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Biomedical subjects

Y Kamata

Publications and source records attributed to Y Kamata.

At least 37 records · Page 2Linked to original sources
PubMed2001

NGF induces neurite outgrowth via a decrease in phosphorylation of myosin light chain in PC12 cells.

The relationship between phosphorylation of myosin light chain (MLC) and neurite outgrowth induced by nerve growth factor (NGF) was studied in PC12 cells. Inhibitors of Rho kinase, HA-1077 or Y-27632 also induced neurite outgrowth. As already reported botulinum exoenzyme C3 which inactivates Rho protein also induced neurite outgrowth. Calyeulin A, an inhibitor of phosphatase counteracted both NGF- and C3- induced neurite outgrowth. Treatments of both NGF and C3 resulted in significant and transient decrease in phosphorylated MLC. These results suggest that NGF induces neurite outgrowth of PC12 by a transient decrease in phosphorylated MLC which is brought about by activation of MLC phosphatase via inhibition of Rho-Rho kinase pathway.

Animals↗
PubMed2001

Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography.

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.

Amino Acid Sequence↗
PubMed2001

Engraftment of genetically engineered amniotic epithelial cells corrects lysosomal storage in multiple areas of the brain in mucopolysaccharidosis type VII mice.

Cell-mediated gene therapy for visceral lesions of lysosomal storage diseases is promising; however, the treatment of central nervous system (CNS) lesions remains a challenge. In this study, we generated rat amniotic epithelial cells (AEC) that overexpress and secrete human beta-glucuronidase (GUSB) following transduction with an adenoviral vector encoding human GUSB. The AEC were used as donor cells for cell-mediated gene therapy of CNS lesions in mice with mucopolysaccharidosis type VII (MPSVII), a lysosomal storage disorder caused by an inherited deficiency of GUSB activity. After confirmation that the secreted GUSB was taken up mainly via mannose 6-phosphate receptors in primary cultured neurons, the AEC were transplanted into the brains of adult MPSVII mice. Histochemical analysis showed extensive GUSB activity throughout the ipsilateral hemisphere of the recipient brains, and pathological improvement of the lysosomal storage was observed even in regions far from the site of injection. These results suggest that intracerebral transplantation of genetically engineered AEC has therapeutic potential for the treatment of CNS lesions in lysosomal storage disorders.

Adenoviridae↗
PubMed2001

Adenovirus-mediated gene therapy for corneal clouding in mice with mucopolysaccharidosis type VII.

Recent advances in systemic treatments for mucopolysaccharidosis have led to therapies that improve the multiple somatic features of this disease, but the therapeutic effect on ocular manifestations such as corneal clouding is not satisfactory. Here, we administered an adenovirus expressing human beta-glucuronidase (AxCAhGUS) into the anterior chamber or intrastromal region of the cornea in mice with mucopolysaccharidosis type VII (B6/MPSVII), and successfully treated corneal clouding of MPSVII. When we injected AxCAhGUS into the anterior chamber of the eyes, cells expressing beta-glucuronidase (GUSB) were located mainly in the trabecular meshwork as well as in all corneal regions, and subsequent pathological corrections in the cornea were achieved. Widespread transgene expression was also observed when we administered AxCAhGUS inside the cornea after lamellar keratotomy, and rapid elimination of the lysosomal storage in the corneal keratocytes occurred. Furthermore, intrastromal vector administration did not generate significant levels of anti-adenovirus neutralizing antibodies, and secondary vector administration was effective. Based on these observations, we conclude that it is worth developing a treatment strategy for corneal clouding in mucopolysaccharidosis based on direct intraocular administration of adenoviral vectors.

Adenoviridae↗
PubMed2001

Effects of Ca2+ on flavin-linked complex enzymes in mitochondria isolated from eggs and embryos of sea urchin.

Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.

Animals↗
PubMed2001

Epithelial-myoepithelial carcinomas of the bronchus.

