[Experience in modified maze operation preserving the anterior internodal tract (AINT)].
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Aged↗
Biomedical subjects
M Shibuya
Publications and source records attributed to M Shibuya.
A new method for analyzing the cell kinetics of human brain tumors by double labeling with bromodeoxyuridine in situ and with iododeoxyuridine in vitro.
BACKGROUND: Cell kinetics studies performed with immunohistochemical techniques to estimate the S-phase fraction have elucidated the proliferative potential of individual brain tumors. METHODS: The authors developed a new double-labeling method that enables other cell kinetics variables, including the duration of the S-phase (Ts) and the potential doubling time (Tp), to be measured from a single biopsy specimen. Using this method, 100 brain tumors were labeled with bromodeoxyuridine (BUdR) in situ and with iododeoxyuridine in vitro; labeled cells were identified by double staining with immunogold-silver and alkaline phosphatase techniques. RESULTS: Ts was fairly uniform (mean, 9.2 +/- 2.1 hour [+/- standard deviation]); range, 6.0-13.7 hours), but Tp varied from 1 day to more than 2 months. The Tp values correlated closely with the BUdR labeling index (LI), or S-phase fraction, and can be calculated from the equation: Tp = 26.9/LI1.02 (r = 0.98, P < 0.005). CONCLUSIONS: This new method facilitates the quantitation of the proliferative potential of individual brain tumors. The S-phase fraction, Ts, and Tp can be calculated from analysis of a single biopsy specimen. This method can be used to estimate the prognosis of individual patients with brain tumors and to select treatment modalities more directly than is possible with single-labeling studies with BUdR.
Biopsy↗
Proliferative potential of brain tumors. Analyses with Ki-67 and anti-DNA polymerase alpha monoclonal antibodies, bromodeoxyuridine labeling, and nuclear organizer region counts.
Histochemical analyses with Ki-67 and anti-DNA polymerase alpha monoclonal antibodies were done, and argyrophilic nucleolar organizer regions (Ag-NOR) were counted to estimate the proliferative potential of 200 brain tumors. The findings were compared with the bromodeoxyuridine labeling index (BrdU LI), or S-phase fraction. The proliferating cell indexes (PCI), determined by Ki-67 and anti-DNA polymerase alpha monoclonal antibodies were higher and Ag-NOR more numerous in medulloblastomas, glioblastomas, and metastatic carcinomas than in astrocytomas or meningiomas. The Ki-67 and DNA polymerase alpha PCI correlated with the BrdU LI (r = 0.87 and r = 0.84, respectively) and with each other (r = 0.94). The number of Ag-NOR correlated less strongly with these indexes in some tumors. These findings suggest that Ki-67 and anti-DNA polymerase alpha monoclonal antibodies may be useful for estimating the proliferative potential of individual tumors in routine clinical practice. The number of Ag-NOR, however, does not always reflect the growth potential of each tumor.
Antibodies, Monoclonal↗
A newly designed nerve monitor for microneurosurgery: bipolar constant current nerve stimulator and movement detector with a pressure sensor.
The authors describe a newly designed nerve monitor which is useful for numerous microneurosurgical procedures. Standard bipolar forceps are used to apply constant current stimulation. Muscle contraction evoked by the stimulation is detected by a small disc-shaped pressure sensor taped to the overlying skin. The responses are monitored both quantitatively on a liquid crystal display and qualitatively through an on-off auditory signal. Surgery can proceed without interruption. This apparatus can safely and reliably monitor the facial nerve, nerves involved in eye movements, lower cranial nerves and spinal nerves. This portable system weights only 1.8 kg and can easily be used by a neurosurgeon.
Cranial Nerve Neoplasms↗
Investigation of a synthetic peptide as immunogen for a variant epidermal growth factor receptor associated with gliomas.
We have previously demonstrated antibody production to a glioma-associated variant form of the human epidermal growth factor receptor in rabbits that had received a synthetic peptide mimicking the unique primary structure of the variant protein as immunogen. We report here the response of mice, rabbits, goats, and macaques immunized by various protocols to this peptide. Titers to both peptide- and cell-elaborated variant receptor were measured, and the capacity to recognize the variant receptor in human tumor samples was determined. Within the range of species and strains investigated, we demonstrated a variable species-associated response to the peptide (rabbits > mice > goats > rats > macaques). Rabbits and a single goat produced specific, high titer antibody activity to the variant receptor protein following immunization with peptide alone. Murine titers to the parent protein were not appreciable following peptide immunization alone; additional immunization with variant receptor as expressed on cell membranes was used to boost this response.
