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Somatic mammalian cells possess well-established S-phase programs with specific regions of the genome replicated at precise times. The ATR-Chk1 pathway plays a central role in these programs, but the mechanism for how Chk1 regulates origin firing remains unknown. We demonstrate here the essential role of cyclin A2-Cdk1 in the regulation of late origin firing. Activity of cyclin A2-Cdk1 was hardly detected at the onset of S phase, but it was obvious at middle to late S phase under unperturbed condition. Chk1 depletion resulted in increased expression of Cdc25A, subsequent hyperactivation of cyclin A2-Cdk1, and abnormal replication at early S phase. Hence, the ectopic expression of cyclin A2-Cdk1AF (constitutively active mutant) fusion constructs resulted in abnormal origin firing, causing the premature appearance of DNA replication at late origins at early S phase. Intriguingly, inactivation of Cdk1 in temperature-sensitive Cdk1 mutant cell lines (FT210) resulted in a prolonged S phase and inefficient activation of late origin firing even at late S phase. Our results thus suggest that cyclin A2-Cdk1 is a key regulator of S-phase programs.

Chk1 is an essential kinase for maintaining genome integrity and cell cycle checkpoints through phosphorylating several downstream targets. Recently, we demonstrated that Chk1 is also required for cell proliferation in somatic cells under unperturbed condition through regulating transcription of several genes. Here, we show that Chk1 is required for S phase progression and RNR2 is a critical downstream target of genes transcriptionally regulated by Chk1. Hence, although RNR2 expression reached maximum at S phase in the presence of Chk1, Chk1 depletion arrested the cell cycle at S phase and reduced RNR2 expression at both mRNA and protein levels. Ectopic expression of RNR2 failed to rescue the S phase arrest observed in Chk1 depleted cells, suggesting the presence of an additional Chk1-target(s) for completion of S phase other than RNR2. Therefore, our results suggest that Chk1 is required for DNA replication at least through regulating RNR2 gene transcription.

DNA damage results in activation or suppression of transcription of a large number of genes. Transcriptional activation has been well characterized in the context of sequence-specific DNA-bound activators, whereas mechanisms of transcriptional suppression are largely unexplored. We show here that DNA damage rapidly reduces histone H3 Threonine 11 (T11) phosphorylation. This correlates with repression of genes, including cyclin B1 and cdk1. H3-T11 phosphorylation occurs throughout the cell cycle and is Chk1 dependent in vivo. Following DNA damage, Chk1 undergoes rapid chromatin dissociation, concomitant with reduced H3-T11 phosphorylation. Furthermore, we find that loss of H3-T11 phosphorylation correlates with reduced binding of the histone acetyltransferase GCN5 at cyclin B1 and cdk1 promoters and reduced H3-K9 acetylation. We propose a mechanism for Chk1 as a histone kinase, responsible for DNA-damage-induced transcriptional repression by loss of histone acetylation.

Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy.

DNA damage is a common event and probably leads to mutation or deletion within chromosomal DNA, which may cause cancer or premature aging. DNA damage induces several cellular responses including DNA repair, checkpoint activity and the triggering of apoptotic pathways. DNA damage checkpoints are associated with biochemical pathways that end delay or arrest of cell-cycle progression. These checkpoints engage damage sensor proteins, such as the Rad9-Rad1-Hus1 (9-1-1) complex, and the Rad17-RFC complex, in the detection of DNA damage and transduction of signals to ATM, ATR, Chk1 and Chk2 kinases. Chk1 and Chk2 kinases regulate Cdc25, Wee1 and p53 that ultimately inactivate cyclin-dependent kinases (Cdks) which inhibit cell-cycle progression. In this review, we discuss the molecular mechanisms by which DNA damage is recognized by sensor proteins and signals are transmitted to Cdks. We classify the genes involved in checkpoint signaling into four categories, namely sensors, mediators, transducers and effectors, although their proteins have the broad activity, and thus this classification is for convenience and is not definitive.

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.

Checkpoint activation by DNA damage during G(2) prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G(2) arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G(2)/M arrest in HeLa S2 cells. Furthermore, UV-induced G(2)/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK.