Epithelial-myoepithelial carcinomas are very rare in the lung, and little is known about the relationship of their histologic features to prognosis. We describe five primary pulmonary epithelial-myoepithelial carcinomas with details on clinical presentation, histology, and immunohistochemical profiles. We also reviewed the literature to detail further their prognosis. The patients' ages ranged from 33 to 57 years (average 51 years). The tumors were all endobronchial and the patients presented with symptoms or imaging features of airway obstruction. The tumors were completely resected; none showed nodal involvement. All five patients are alive and free of disease 4 months to 8 years (average 4.2 years) after surgery. Four tumors showed a mixed pattern of glands lined by a dual layer of cells and solid sheets of either spindle cells or clear cells, the glandular and solid components being present in variable proportions. The fifth tumor comprised purely spindle cells. The mitotic rate was <1/20 high power fields in both the glandular and spindle/clear cell components. In one case there was focal nuclear pleomorphism. The inner layer of the glands stained for cytokeratins and epithelial membrane antigen, and the outer layer for S-100 and smooth muscle actin. In one case the spindle cells stained for CD34. A review of published cases shows the majority of tumors behave in an indolent fashion, the rare aggressive tumors being predominantly myoepitheliomatous. Nevertheless, the term epithelial-myoepithelial carcinoma is preferred because of their malignant potential. A high mitotic rate, tumoral necrosis, and nuclear pleomorphism appear to be adverse prognostic factors.

Actins↗
PubMed2001

Transdermal drug delivery by electroporation applied on the stratum corneum of rat using stamp-type electrode and frog-type electrode in vitro.

Transdermal enhancement effects of electroporation applied only on the stratum corneum by two electrode types, the stamp-type electrode and the frog-type electrode, were investigated in vitro using excised rat skin. Carboxyfluorescein (CF) was selected as a model compound. The excised skin was set in a Franz type diffusion cell and a square wave electric pulse was applied to the stratum corneum under various electric pulse conditions. We determined the permeability of CF to the receptor compartment under these conditions. Voltage, electric pulse length, and number of electric pulses, were varied from 10 to 1000 V, 50 micros to 15 ms and 5 to 30 pulses, respectively. Flux rate was enhanced as the electric pulse condition strengthened. However, the maximum value was attained in the flux rate, above which no increase was observed despite strengthening of the electric pulse. Although at low electric pulses, the enhancement effect of the frog-type electrode was superior to that of the stamp-type electrode, the maximum flux rates were the same. These results indicate that electroporation on the stratum corneum using the stamp-type electrode or frog-type electrode, is useful for transdermal drug delivery.

Administration, Cutaneous↗
PubMed2000

Difference in hydrophobicity between botulinum type B activated and non-activated neurotoxins under low pH conditions.

We showed that botulinum type B activated neurotoxin with a di-chain structure became hydrophobic more quickly and extensively than did the non-activated toxin with a single-chain structure on low pH exposure. The activated toxin possessed 50-fold higher toxicity than did the non-activated type. The difference in the susceptibility to hydrophobic change may be one clue to answering the question of why the activated toxin possesses a higher toxicity than does the non-activated type.

Botulinum Toxins↗
PubMed2000

Relationship between ATP level and respiratory rate in sea urchin embryos.

In sea urchin embryos, the rate of respiration, as a result of electron transport through the mitochondrial respiratory chain, was enhanced after hatching without any change in the intrinsic capacity of electron transport in mitochondria. The increase in respiratory rate after hatching was accompanied by an evident decrease in intracellular adenosine triphosphate (ATP) concentration without any change in intracellular levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Adenosine triphosphate is proposed to fortify acceptor control of respiration at high concentrations and to reduce the respiratory rate even in the presence of ADP, the acceptor. The relationships between the respiratory rate and intracellular ATP concentration in embryos were the same as those in mitochondria isolated from embryos, obtained in the presence of ADP at the same concentration as in the embryos. Probably, the respiratory rate is enhanced after hatching because of the decrease in the level of ATP. In embryos kept in a medium containing adenosine, intracellular ATP concentration increased especially after hatching, without any change in the ADP level, and the respiratory rate after hatching was made as low as the rate expected, based on the relationships obtained on isolated mitochondria. The respiratory rate in embryos probably depends on intracellular ATP concentration, irrespective of the developmental stage in early development.

Adenosine Triphosphate↗
PubMed2000

Evidence for GABA(A) receptor agonistic properties of ketamine: convulsive and anesthetic behavioral models in mice.

UNLABELLED: We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) (GABA(A)) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABA(A) receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10-22) mg/kg. Even large anesthetic doses (100-150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABA(A) receptor agonist, muscimol (0.32-1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1-3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2-5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2-20 mg/kg), nor the GABA synthesis inhibitor, L-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABA(A) receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABA(A) receptors. IMPLICATIONS: We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the gamma-aminobutyric acid(A) receptor antagonist bicuculline. The gamma-aminobutyric acid(A) receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the gamma-aminobutyric acid synthesis inhibitor, L-allyglycine, did not. The effects of ketamine on the gamma-aminobutyric acid(A) receptors appear to correlate with its anesthetic actions.

