative T-cell receptor alpha beta+ cell population in the lymph nodes than did normal mice, and the increase of this cell population was observed in CD4(-) mice after sensitization. Draining lymph node cells from sensitized normal mice restored the responsiveness in CD4(-) mice, but those from sensitized CD4(-) mice were less effective in restoring the CHS response in normal mice. Langerhans cell numbers were normal, and function, as assessed by the ability to present soluble hapten, was not impaired in CD4(-) mice. Skin cytokine profiles demonstrated an increase in interferon-gamma, interleukin-2, and interleukin-4 mRNA levels after challenge in normal mice, whereas this response was blunted in CD4(-) mice. CD4(-) mice also showed hyporesponsiveness in inflammatory reaction to irritant chemicals. These results suggest that the CD4 molecule is required for optimal induction of CHS as well as irritant contact dermatitis and may influence the development of CHS by modulating the cytokine profiles in the skin.

Animals↗
PubMed1996

Tumour necrosis factor receptor II (p75) signalling is required for the migration of Langerhans' cells.

Langerhans' cells (LC) represent the major antigen-presenting cells within the epidermis. Following exposure of the skin to antigen, LC take up antigen, migrate into draining lymph nodes (DLN) and present processed antigen to T lymphocytes, thereby initiating an immune response. The molecular mechanisms responsible for LC migration remain unclear. Cytokines, in particular tumour necrosis factor-alpha (TNF-alpha) have been suggested to influence LC migration. There are two distinct membrane receptors for TNF-alpha, TNF receptor I (TNF-R1, p55) and TNF receptor II (TNF-R2, p75), thought to be responsible for distinct TNF-alpha activities. It is believed that most of TNF biological activities are mediated via TNF-R1. In order to examine the role of TNF-R1 signalling in LC migration, we utilized TNF-R1 gene-targeted mutant mice. Following application of the hapten fluorescein isothiocyanate (FITC), FITC-bearing cells in DLN were examined by flow cytometry. A normal number of FITC+/Ia+ cells (LC) were found in DLN from TNF-R1-deficiency mice, suggesting that TNF-R1-dependent signalling is not crucial for LC migration. To investigate the possibility of signalling through TNF-R2, blocking studies using a neutralizing anti-TNF-alpha antibody were performed. The results revealed that anti-TNF-alpha antibody significantly inhibited LC accumulation in DLN in TNF-R1-deficient mice, thus suggesting that TNF-R2 signalling is involved in LC migration from skin to DLN and that murine LC express TNF-R2.

Animals↗
PubMed1996

Effects of UVB irradiation on keratinocyte growth factor (KGF) and receptor (KGFR) expression in cultured human keratinocytes.

Keratinocyte growth factor (KGF) and its receptor (KGFR) are thought to play important roles in normal keratinocyte growth and differentiation. Since UVB radiation is known to influence keratinocyte growth, we sought to determine whether UVB would alter the expression of KGF and KGFR. Using a reverse-transcription coupled polymerase chain reaction (RT-PCR), the present study examined the expression of KGF and KGFR mRNA in cultured normal human keratinocytes exposed to UVB irradiation. Total cellular RNA was extracted from cultured keratinocytes at various time points after irradiation, reverse transcribed and used for PCR amplification using primers specific for KGF and KGFR. Constitutive expression of KGFR mRNA, but not KGF mRNA, was detected in normal cultured human keratinocytes. After UVB irradiation at 300 J/m2, the KGF mRNA remained undetectable while the KGFR mRNA level was significantly decreased. The down-regulation of KGFR mRNA expression was also confirmed by Northern blot analysis. Immunohistochemical studies demonstrated a decreased positive signal of KGFR in human keratinocytes after UVB irradiation. Our results suggest a possible role for the KGF-KGFR signalling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of keratinocytes in hyperproliferative skin disorders may be related to downregulation of KGFR.

Cells, Cultured↗
PubMed1996

Differential distribution of CaM kinases and induction of c-fos expression by flashing and sustained light in rat retinal cells.

PURPOSE: To examine the expression of c-fos proto-oncogene and phosphorylation of cAMP responsive element binding (CREB) protein in the rat retina after changes in the light-dark condition. METHODS: Rats were exposed to both steady light and flashing light and were killed at the end of light exposure. The retinas were analyzed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using phosphoSer-133 specific CREB antiserum, anti-calcium calmodulin dependent protein (CaM) kinase II, and anti-CaM kinase IV. RESULTS: c-fos mRNA was expressed in the outer half of the inner nuclear layer (INL) and in the ganglion cell layer (GCL) after 30 minutes of sustained light. After 30 minutes of flashing light, c-fos expression also was detected in the inner border of the INL. Phosphorylated CREB immunoreactive nuclei had similar distribution after steady and flashing light. Both CaM kinase II and CaM kinase IV, which phosphorylate CREB at Ser 133 in vitro, were expressed in the GCL and in the INL. CaM kinase II, however, was localized in the inner border of the INL, whereas CaM kinase IV was distributed in the outer half of the INL. CONCLUSIONS: These results suggest that the differential expression of c-fos mRNA induced by flashing and sustained light may reflect the CREB phosphorylation by CaM kinases in a different subpopulation of retinal cells.

