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Biomedical subjects

H Kitasato

Publications and source records attributed to H Kitasato.

At least 37 records · Page 2Linked to original sources
PubMed1999

Inhibition of neutrophil apoptosis by verotoxin 2 derived from Escherichia coli O157:H7.

In order to evaluate the pathological role of verotoxin 2 (VT2), we investigated the effects of VT2 on neutrophil apoptosis in vitro. The results showed that VT2 caused a significant delay in spontaneous neutrophil apoptosis and that the effect was abrogated by a protein kinase C inhibitor. These data indicate that longer survival of neutrophils may aggravate neutrophil-mediated tissue damage in VT2-associated diseases.

Apoptosis↗
PubMed1998

Glucagon induces suppression of ATP-sensitive K+ channel activity through a Ca2+/calmodulin-dependent pathway in mouse pancreatic beta-cells.

Glucagon is known to increase intracellular cAMP levels and enhance glucose-induced electrical activity and insulin secretion in pancreatic beta-cell perfused with Krebs-Ringer bicarbonate solution. The present experiments were aimed at evaluation of the hypothesis that changes in beta-cells ATP-sensitive K+ (K(ATP)) channel activity are involved in the glucagon-induced enhancement of electrical activity. Channel activity was recorded using the cell-attached configuration of the patch-clamp technique. Addition of glucagon (2.9 x 10(-7) m) in the presence of 11.1 mm glucose caused closure of K(ATP) channels followed by an increase in the frequency of biphasic current transients (action currents) due to action potential generation in the cell. Three calmodulin-antagonists (W-7, chlorpromazine, and trifluoperazine) restored with similar efficacy K(ATP) channel activity in cells being exposed to glucagon. At 2.8 mm glucose, glucagon did not affect K(ATP) channel activity until Ca2+ was released from Nitr-5 by flash photolysis, at which point channel activity was transiently suppressed. Similar effects were seen when db-cAMP was used instead of glucagon. These results support the view that glucagon and other cAMP-generating agonists enhance glucose-induced beta-cell electrical activity through a Ca2+/calmodulin dependent-closure of K(ATP) channels.

Action Potentials↗
PubMed1998

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells.

From K3/II, which is a highly oncogenic HPV16-transformed Syrian hamster cell line, thymidine-kinase(TK)-less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes-simplex-virus TK gene (HSV TK) and several sub-lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti-viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co-cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by-stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6-induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/11 cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells.

Animals↗
PubMed1998

Phylogenic study of calcitonin gene-related peptide-immunoreactive structures in the pancreas.

The immunohistochemical localization of calcitonin gene-related peptide was examined, at both light and electron microscopic levels, in the pancreas of various vertebrates, including the eel, bullfrog, turtle, chicken, mouse, rat, guinea pig, dog, monkey, and human. Immunoreactive staining was observed in nerve fibers in every animal species examined, but positive endocrine cells were limited to the rat, monkey, and human. The density of the positive endocrine cells varied considerably among the three species (monkey > rat > human). Positive nerve fibers were distributed throughout the parenchyma, being particularly rich around pancreatic ducts, and near large or small blood vessels. In four species (eel, mouse, rat, and dog), positive nerve fibers formed a dense network in the islet region. There were positive varicose nerve fibers around exocrine cells. These fibers, varying in density in different species (relatively high in the eel, bullfrog, and rat), were sometimes adjacent to acinar cells. At the electron microscopic level, positive nerve terminals were often demonstrated in close apposition to the outer membrane of acinar cells. The eel pancreas revealed an exceptional pattern of staining in neuronal cell bodies that were scattered in the interlobular connective tissue. Despite these anatomical differences, the omnipresence of this peptide suggests its essential role(s) in the pancreas.

Animals↗
PubMed1998

Human papillomavirus 57 identified in a plantar epidermoid cyst.

We report a 23-year-old Japanese man who had plantar warts on the right sole, beneath one of which an epidermoid cyst developed. On microscopic examination, an acanthotic epidermis markedly invaginated into the underlying dermis, resulting in an open epidermoid cyst. Not only the polymerase chain reaction but also an in situ hybridization detected HPV 57 DNA in the cyst. HPV 60 is the only type of HPV that has been identified in epidermoid cysts. To our knowledge, this is the first case report of an epidermoid cyst, in which a different type of virus from HPV 60 was identified. Histological features of the cyst were also different those of HPV 60-associated epidermoid cysts.

