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Biomedical subjects

H Fujisawa

Publications and source records attributed to H Fujisawa.

At least 217 records · Page 12Linked to original sources

Growth-associated expression of a membrane protein, neuropilin, in Xenopus optic nerve fibers.

Neuropilin (previously referred to as A5) is a neuronal cell surface protein which is widely distributed in such vertebrate species as Xenopus, chicken and mouse. In these vertebrate species, neuropilin is expressed in particular neuron circuits at particular developmental stages when axonal growth is active. To test whether the expression patterns of neuropilin is associated with axonal growth, we examined expression patterns of neuropilin in developing and regenerating Xenopus optic nerves. In embryos, a neuropilin-specific monoclonal antibody A5 (MAbA5) bound strongly to the optic nerves, and in situ hybridization signals for neuropilin mRNA were prominent in the retinal ganglion cells (RGCs), indicating that developing optic nerve fibers express neuropilin. The binding of MAbA5 to the optic nerve fibers was maximal at stages 41-43, then decreased in the subsequent tadpole life. In tadpoles after stage 50, the binding of MAbA5 to the optic nerves was extremely weak or almost nil, although RGCs expressed considerable amounts of neuropilin mRNA. When the tadpole optic nerves were crushed and prompted to regenerate, however, neuropilin protein reappeared in the optic nerve fibers. The binding of MAbA5 to the regenerating optic nerve fibers was detectable as early as the 5th day and maximal at the 2nd or 3rd weeks after the optic nerve crush, and declined thereafter. These findings suggest that the expression of neuropilin in the optic nerve fibers is regulated in an axonal growth-associated manner.

Animals↗

Overexpression of a membrane protein, neuropilin, in chimeric mice causes anomalies in the cardiovascular system, nervous system and limbs.

Neuropilin is a type 1 membrane protein, which is highly conserved among Xenopus frog, chicken and mouse. The extracellular part of the neuropilin protein is composed of three unique domains, each of which is thought to be involved in molecular and/or cellular interactions. In mice, neuropilin is expressed in the cardiovascular system, nervous system and limbs at particular developmental stages. To clarify the roles of neuropilin in morphogenesis in vivo, we generated mouse embryonic stem (ES) cell clones that constitutively expressed exogenous neuropilin, then produced chimeras using these ES cell clones. The chimeras overexpressed neuropilin and were embryonic lethal. The chimeric embryos exhibited several morphological abnormalities; excess capillaries and blood vessels, dilation of blood vessels, malformed hearts, ectopic sprouting and defasciculation of nerve fibers, and extra digits. All of these abnormalities occurred in the organs in which neuropilin is expressed in normal development. The variety of abnormalities occurring in these chimeric embryos suggested diverse functions of neuropilin in embryonic morphogenesis, which may be ascribed to multiple interaction domains identified in the molecule. Correct spatiotemporal expression of neuropilin seems to be essential for normal development of the cardiovascular system, nervous system and limbs.

Animals↗

[Antigenicity study on montirelin hydrate (NS-3)].

An antigenicity study of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in Hartley guinea pigs. Animals were sensitized twice by subcutaneous injections of montirelin hydrate in combination with Freund's complete adjuvant and twice by intramuscular injections of montirelin hydrate alone. Montirelin hydrate was found to be negative in the homologous passive cutaneous anaphylaxis test carried out with sera from the sensitized guinea pigs. Negative results were also obtained in the active cutaneous anaphylaxis test and the active systemic anaphylaxis test in guinea pigs sensitized with montilerin hydrate. These results show that montirelin hydrate has no antigenicity under the present experimental conditions.

Animals↗

[Acute pulmonary embolism after radical operation for renal cell carcinoma with vena caval extension: a case cured by thrombolytic therapy].

A 54-year-old man was hospitalized for a right renal tumor with intraluminal extension into the vena cava. He underwent radical nephrectomy with thrombectomy and regional lymphadenectomy. On the 8th postoperative day, he suddenly complained of dyspnea with tachypnea and cyanosis. Arterial blood gas analyses under an oxygen flow of 4L/min revealed PaO2 32.1 mmHg. Pulmonary angiography revealed filling defects in the right main pulmonary artery and left descending branch. Under the diagnosis of acute pulmonary embolism, thrombolytic and anti-coagulation therapy was performed and the patient recovered from the disease. We should be aware of pulmonary embolism as a postoperative complication of urological surgery.