Animals↗
PubMed1996

Magnetic resonance imaging of thymic epithelial tumors.

The authors review their experience with magnetic resonance imaging (MRI) of the thymus and discuss the appearance of thymic epithelial tumors where MRI is clinically useful. Detailed descriptions of MRI findings in benign thymomas, invasive thymomas, and thymic carcinomas are provided. Most benign (noninvasive) thymomas appear with a slightly higher signal intensity than that of muscle on T1-weighted images. On T2-weighted images, thymomas have an increase in signal intensity on both T1- and T2-weighted images. Neither fibrous septa nor lobulated internal architecture can be detected on MRI. Gd-DTPA-enhanced MR images show homogeneous enhancement. On the other hand, invasive thymomas show the same signal intensity as benign thymomas, both on T1- and T2-weighted images. However, invasive thymomas appear inhomogeneous in signal intensity on T2-weighted images. T2-weighted images also show a lobulated border, fibrous septa, and lobulated internal architecture, characteristic of most invasive thymomas. Irregularity of tumor margins indicating invasion into surrounding structures is noted in some cases of invasive thymomas. Exceptionally minute thymomas (< 1 cm in diameter) show a different signal intensity on MRI as compared to those of usual thymomas: both T1- and T2-weighted MR images show a low signal intensity mass with irregular or unclear borders. Histopathologically, these minute thymomas contain numerous tiny cysts and/or abundant collagenous tissues. Generally, thymic carcinomas, except carcinoid tumors, appear with a relatively low signal intensity on T1- and T2-weighted MR images in comparison to those of thymomas. In particular, well-differentiated squamous cell carcinomas appear with a low signal intensity on both T1- and T2-weighted images. Abundant collagenous tissue may be a causative factor for the low signal intensity on T2-weighted MR images. Thymic carcinomas appear slightly inhomogeneous on both T1- and T2-weighted images. Neither fibrous septa nor lobulated internal architecture can be detected in any thymic carcinoma. If MRI is performed on a patient with anterior mediastinal tumors, thymic carcinoma may be precisely diagnosed when characteristic MR findings are demonstrated.

Adult↗
PubMed1996

[Far lateral lumbar disc herniation: clinical and radiographical features of three cases].

The authors report three operated cases of far lateral lumbar disc herniation (FLLDH) during the past two years and discuss their diagnostic pitfalls. Until recently FLLDH was hardly ever diagnosed because the myelography was negative in almost all cases. Since the advent of CT and/or MRI, FLLDH has been found to be not such a rare entity. FLLDH has also been found to reveal characteristic clinical features and radiographical findings. Usual lumbar disc herniations occur at L4/5 or L5/S1 levels, producing low back pain with the pain or sensory disturbance from the posterolateral thigh down to the foot. In contrast, FLLDH affects upper lumbar levels and produces severe anterolateral thigh pain, dysesthesia resulting from nerve root or dorsal root ganglion (DRG) compression in the foraminal or extraforaminal region. The level predilection of these two groups can be attributed to the difference of the facet joint planes between the upper and lower lumbar levels. The facets with a coronal plane are resistant to lateral bending and rotational forces, but those with a sagittal plane are unstable resulting in more shearing stress to the intervertebral discs. A patient with definite neurological signs but a negative myelography should be examined for FLLDH by using a high-resolution CT or MRI. MRI clearly shows the detailed anatomical relationships between herniated disc and nerve root or DRG in the foraminal and extraforaminal regions. As well as thin-sliced axial images, sagittal MR images that include the foraminal zone are useful for detecting a direct nerve root compression from FLLDH. The authors conclude that gait disturbance due to severe leg pain, antero-lateral thigh pain or dysesthesia are characteristic of FLLDH, and that either a foraminal or extraforaminal herniated disc or both on a CT and/or MRI are diagnostic radiographical findings of FLLDH.

Adult↗
PubMed1995

Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2+ mobilization.

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca2+ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca2+ mobilization. Western blot analysis revealed that the microsomes contain the alpha isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca2+ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca2+ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca2+ release.

Adenosine Diphosphate Ribose↗
PubMed1995

Effect of gene-targeted mutation in TNF receptor (p55) on contact hypersensitivity and ultraviolet B-induced immunosuppression.