Adult↗
PubMed1998

Cyclic AMP-elevating agents prevent oligodendroglial excitotoxicity.

Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca2+ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca2+ influx (approximately 5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca2+ influx, leading to a less prominent response of intracellular Ca2+ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca2+ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca2+ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca2+ influx.

Animals↗
PubMed1998

A putative human papillomavirus type 57 new subtype isolated from plantar epidermoid cysts without intracytoplasmic inclusion bodies.

Human papillomavirus type 60 (HPV-60) is the only virus type that has been identified in epidermoid cysts. In this study, HPV-57 DNA was found in three out of 18 plantar epidermoid cysts with different histological features from HPV-60-associated cysts, using PCR and Southern hybridization. The HPV-57-associated cysts had features resembling an HPV-2-specific cytopathic effect. The sequences of two HPV-57 DNA clones isolated from two patients were identical, but differed at some positions from those of HPV-57a and HPV-57b. This putative new subtype was tentatively designated as HPV-57c, and may be associated with plantar epidermoid cysts showing histological features resembling the HPV-2 cytopathic effect.

Amino Acid Sequence↗
PubMed1997

Development of neuropeptide Y innervation in the liver.

Hepatic neuropeptide Y (NPY) innervation was studied by immunohistochemistry in various mature vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea pig, dog, monkey, and human. In addition, an ontogenetic study on hepatic NPY was made in developing mice and guinea pigs. In all species examined except the eel, NPY-like immunoreactivity was detected in nerve fibers. In the carp, bullfrog, turtle, chicken, mouse, and rat, NPY-positive fibers were distributed around the wall of hepatic vessels and the bile duct of the Glisson's sheath. The density of NPY-positive fibers increased with evolution. However, in the guinea pig, dog, monkey, and human, numerous NPY-positive fibers were observed not only in the Glisson's sheath but also in the liver parenchyma. Positive fibers formed a dense network that surrounded the hepatocytes. The present immunoelectron microscopic study has confirmed that NPY-positive terminals are closely apposed to hepatocytes. Ontogenically, NPY-positive fibers were first found in the embryonic liver of 19-day-old mice. Positive fibers increased with age, and the highest peak was seen 1 week after birth. However, NPY-positive nerve fibers were present abundantly in Glisson's sheath and in the hepatic parenchyma of neonatal (3 and 7 days old) guinea pigs in a distribution similar to that in mature animals. This ontogenetic pattern suggests that NPY plays a certain role in the developing liver.

Animals↗
PubMed1997

Immunohistochemical localization of cellubrevin on secretory granules in pancreatic B-cells.

Cellubrevin is one of the proteins involved in the docking and fusion of secretory granules to the plasma membrane. It has been reported that cellubrevin is widely distributed in both neural and non-neural cells, including insulin-secreting B-cells. This study aims to demonstrate by immunohistochemical techniques that cellubrevin is localized in insulin-secreting cells and further to examine whether it might occur in glucagon- and somatostatin-secreting cells in the pancreatic islet in the rat and mouse. We used the polyclonal antibody against the N-terminal peptide whose specificity was confirmed by Western blot analysis. Double immuno-staining demonstrated that cellubrevin was localized in insulin-containing cells, but both glucagon-containing and somatostatin-containing cells lacked the immuno-reactivity. Immuno-electron microscopic analysis revealed the localization of cellubrevin on the margin of secretory granules near the plasma membrane but not in the granules closer to the nucleus. These observations support the view that cellubrevin in the pancreatic islet is expressed on the membrane of the secretory granules in B-cells at the stage of exocytosis.

Animals↗
PubMed1997

Suppressing Na+ influx induces an increase in intracellular ATP concentration in mouse pancreatic beta-cells.