Amino Acid Sequence↗
Vasodilatory effects of okadaic acid on the canine cerebral artery.
We investigated the in vivo and in vitro vasodilatory effects of okadaic acid, an inhibitor of protein phosphatases, in canine basilar arteries. Angiography revealed that the intracisternal injection of okadaic acid produced a long-lasting increase in the internal diameter of the canine basilar artery. The maximal increases in diameter induced by 1 and 10 nmol of okadaic acid were 23.3 +/- 13.5 and 33.8 +/- 11.9%, respectively. Okadaic acid in the concentrations of 10(-7) and 10(-6) M also exerted a dose-dependent, long-lasting relaxation without any contraction in isolated basilar arteries, even in the resting condition. Similar effects (ED50 values and maximal relaxation) were observed in arterial strips precontracted with K+, prostaglandin F2 alpha, and phorbol 12-myristate 13-acetate. These in vito and in vivo results suggest that inhibition of protein phosphatases by okadaic acid produces a vasodilation in the cerebral artery.
Angiography↗
Increased Ca2+ influx in the resting state maintains the myogenic tone and activates charybdotoxin-sensitive K+ channels in dog basilar artery.
We examined whether Ca2+ channel function in the resting state alters the resting tone and Ca(2+)-activated K+ (KCa) channel function in dog basilar artery: data were compared with findings in the mesenteric artery. Isolated dog basilar artery maintained a myogenic tone; that is, the resting tone decreased when either the Krebs solution was replaced with a Ca(2+)-free solution or nifedipine was added. The basal 45Ca influx in the resting state of the basilar artery was significantly increased compared with that in the mesenteric artery, and this increase in the basilar artery was reduced by nifedipine. The addition of charybdotoxin (ChTX), a blocker of large-conductance KCa channels, to the resting strips caused a concentration-dependent contraction in the basilar artery but not in the mesenteric artery. The ChTX-induced contraction in the basilar artery was abolished by nifedipine. In resting strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in the basilar artery than in the mesenteric artery. The addition of nifedipine to the resting strips decreased the basal 86Rb efflux rate constant only in the basilar artery. These results suggest that the transmembrane Ca2+ influx via L-type voltage-dependent Ca2+ channels was significantly increased in the resting state of the basilar artery and that the myogenic tone was therefore maintained and the ChTX-sensitive KCa channels were highly activated.
Animals↗
Role of nitric oxide in the cerebral vasodilatory responses to vasopressin and oxytocin in dogs.
We angiographically assessed the vasodilatory effects of vasopressin and oxytocin on the basilar arteries in dogs. Intracisternal bolus injections of vasopressin (100 pmol and 1 nmol) and oxytocin (1 and 10 nmol) produced dose-dependent increases in the internal diameter of the basilar arteries without affecting mean arterial blood pressure. The maximal dilatations of the basilar arteries induced by 1 nmol vasopressin and 10 nmol oxytocin were 142.3 +/- 19.9 and 136.8 +/- 25.5% of the baseline, respectively. When the same peptides were injected into the vertebral artery, the maximal dilatations were similar, but the duration of response was shorter. Pretreatment with intracisternal injection of 10 mumol NG-monomethyl-L-arginine (L-NMMA), which inhibits the synthesis of nitric oxide from L-arginine, suppressed the vasodilatory responses induced by intracisternal injection of vasopressin and oxytocin and by intraarterial injection of vasopressin. Calcitonin gene-related peptide also caused dilatation of the basilar artery when injected into the cisterna magna, but its effect was not blocked by L-NMMA. L-NMMA reduced the basal diameter of the basilar artery in a dose-dependent manner; L-arginine produced dose-dependent increases in diameter. The vasoconstriction induced by L-NMMA was reversed by high concentrations of L-arginine. These results suggest that vasopressin and oxytocin dilate the basilar arteries via the release of nitric oxide from both the intraluminal and the extraluminal sides and that synthesis and release of nitric oxide in the vascular wall contribute to maintenance of basal vascular tonus.