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic proinflammatory cytokine. TNF-alpha has been implicated in the pathogenesis of delayed-type hypersensitivity reactions such as allergic contact hypersensitivity and has been suggested as a mediator of ultraviolet B (UVB)-induced immunosuppression. Conflicting reports, however, exist concerning the effects of TNF-alpha on contact hypersensitivity (CHS). To determine the role of TNF-alpha in the generation and regulation of CHS, gene-targeted mutant mice lacking TNF-receptor (p55) gene (TNF-R1(-) mice) were treated with dinitrofluorobenzene (DNFB) to induce CHS. TNF-R1(-) mice showed significant hyperresponsiveness in CHS (152.8 +/- 20.9%, p < 0.025) compared with normal syngeneic mice (C57BL/6) assessed by ear swelling. To determine whether UVB can induce suppression in TNF-R1(-) mice, mice were irradiated on the shaved abdomen with 96 mj/cm2 UVB and 3 days later they were painted with 0.5% DNFB (sensitization dose), followed 5 days later with 0.2% DNFB to the left ear (challenge dose). Significant suppression of CHS was observed both locally (sensitization on irradiated site) and systemically (sensitization on unirradiated site) in UVB-irradiated TNF-R1(-) mice as well as in normal mice. To rule out possible signaling through p75 TNF-R, the mice were treated with anti-TNF-alpha Ab (V1q), which can neutralize any TNF effects through either receptor. V1q had no effect on these phenomena observed in TNF-R1(-) mice. These results suggest that TNF-alpha plays a regulatory role in CHS but is not required to induce UVB-mediated immunosuppression.

Animals↗
PubMed1995

Analysis of the fine structure of the prohead binding domain of the packaging protein of bacteriophage T3 using a hexapeptide, an analog of a prohead binding site.

A large subunit of bacteriophage T3 packaging enzyme, a product of gene 19 (gp19, 586 amino acid residues), binds a prohead prior to DNA translocation in DNA packaging. Its C-terminal region (571 to 576, Region I) is of crucial importance for prohead binding. To elucidate the functional role(s) of Region I in DNA packaging, a hexapeptide (6pT3) corresponding to the Region I sequence and its variants were synthesized and their effects on DNA packaging in a defined in vitro system were examined. 6pT3 did not inhibit gp19wt (wild type)-prohead binding but interfered with their functional interaction, resulting in inhibition of DNA packaging. The inhibitory effect of 6pT3 on gp19wt was reversible. The effect of 6pT3 was examined with gp19 delta C10, which was active in DNA packaging in spite of lacking the extreme C-terminal 10 amino acids (Region II). The inhibitory effect on gp19 delta C10 was more severe than that on gp19wt and was irreversible. From these results, we concluded that the prohead binding domain is composed of two subdomains: Region I is a "core" domain, and its binding to the prohead is crucial for DNA packaging, and Region II is an "anchor" domain stabilizing the binding by Region I.

Amino Acid Sequence↗
PubMed1995

A novel highly specific and potent inhibitor of calmodulin-dependent protein kinase II.

A novel synthetic peptide AIP (autocamtide-2-related inhibitory peptide), a nonphosphorylatable analog of autocamtide-2, was found to be a highly specific and potent inhibitor of calmodulin-dependent protein kinase II (CaM-kinase II). It was 50 and 500 times more potent than CaMK-(281-302Ala286) and KN-93, respectively, under the assay conditions used. The inhibition was unaffected by the presence or absence of Ca2+/calmodulin, and it was competitive with autocamtide-2 and noncompetitive with syntide-2. AIP (1 microM) completely inhibited CaM-kinase II activity, but did not affect cyclic AMP-dependent protein kinase, protein kinase C, calmodulin-dependent protein kinase IV, and unidentified protein kinases occurring in a rat brain extract. These results indicate that AIP is a useful tool for studying the physiological roles of CaM-kinase II.

Amino Acid Sequence↗
PubMed1995

Characterization of c-kit-positive neurons in the dorsal root ganglion of mouse.

Previously, we showed that c-kit receptor tyrosine kinase is expressed by a subpopulation of dorsal root ganglion (DRG) neurons, and that the ligand for the c-kit receptor, stem cell factor (SCF), induces the neurite outgrowth and supports the survival of these neurons in culture [16]. However, it is unknown which class of DRG neurons express c-kit receptor and which factor regulates differentiation and survival of c-kit-positive neurons. In the present study, we attempted to characterize c-kit positive neurons in the mouse DRG. The c-kit-positive neurons were small or medium in size, and 44% of these neurons contained substance P. Central fibers of the c-kit-positive neurons terminated in laminae I and II of the gray matter of the spinal cord. These results suggest that c-kit-positive neurons in the DRG belong to a functional subpopulation. The c-kit receptor protein was presented on the membrane of processes and growth cones in neurons. When DRG cells of embryonic day 15.5 or 17.5 were cultured, the survival of c-kit-positive neurons was supported by SCF, nerve growth factor (NGF) or leukemia inhibitory factor. SCF and NGF synergistically supported the survival of c-kit-positive neurons at submaximal concentrations. c-kit-positive DRG neurons from neonatal mice survived without addition of any factor in culture, suggesting that the requirement for trophic support in c-kit-positive neurons changes during development.

Age Factors↗