The effects of suppressing Na+ influx on the activity of ATP-sensitive K+ channels (K(ATP) channel) and intracellular ATP concentration in mouse pancreatic beta-cells were studied. Lowering extracellular Na+ concentration brought about a closing of K(ATP) channels. The activity of K(ATP) channels was markedly inhibited by the addition of amiloride, a blocker of Na+/H+-counter transporter. Mannoheptulose completely eliminated the inhibition otherwise induced by amiloride. Monensin, an electroneutral Na+/H+ antiporter, remarkably increased the activity of K(ATP) channels. Removing extracellular Ca2+ also caused inhibition of the channel activity. ATP measurement experiments using isolated islets revealed that the intracellular ATP concentration of islet cells was significantly increased by incubating either with amiloride or a low Na+ solution. The measurement of fluorescence excited at 360 nm demonstrated that both suppressing Na+ influx and inhibition of Na+/K+-pumps caused a transient increase in the reduced form of pyridine nucleotide. These findings indicate that a decrease in Na+ influx could cause an elevation in intracellular ATP concentration probably through inducing a fall in ATP consumption at the Na+/K+-pump sites.

Adenosine Triphosphate↗
PubMed1997

Putative regulatory sequence in human papillomavirus type 16 E2 open reading frame.

A 114 bp fragment of the human papillomavirus type 16 (HPV16) E2 open reading frame (nt. 3142-3255) containing a putative estrogen responsive element (ERE) was amplified and cloned into pBLCAT2 plasmid in both sense (p159-4) and anti-sense (p164) orientation. The plasmids were transfected into human breast-cancer cell line MCF-7 containing estrogen receptor and the cultures were kept in the presence or absence of beta-estradiol. The chloramphenicol acetyltransferase (CAT) activity was not influenced by estrogen. However, a silencer effect was observed both in cultures transfected with p159-4 and p164 plasmids. We prepared and cloned synthetic fragments containing the putative ERE and failed to prove that the palindrome in the putative ERE was responsible for the silencer activity.

Base Sequence↗
PubMed1996

A possible role of the ATP-sensitive potassium ion channel in determining the duration of spike-bursts in mouse pancreatic beta-cells.

The pancreatic beta-cell displays an electrical activity consisting of spike bursts and silent phases at glucose concentrations of about 10 mM. The mechanism of initial depolarization induced by glucose is well defined. However, the mechanism inducing the silent phase has not been fully elucidated. In the present study, the possibility of involvement of ATP-sensitive K+ channels in repolarization was examined using the patch-clamp technique in the cell-attached recording configuration. Ouabain (0.1 mM), an inhibitor of Na+/K+-ATPase, caused a complete suppression of ATP-sensitive K+ channel activity followed by typical biphasic current deflections, which were due to action potentials. The channel activity was also inhibited by removal of K+ from a perifusion solution. Furthermore, the activity of ATP-sensitive K+ channels was markedly inhibited either by replacement of external NaCl with LiCl or by addition of amiloride (0.2 mM), a blocker of Na+/H+ antiport. Addition of L-type Ca2+ channel blockers such as Nifedipine for Mn2+ induced the complete suppression of K+ channel activity. These findings strongly suggest that a fall in ATP consumption results in sustained depolarization, and that the repolarizations interposed between spike-bursts under normal ionic conditions are due to the periodical fall of ATP concentration brought about by periodical acceleration of ATP consumption at Na+/K+-pumps. It is concluded that the elevation of intracellular Na+ concentration as a consequence of accelerated Na+/Ca2+-countertransport during the period of spike-burst enhances ATP consumption, leading to a fall in ATP concentration which is responsible for termination of spike-burst and initiation of repolarization.

Action Potentials↗
PubMed1996

Glucagon induces Ca2+-dependent increase of reduced pyridine nucleotides in mouse pancreatic beta-cells.

Glucagon enhances the electrical activity of pancreatic beta-cells. The mechanism of the glucagon-evoked enhancement of electrical activity was investigated in terms of glucose metabolism. ICR mice aged 6-12 weeks were used for experiments. Intracellular Ca2+ increased in parallel with the enhancement of electrical activity. The stimulating effect of glucagon on Ca2+ oscillation was suppressed by calmodulin-antagonists (Chlorpromazine, W-7, and trifluoperazine). To trace the glucagon-evoked change in glucose metabolism, the reduced pyridine nucleotide (NAD(P)H) fluorescence was monitored using the microfluorometry with the excitation of 360 nm and the emission of 465 nm in islet cell clusters mainly consisting of beta-cells. In the presence of 2.5 mM Ca2+ glucagon (8.6 X 10(-8) M) increased the NAD(P)H fluorescence, while in the absence of Ca2+ the hormone had no effect on the fluorescence. Extracellular Ca2+ removal from the glucagon-containing perifusion solution decreased the fluorescence to the level which had been attained before glucagon was added. Chlorpromazine (10 microM) reversed the glucagon-induced increase of NAD(P)H fluorescence as well as removing Ca2+ W-7 (15 microM) and trifluoperazine (30 microM) also suppressed the glucagon-induced increase of NAD(P)H. These results suggest that Ca2+/calmodulin system is involved in the acceleration of glycogenolysis by glucagon in beta-cells. On the basis of these observations, the mechanism of glucagon-induced enhancement of electrical activity and the relative ineffectiveness of glucagon at low glucose concentrations were discussed.