Animals↗
Triphasic response of rat intracerebral arterioles to increasing concentrations of vasopressin in vitro.
To determine how vasopressin affects the vascular tone of the smaller cerebral arterioles, we carried out an in vitro study of isolated and cannulated intracerebral arterioles of rats. We found that increasing concentrations of vasopressin induced a triphasic response of vasodilation (10(-12)-10(-11) M), vasoconstriction (10(-10)-10(-8) M), and vasodilation stabilizing to control diameter (10(-7)-10(-6) M) and that the maximum constriction was twice the maximum dilation in these smaller arterioles [21.2 +/- 13.1% (mean +/- SD) decrease in diameter vs. 11.2 +/- 5.7% increased]. Pretreatment of the arterioles with NG-monomethyl-L-arginine (10(-4) M), a specific inhibitor of endothelium-derived relaxing factor, abolished the vasopressin-induced vasodilation and significantly increased the vasoconstriction. These results suggest that these arterioles were maintained in a dilated state by an endothelium-derived relaxing factor activated by vasopressin. Both vasodilation and vasoconstriction were found to be mediated through vasopressin V1 receptors in a study of arterioles pretreated with d(CH2)5Tyr(Me)arginine vasopressin (10(-6) M), a vasopressin V1 receptor antagonist. These results support the hypothesis that vasopressin may constrict smaller cerebral arterioles while simultaneously dilating larger cerebral arteries. Our results also suggest that vasopressin may aggravate cerebral ischemia in pathological conditions, such as subarachnoid hemorrhage, when the arteriolar response to vasopressin shifts from vasodilation to vasoconstriction due to increased vasopressin levels in plasma and CSF and impaired endothelium-derived relaxation.
Animals↗
Establishment of a human small cell lung carcinoma cell line carrying amplification of c-myc gene and chromosomal translocation of t(3p;6p) and t(12q;17p).
A transplantable tumor and an in vitro culture cell line (GK-T3) were established from metastatic liver tissue of human small cell lung carcinoma (SCLC). Southern blot analysis revealed about 30-fold amplification of c-myc gene in the tumor cells in liver, xenografts, and in vitro cell line. The degree of c-myc amplification was essentially conserved through serial passages in nude mice and cultivation in vitro. The level of c-myc mRNA was significantly increased in these cells. Cytogenetically, numerical and complex structural abnormalities were observed in GK-T3 cells, including t(3p;6p), t(12q;17p), two homogeneously staining regions (hsrs) and several double minutes (dmins). These results suggest that activation of c-myc gene and alteration of gene(s) around these chromosomal breakpoints may play a role in tumorigenesis of GK-T3 SCLC.
Aged↗
Characterization of the promoter region of the human c-kit proto-oncogene.
The c-kit proto-oncogene encodes a tyrosine kinase receptor for stem cell factor and plays a critical role in the growth and differentiation of various types of cells including hematopoietic stem cells. To investigate the mechanisms of its transcriptional regulation, we isolated the 5' flanking region of the human c-kit gene and characterized its promoter activity in hematopoietic cells. Nucleotide sequence analysis revealed that the 1.2 kb 5' flanking region lacked a typical "TATA box," but had a relatively high G + C content and four potential Sp1-binding sites. Putative binding sites for AP-2, basic helix-loop-helix proteins, Ets-domain proteins, Myb and GATA-1 were also found. Primer extension and S1 nuclease protection analyses of hematopoietic cells indicated that the major transcription start sites are 62 bp and 58 bp upstream of the translation start site. Essentially the same start sites were detected in non-hematopoietic cells such as small cell lung carcinoma and glioblastoma: this single promoter in c-kit is different from the multiple promoter system of c-fms, a c-kit-related gene, in which at least two promoters are differently used in hematopoietic and non-hematopoietic cells. An analysis of the c-kit 5' flanking region using the bacterial chloramphenicol acetyltransferase gene (CAT assay) in human erythroleukemia HEL cells, which express the endogenous c-kit mRNA at high levels, showed that a region from -180 to -22 is important for the expression of the c-kit gene. In addition, a negative regulatory element(s) is suggested to be involved in the regulation of the c-kit gene expression in mammals.
Amino Acid Sequence↗
Percutaneous cardiopulmonary support as the second generation of venoarterial bypass: current status and future direction.