Acute Disease↗

Differential expression of two cell surface proteins, neuropilin and plexin, in Xenopus olfactory axon subclasses.

Immunohistochemistry by using monoclonal antibodies named A5 and B2, which specifically recognize cell surface proteins the neuropilin and the plexin, respectively, revealed that olfactory axons in Xenopus tadpoles were classified into several subgroups by virtue of the expression levels of these two cell surface molecules. The vomeronasal axons expressed the plexin but not the neuropilin. The plexin-positive and neuropilin-negative vomeronasal axons form a discrete fiber bundle, even after they joined with the principal olfactory axons. However, the principal olfactory axons were divided into at least two subclasses; the neuropilin-predominant axons which expressed high levels of the neuropilin and low levels of the plexin, and the plexin-predominant axons which expressed high levels of the plexin and low levels of the neuropilin. Within the olfactory nerve the pathways for these two principal olfactory axon subclasses were initially intermingled with each other, but were gradually segregated throughout their courses from the nose to the cerebrum. Eventually, the neuropilin-predominant and the plexin-predominant principal olfactory axon subclasses projected to specified glomeruli in topographically related regions within the main olfactory bulb. Neuroanatomical tracings of the olfactory projection also confirmed the gradual segregation of the pathways for the principal olfactory axons. These results allow us to speculate that both the neuropilin and the plexin are involved in axon interactions, and play roles in the organization of the precise patterns of the olfactory pathway and projection.

Animals↗

Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. We have examined here whether Rabphilin-3A is phosphorylated by calmodulin-dependent protein kinase II (CaMKII). Recombinant Rabphilin-3A was phosphorylated by CaMKII purified from rat brain. Two moles of phosphate were maximally incorporated into one mole of Rabphilin-3A. The phosphorylation sites were Ser34, Thr205, Thr209, and Thr537. These results suggest that the CaMKII-catalyzed phosphorylation of Rabphilin-3A may modulate neurotransmitter release.

Adaptor Proteins, Signal Transducing↗

Staurosporine induces the outgrowth of neurites from the dorsal root ganglion of the chick embryo and PC12D cells.

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40-100 nM) seemed to have different characteristics, possibly as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contrast microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.

Alkaloids↗

Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.

Adenosine Triphosphatases↗

Massive astrocytic swelling in response to extracellular glutamate--a possible mechanism for post-traumatic brain swelling?

Little attention has been paid to the responses of astrocytes in the brain to the application of neurotoxic excitatory transmitters such as glutamate. We have developed a simple model to study the responses of cells within the cerebral cortex to neurotoxic levels of glutamate. After short periods of perfusion with glutamate, perivascular and interstitial astrocytes swell and become electron lucent. The astrocyte swelling extends, with increasing periods of perfusion, up to 400 um into the adjacent, intact neuropil. After 90 minutes of glutamate perfusion, intermediate filament bundles and glycogen granules occur within the astrocyte cytoplasm. We obtained no evidence for compromised blood flow. We suggest that astrocyte swelling serves to limit the diffusion of glutamate from the site of the lesion.

Animals↗

Focal microvascular occlusion after acute subdural haematoma in the rat: a mechanism for ischaemic damage and brain swelling?

Using a rat model of subdural haematoma (SDH) which is associated with ischaemic damage in the ipsilateral hemisphere, we have studied the microcirculation and the time course of development of ischaemia beneath the subdural haematoma. The latter was investigated by measuring cerebral blood flow (CBF) using the hydrogen clearance technique, the former by electron microscopy (EM) and carbon black angiography. CBF fell below 5 ml/100g/min within five minutes of SDH and remained low (< or = 7 ml/100g/min) for over two hours. Contralateral CBF fell to 54% of control but normalized by 1 hour. Carbon black angiography showed absent perfusion beneath the SDH. EM demonstrated: 1) normal cortical vessel traversing the haematoma; 2) occlusion of cortical vessels under SDH by clotted red cells and platelets; 3) massively enlarged perivascular spaces due to swelling of astrocytes. No vasospasm was seen. The "ischaemic core" may develop where microvascular occlusion occurs, and this microcirculatory disturbance initiates ischaemic cell damage with release of glutamate, which spreads outward from the core. This causes further damage of neurons and swelling of astrocytes, resulting in severe ischaemic necrosis, and progressive brain edema. The cause of microvessel occlusion remains unknown.