Animals↗
PubMed1996

The intrinsic rhythmicity of spike-burst generation in pancreatic beta-cells and intercellular interaction within an islet.

The pancreatic beta-cell has four types of Ca2+ channel (L-type, T-type, low-threshold slowly inactivating, and low-threshold non-inactivating Ca2+), although the low-threshold non-inactivating Ca2+ channel has not yet been confirmed experimentally. Beside these, there are at least three types of K+ channels (K(ATP), K(Ca,V), and K(V)), and transporters (GLUT-2, Na+/Ca(2+)-countertransporter, and Na+/K(+)-pump) as schematically shown in Fig.4. Opinions on the mechanism of spike-burst are converging to the following view: At intermediate glucose concentrations, the intracellular ATP/ADP ratio oscillates in the following way. A gradual rise in the ATP/ADP ratio causes gradual progression of depolarization to the threshold for the low-threshold Ca2+ channels, of which the opening causes regenerative depolarization to the plateau potential on which spikes (the L-type Ca2+ channel contributes to spike firing) are superimposed. During the active phase, a fall in the ATP/ADP ratio follows a gradual rise in ATP consumption. Slight repolarization due to the opening of a small fraction of K(ATP) channels triggers regenerative repolarization. With the progress of repolarization, a residual fraction of voltage-gated Ca2+ channels (low-threshold non-inactivating) are deactivated. During the silent phase, a gradual rise in the ATP/ ADP ratio leads to gradual depolarization back to the threshold for the next spike-burst. There are still a diversity of views regarding the mechanism of the initial spike-train. On the basis of observations made in various laboratories including ours, we propose the following working model: At low concentrations of glucose, alpha-cells secret glucagon which induces a rise in cAMP in beta-cells lodged in the same islet. A rise in cAMP itself does not activate the enzymes relevant to glycogenolysis, but merely prepares to activate the enzymes. When extracellular glucose increases, Ca2+ spikes are elicited. Influxed Ca2+ ions, together with cAMP, work to activate the enzymes, resulting in an additional supply of fuel for ATP synthesis. After sometime, the cAMP level falls back to a low level and the additional glucose supply from stored glycogen stops. This reaction sequence may be the mechanism behind the initial spike-train. To substantiate this working model, it may be important to elucidate the dependence of the phosphorylasekinase and glycogenphosphorylase activities on the Ca2+ in beta-cells.

Acetylcholine↗
PubMed1995

Antibodies against GM1 ganglioside affect K+ and Na+ currents in isolated rat myelinated nerve fibers.

High titers of anti-GM1 ganglioside antibodies (anti-GM1 antibodies) may be implicated in lower motor neuron disease. We studied the pathogenic role of anti-GM1 antibody using the petroleum jelly-gap voltage clamp technique on isolated single myelinated rat nerve fibers. Anti-GM1 antisera were obtained from rabbits immunized with GM1 ganglioside. Extracellularly applied anti-GM1 antisera without complement activity increased both the rate of rise and the amplitude of the K+ current elicited by step depolarization, with little effect on Na+ current. In the presence of active complement, however, anti-GM1 antibodies decreased the Na+ current, and caused a progressive increase of nonspecific leakage current. Neither complement alone nor complement-supplemented antisera from which anti-GM1 antibodies were depleted by affinity chromatography had any effect on ionic current. These observations indicate that anti-GM1 antibodies themselves can uncover K+ channels in the paranodal region, while anti-GM1 antibodies bound to the nodal membrane in the presence of complement may form antibody-complement complexes that block Na+ channels and disrupt the membrane at the node of Ranvier.

Action Potentials↗