We have developed a new percutaneous cardiopulmonary support (PCPS) system in cooperation with Terumo Corporation, called the Emergency Bypass System (EBS). This preassembled system is unique in terms of its small priming volume (470 cc), fully automatic priming function, originally developed membrane oxygenator, and Capiox straight path centrifugal pump. The priming process takes only 5 min. We have used this system in 4 patients (all were male; mean age 56 years, range 35-73 years). The duration of assist ranged from 15 min to 210 h. Maximum bypass flow ranged from 2.2 to 3.6 L/min, systemic circulation was very well maintained, and urinary output was acceptable. Although the centrifugal pump was in good working condition, we had to change the oxygenator because of serum leakage 36 to 48 h after initiation of the assist. One patient was weaned from the PCPS, but died of brain death. The other patients could not be weaned from the PCPS. The cause of death in these patients was irreversible myocardial damage. Disseminated intravascular coagulopathy triggered by surgical trauma related to various procedures developed in 2 patients during assist and was the cause of cessation of the assist. Problems to be ratified and future directions in the EBS and PCPS were earlier introduction through the establishment of clear indications and endpoint, heparin coating, improvement in durability of the oxygenator, and cost.
Adult↗
Testis-specific mak protein kinase is expressed specifically in the meiotic phase in spermatogenesis and is associated with a 210-kilodalton cellular phosphoprotein.
The mak gene encodes a new protein kinase distantly related to cdc2 kinase, and its transcripts are expressed exclusively in testicular germ cells at and after meiosis (H. Matsushime, A. Jinno, N. Takagi, and M. Shibuya, Mol. Cell. Biol. 10:2261-2268, 1990). In this study, we prepared a series of antibodies against synthetic peptides and fusion products of the mak gene and characterized the subcellular localization, protein kinase activity, and association with other cellular proteins of Mak. Mak products were identified as 66- and 60-kDa proteins that specifically appeared in rat testes after puberty. Testicular germ cell fractionation revealed that Mak products were most abundant in the fraction of the late pachytene stage and that their levels were dramatically decreased in postmeiotic haploid cells. Mak products were localized mostly in the cytoplasm as a soluble form. [35S]methionine labelling demonstrated that Mak products were associated with a 210-kDa cellular protein; in an in vitro kinase assay with immunoprecipitates of Mak products, the 210-kDa cellular protein was efficiently phosphorylated on serine and threonine residues. Furthermore, in a testicular cell culture system with 32Pi, the 210-kDa molecule associated with Mak was phosphorylated in vivo on serine and threonine residues. These results strongly suggest that the Mak complex may play a role in meiosis during spermatogenesis and that a phosphorylated 210-kDa protein is one of the physiological substrates for this protein kinase.
Amino Acid Sequence↗
Regional differences in the vasodilator response to vasopressin in canine cerebral arteries in vivo.
BACKGROUND AND PURPOSE: The aim of this study was to investigate the regional differences in the in vivo vasodilator responses to vasopressin, which is thought to stimulate the release of nitric oxide from the endothelium, in canine cerebral arteries by angiography. METHODS: Angiograms were performed through a catheter inserted directly into the right vertebral artery and were taken periodically after the infusion of vasopressin. The diameters of various segments of the major arteries were measured using a computerized image analysis system. RESULTS: The bolus administration of vasopressin (10 pmol to 1 nmol) into the vertebral artery produced a long-lasting, dose-dependent vasodilation in the major cerebral arteries centering around the circle of Willis. One nanomole of vasopressin appeared to be the optimal dose for producing maximal vasodilation. The internal diameters of the basilar, posterior communicating, and internal carotid arteries experienced the most dilation (approximately 150% that of control) 2 minutes after the infusion of 1 nmol of vasopressin, followed by those of the middle cerebral, the intracranial portion of the vertebral, and the anterior spinal arteries (approximately 130% that of control). The extracranial portion of the vertebral artery (109.8 +/- 4.8% that of control, n = 4) was less sensitive to 1 nmol of vasopressin. Pretreatment with an intracisternal injection of 10 mumol of NG-monomethyl L-arginine suppressed the vasodilator effect of vasopressin and substance P, whereas it did not affect the response to vasoactive intestinal peptide. CONCLUSIONS: These results suggest that the arteries composing the circle of Willis at the base of the brain are more sensitive to nitric oxide release induced by vasopressin compared with other intracranial and extracranial arteries.