Allylglycine↗
PubMed2000

Increased sensitivity to stress in spontaneous recurrence of methamphetamine psychosis: noradrenergic hyperactivity with contribution from dopaminergic hyperactivity.

The significance of increased sensitivity to stress associated with noradrenergic hyperactivity involving dopaminergic change in spontaneous recurrences of methamphetamine (MAP) psychosis (flashbacks) was examined. Plasma monoamine metabolite levels were assayed in 18 subjects with flashbacks who had been exposed to stressful events plus MAP-induced frightening psychotic symptoms (N = 11) or frightening psychotic symptoms alone (N = 7) during previous MAP use, in 15 nonflashbackers with a history of MAP psychosis, in 8 subjects with persistent MAP psychosis, and in 27 control subjects. Monoaminergic values were subjected to a square-root transformation, rendering the distribution normal. The numbers of stressful events (mostly threatening events) and frightening psychotic symptoms were significantly higher in the flashbackers than in the nonflashbackers. Factors triggering flashbacks were mild psychosocial stressors (mostly a mild fear of other people). During flashbacks, plasma norepinephrine levels increased, and the flashbackers, 11 of whom had experienced stressful events plus frightening psychotic symptoms, had an additional small increase in plasma levels of 3-methoxytyramine, which is indicative of dopamine release. Thus, threatening stressful events, together with MAP use, may induce noradrenergic hyperreactivity to subsequent mild stressors. Threatening, stressful events plus frightening psychotic symptoms may further induce increased dopamine release in response to mild stressors. Increased sensitivity to stress associated with noradrenergic hyperactivity involving increased dopamine release may have elicited memories of MAP psychosis related to frightening, stressful experiences. The increased sensitivity may be critical for the development of flashbacks.

Adult↗
PubMed2000

Use of electroporation to accelerate the skin permeability enhancing action of oleic acid.

Rat skin permeability after treatment by electroporation (newly developed frog type electrode, 100V, 10 pulses), oleic acid/propylene glycol (PG) and a combination of both were investigated using Fourier transformed infrared attenuated total reflectance (FT-IR/ATR) analysis. Electroporation immediately disordered the stratum corneum lipid structure up to a certain threshold level. This action lasted throughout the experiment. This may be attributed to the formation of long lifetime of metastable lipid structures, which may allow molecules to pass to the inside of the stratum corneum due to the electroporation-induced fluidized lipid membranes. Electroporation also altered the protein structure of the stratum corneum. When electroporation was combined with 0.05 M oleic acid/PG, uptake of oleic acid and PG into the stratum corneum was remarkably accelerated compared to the application of only 0.05 M oleic acid/PG to the skin. This indicates that electroporation enables oleic acid and PG to penetrate the stratum corneum easily by disrupting the structure of the latter. PG transfer into the dermis from the epidermis was accelerated, not because of the direct action of electroporation on the dermis, but because electroporation induced the rapidly disordering action of oleic acid on the stratum corneum. Lipid-soluble indomethacin permeated the skin more rapidly when the skin was treated with electroporation plus oleic acid/PG than with 0.05 M oleic acid/PG in vitro.

Animals↗
PubMed2000

Endocytosis of Clostridium botulinum type B neurotoxin into rat brain synaptosomes.

Clostridium botulinum type B neurotoxin cleaves VAMP (vesicle-associated membrane protein)/synaptobrevin into two fragments, which results in inhibition of neurotransmitter release. The induced fragment did not react to the antibody raised against the synthetic peptide of the amino-terminal 20 residues of VAMP-2, suggesting that the toxin treatment has caused antigenical alteration in the amino-terminal region of VAMP-2. In rat brain synaptosomes, type B neurotoxin was reduced presumably with sulfhydryls in the membrane and detected in the synaptic vesicle fraction which involved the degradation of VAMP-2 and the inhibition of neurotransmitter release. The light chain in a free form was present in the cytosol fraction. These findings suggest a possibility that type B neurotoxin endocytoses into synaptic vesicles by the recycling pathway and the light chain is penetrable through synaptic vesicle membrane. However, the amount of type B neurotoxin entrapped into synaptic vesicles appears to be extremely small, which may be attributed to a lower sensitivity of the toxin to brain synaptosomes than to peripheral nerve endings.

Animals↗