Animals↗

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes, neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland.

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intra-adrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Adrenal Glands↗

Effect of thermal stress on glutathione metabolism in human erythrocytes.

This is the first experiment to investigate the effect of heat and cold stress on glutathione metabolism in human erythrocytes. We immersed men at three different water temperatures for 10 min. At 39 degrees C, no remarkable changes were observed. Levels of glutathione (GSH) decreased from 2.44 (0.14) to 1.80 (0.10) mumol.ml red blood cells-1 [mumol.ml RBC-1; mean (SEM); P < 0.0005] and those of lipid peroxides increased from 1.87 (0.03) to 2.06 (0.04) nmol.ml RBC-1 (P < 0.01) after the immersion at 42 degrees C. In contrast, levels of GSH increased from 2.46 (0.17) to 2.91 (0.17) mumol.ml RBC-1 (P < 0.05) and those of lipid peroxides did not change after the immersion at 25 degrees C. The activities of glutathione peroxidase decreased from 35.90 (1.83) to 34.33 (1.66) IU.g Hb-1 (P < 0.01) after the immersion at 42 degrees C; however, these activities did not change after the immersion at 25 degrees C. The activities of glutathione reductase (both active and inactive forms) showed no changes at any temperatures. These changes indicate that heat stress causes oxidative stress in the human body; however, cold stress is thought to augment the activity of the antioxidative defence system. It is suggested that body exposure to hot environmental conditions should not be recommended for patients suffering from a damaged antioxidative defence system.

Adult↗

Prognostic implications of meningiomas in the elderly (over 70 years old) in the era of magnetic resonance imaging.

During the 5 years from 1987 to 1991, 89 elderly patients, aged 70 years and over, were admitted to departments of neurosurgery in Yamaguchi prefecture with meningioma. The clinical features and prognostic implications of meningioma in the elderly were assessed retrospectively. Seventy-eight (88%) of the 89 patients underwent surgery, which was a higher rate than has been previously reported. The length of clinical history was also shorter than in previous studies, and was partly due to the recent introduction of magnetic resonance imaging (MRI). The incidence of poor prognosis (severe disability, vegetative or dead) in the elderly and a younger group aged less than 70 years was 13% and 7%, respectively, but the difference was not statistically significant. In the surgically treated elderly group, age did not influence the patient's outcome. The factors affecting the outcome were pre-operative neurological deficit (p < 0.05), histological malignancy (p < 0.05), and multiple operations (p < 0.05). Twenty-seven of the elderly meningioma patients were in good physical condition with minimal neurological involvement. They underwent total removal of the tumour at the first operation, and the histological diagnosis was benign. Twenty-five of these 27 patients fell into the best outcome category. Therefore, age alone was not a factor preventing proper surgical treatment of meningioma in the elderly.

Aged↗

Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines.

Expression of nitric oxide synthase (NOS) was studied in nine human neuroblastoma and two human glioblastoma cell lines. Neuronal NOS (n-NOS) mRNA of approximately 10 kb was detected in four of the nine neuroblastoma cell lines by northern blot analysis using human n-NOS cDNA as a probe. Expression of the n-NOS mRNA was also detected in another neuroblastoma cell line in a subsequent reverse transcriptase polymerase chain reaction (RT-PCR) study, but no n-NOS mRNA expression was observed in the other four neuroblastoma cell lines or in the glioblastoma cell lines. The level of NOS activity correlated well with that of n-NOS mRNA expression in neuroblastoma cell lines expressing n-NOS mRNA. Western blot analysis showed that the n-NOS expressed in neuroblastoma cells was a 160-kDa protein reacted with anti-n-NOS antibody. By using the RT-PCR method, a short n-NOS (n-NOS-2) mRNA with a 315-bp inframe deletion from the entire n-NOS (n-NOS-1) mRNA was detected in the human neuroblastoma cells. The structural diversity of human n-NOS mRNA was demonstrated for the first time.

Amino Acid Oxidoreductases↗

A novel method for generating nested deletions using the in vitro bacteriophage T3 DNA packaging system.