Angiography↗
Immunohistochemical expression of PIT-1 protein in pituitary glands of human GRF transgenic mice: its relationship with hormonal expressions.
It has been suggested that pit-1 protein may play a role in the differentiation of the anterior pituitary cells. The present immunohistochemical studies were designed to elucidate the relationship between functional differentiation of pituitary adenoma and expression of pit-1 protein in human (h) GRF transgenic mice. Pituitaries from a 10 month old and a 6 month old transgenic mice were fixed in 4% paraformaldehyde and embedded in paraffin. The indirect immunoperoxidase method was performed using antibodies against hGRF, GH, PRL, ACTH, alpha subunit (SU), FSH beta SU, LH beta SU, TSH beta SU, and pit-1 protein. Immunohistochemical double staining was performed at light and electron microscopic levels. The pituitary glands of hGRF transgenic mice (both 10 month and 6 month old) demonstrated diffuse hyperplasia of GH positive cells with coexpression of hGRF within the same cells. There were also scattered cells which were positive for other hormones and hormone subunits in the hyperplastic pituitary. Three discrete nodules were found in the pituitary gland of a 10 month old hGRF transgenic mouse and were identified as adenomas. These adenomas were composed of enlarged round cells which were positive only for GH, hGRF, PRL and TSH beta SU. Pit-1 protein was intensely expressed in the nuclei of the adenoma cells. These results suggest the existence of an autocrine mechanism by hGRF in the formation of somato-lacto-thyrotroph adenoma via constitutive pit-1 expression.
Adenoma↗
Immunohistochemical double staining with immunogold-silver and alkaline phosphatase to identify nuclear markers of cellular proliferation.
To facilitate cell kinetics studies of brain tumors labeled with thymidine analogs, we developed a new method to identify nuclei labeled sequentially with bromodeoxyuridine (BUdR) and iododeoxyuridine (IUdR) by double staining with immunogold-silver and alkaline phosphatase. Patients received an intraoperative infusion of BUdR; excised tumor specimens were immediately labeled with IUdR in vitro, fixed with 70% alcohol, embedded in paraffin, and cut into 6 microns sections. The sections were incubated first with BR-3, a monoclonal antibody that recognizes only BUdR, and then with IU-4, a monoclonal antibody that recognizes both BUdR and IUdR; sections were counterstained with hematoxylin to identify unlabeled nuclei. Nuclei labeled only with IUdR stained red, whereas those labeled with BUdR or with both BUdR and IUdR stained black against a red background; unlabeled nuclei stained blue. This method was the most efficient differential staining technique to identify nuclei labeled only with IUdR and those labeled with BUdR among unlabeled nuclei.
Alkaline Phosphatase↗
Functional role of charybdotoxin-sensitive K+ channels in the resting state of cerebral, coronary and mesenteric arteries of the dog.
To determine the possible role of Ca(++)-activated K+ (KCa) channels in the regulation of resting tone of arteries, the effects of agents that interact with these channels on tension and 86Rb efflux were examined in endothelium-denuded strips of cerebral (middle cerebral, posterior cerebral and basilar), coronary and mesenteric arteries of the dog. Strips of cerebral arteries maintained a myogenic tone; i.e., the resting tone decreased when either the Krebs' solution was replaced with a Ca(++)-free solution or nifedipine was added. The addition of charybdotoxin, a blocker of large conductance KCa channels, to the resting strips (strips at a resting state) caused a concentration-dependent contraction in the cerebral arteries but not in the coronary or mesenteric artery. In resting strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in the cerebral arteries than in the coronary and mesenteric arteries. The addition of nifedipine to the resting strips decreased the basal 86Rb efflux rate constant in the cerebral and coronary arteries. Effects of nifedipine on tension and 86Rb efflux in 20.9 mM K(+)-contracted strips of the mesenteric artery were comparable to the effects of this blocker in the resting strips of the middle cerebral artery. The 86Rb efflux rate constant during the stimulation with 65.9 mM K+ was similar for the middle cerebral and mesenteric arteries. Studies using 1- or 5-min pulse labeling with 45Ca demonstrated increased basal 45Ca influx in the resting state of cerebral arteries compared with the coronary and mesenteric arteries.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Prognostic parameters in axillary node negative breast cancer.
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Axilla↗