To sequence a DNA segment inserted into a cosmid vector under the directed sequencing strategy, we established a simple and rapid method for generating nested deletions which uses the in vitro packaging system of bacteriophage T3 DNA. The principle is based on the previous finding that this system can translocate any linear double-stranded DNA up to 40 kb into the phage capsid in a time-dependent manner and the encapsulated DNA becomes DNase-resistant. For this purpose, we constructed a cosmid vector that carries two different antibiotic selection markers at both sides of the multiple cloning site, and after insertion of a DNA segment, the clone was linearized by lambda-terminase at the cos site. After the packaging reaction in vitro followed by DNase treatment, the encapsulated DNA was introduced into Escherichia coli cells to give clones with unidirectional deletions by differential antibiotic selection. Restriction and sequence analyses of deletion clones demonstrated that an ordered set of clones with nested deletions, ranging from less than 1 kb to 25 kb, was created from either the end of the DNA segment. Thus, nested deletion clones that cover the entire region of a approximately 40-kb cosmid insert can be obtained by a single packaging reaction, and its restriction map can be simultaneously obtained.

Bacteriophage T3↗

cDNA cloning and expression of human calmodulin-dependent protein kinase IV.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) is a Ca(2+)-responsive multifunctional protein kinase which occurs abundantly in the brain and thymus. A human cDNA clone encoding CaM-kinase IV was isolated from a Jurkat cell cDNA library and its nucleotide sequence was determined. The cDNA sequence encoded a protein consisting of 473 amino acids with a molecular weight of 51,925. The nucleotide sequence for the coding region and the deduced amino acid sequence showed 81 and 80% identities with those of the rat enzyme, respectively. Western blot analysis, using a polyclonal antibody raised against the recombinant human CaM-kinase IV, which was expressed in Escherichia coli, revealed two bands corresponding in mobility to molecular weights of 60,000 and 61,000, respectively, in a Jurkat cell extract. The antibody also cross-reacted with both isoforms of CaM-kinase IV from rat cerebellum, the apparent molecular weights being 62,000 and 64,000, respectively.

Amino Acid Sequence↗

Inactivation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin.

Incubation of calmodulin-dependent protein kinase II with Ca2+ and calmodulin resulted in a marked inactivation of the enzyme. Chelation of Ca2+ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation. The concentration required for the half-maximal inactivation, 127 microM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme. The Ca2+/calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca2+ and calmodulin. These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme. The inactivation by Ca2+/calmodulin was temperature-dependent. The addition of both 500 microM ADP and 10 mM MgCl2 markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone. The addition of 5 microM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr286/Thr287 in alpha/beta, gamma, and delta isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca2+/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 microM.

Animals↗

Purification and characterization of Ca2+/calmodulin-dependent protein kinase IV kinase from rat brain.

Calmodulin-dependent protein kinase IV (CaM-kinase IV) kinase was recently discovered in the rat brain by its activity to activate the inactive recombinant CaM-kinase IV expressed in Escherichia coli [Okuno, S. and Fujisawa, H. (1993) J. Biochem. 114, 167-170]. In the present study, CaM-kinase IV kinase was purified approximately 2,000-fold from rat cerebral cortex by purification procedures including calmodulin affinity chromatography, and its properties were examined. The highly purified CaM-kinase IV kinase gave one major protein band corresponding to a molecular weight of about 66,000 upon SDS-PAGE. The purified CaM-kinase IV kinase phosphorylated and concomitantly activated CaM-kinase IV purified from rat brain as well as the recombinant kinase expressed in Escherichia coli in a Ca2+/calmodulin-dependent manner. The phosphorylation of CaM-kinase IV by CaM-kinase IV kinase occurred on only serine residue(s). Among a number of proteins, including several known to be phosphorylated by the various protein kinases tested, CaM-kinase IV was the best substrate for CaM-kinase IV kinase. Since syntide-2, a synthetic peptide known to be a good peptide substrate for calmodulin-dependent protein kinase II (CaM-kinase II), was a fairly good substrate for CaM-kinase IV kinase, some kinetic properties of CaM-kinase IV kinase were examined using syntide-2 as a substrate. The Km value for the peptide substrate in the presence of Ca2+/calmodulin was almost two orders of magnitude lower than that in its absence, although the Vmax value was almost the same in the presence and absence of Ca2+/calmodulin.

Amino Acid Sequence↗