The kinase Chk2 and tumor suppressor p53 participate in an ill defined regulatory interaction in mammalian cells. The abundance of Chk2 mRNA and protein has now been shown to be decreased by the induction of p53 in Saos2 cells. Ionizing radiation also triggered the phosphorylation and subsequent down-regulation of Chk2 in human colorectal HCT116 (p53(+/+)) cancer cells; irradiation of its isogenic mutant HCT116 (p53(-/-)) cells, which lack functional p53, induced Chk2 phosphorylation but not its down-regulation. In addition, HCT116 (p53(+/+)) cells constitutively expressing a dominant negative p53 (V143A) failed to suppress Chk2 expression after irradiation. Reporter gene assays in HCT116 (p53(+/+)) cells revealed that wild-type p53 repressed, whereas a dominant negative p53 mutant increased, the activity of the human Chk2 gene promoter. Mutational analysis showed that a CCAAT box located between nucleotides -152 and -138 of the promoter was responsible for its negative regulation by p53. Electrophoretic mobility shift assays demonstrated that the transcription factor NF-Y binds to this CCAAT sequence. A dominant negative mutant of NF-YA abolished the effect of p53 on Chk2 promoter activity. These results suggest that p53 negatively regulates Chk2 gene transcription through modulation of NF-Y function and that this regulation may be important for reentry of cells into the cell cycle after DNA damage is repaired.

The maximum likelihood expectation maximization (ML-EM) algorithm has become available as an alternative to filtered back projection in SPECT. The actual physical performance may be different depending on the manufacturer and model, because of differences in computational details. The purpose of this study was to investigate the characteristics of seven different types of ML-EM algorithms using simple simulation data. Seven ML-EM algorithm programs were used: Genie (GE), esoft (Siemens), HARP-III (Hitachi), GMS-5500UI (Toshiba), Pegasys (ADAC), ODYSSEY-FX (Marconi), and Windows-PC (original software). Projection data of a 2-pixel-wide line source in the center of the field of view were simulated without attenuation or scatter. Images were reconstructed with ML-EM by changing the number of iterations from 1 to 45 for each algorithm. Image quality was evaluated after a reconstruction using full width at half maximum (FWHM), full width at tenth maximum (FWTM), and the total counts of the reconstructed images. In the maximum number of iterations, the difference in the FWHM value was up to 1.5 pixels, and that of FWTM, no less than 2.0 pixels. The total counts of the reconstructed images in the initial few iterations were larger or smaller than the converged value depending on the initial values. Our results for the simplest simulation data suggest that each ML-EM algorithm itself provides a simulation image. We should keep in mind which algorithm is being used and its computational details, when physical and clinical usefulness are compared.

NAK has been identified as an IkappaB-kinase activating-kinase that plays an important role in NF-kappaB activation in response to several pro-inflammatory cytokines such as TNF-alpha. We describe here the genomic structure of the human NAK gene and analysis of the promoter. The gene spanned 40.5 kb and contained 21 exons with lengths ranging from 39 to 196 bp. Comparison of the phase and position of intron insertions within the human NAK gene with those within IKKalpha, IKKbeta and IKK epsilon indicated that the exon/intron organization of IKK epsilon is more highly conserved than that of IKKalpha or IKKbeta. The transcriptional start site was mapped at a position about 98 bp upstream from the translation start site by means of both an RNase protection assay and a primer extension method. Fluorescence in situ hybridization using full-length human NAK cDNA as a probe showed that the human NAK gene is localized to human chromosome 13q14.2-3, a region in which the loss of heterozygosity is associated with squamous cell carcinoma and leukemia. By using a series of deletion constructs in performing a reporter assay, a minimal 77 bp upstream of the transcriptional initiation site was shown to contribute to the major promoter activity.

We reported a 25-year-old woman with postural and kinetic tremor caused by diffuse axonal injury. The patient demonstrated consciousness disturbance, left oculomotor palsy and tetraparesis because of an automobile accident. T2-weighted and FLAIR MRI showed features of diffuse axonal injury. Hyperintense lesions appeared in the corpus callosum, fornix, dorsal portion of midbrain, right cerebral peduncle, and bilateral internal capsules. About 3 weeks later, head tremor and left hemiparesis appeared with improvement of consciousness. Administration of trihexyphenidyl decreased the tremor. Ten weeks after the accident, a coarse tremor in the head and right upper extremity developed after withdrawal of trihexyphenidyl. Tremor in the right upper limb predominantly occurred while maintaining an upright posture and with intended movements. Re-administration of trihexyphenidyl decreased the tremors. The dentatothalamic pathway is one of the lesions responsible for posttraumatic tremor. Our patient demonstrated lesions of diffuse axonal injury involving the dentatothalamic pathway. We considered that these lesions were associated with postural and kinetic tremor in our case. The tremor occurred at least 3 weeks after the accident. This finding suggested that the tremor was caused by transsynaptic alternations of thalamus or the extrapyramidal system secondary to involvement of the dentatothalamic pathway.

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Based on five-year experience, we have evaluated the electronic anesthesia record keeping system (DS 5300 OR, Fukuda Denshi, Tokyo), which is the first commercially available system combined with an anesthesia monitor in Japan. Although keying in of text with a keyboard on the screen remains to be improved, the user interface of touch screens is easy to use. It seems to be a great advantage that physiologic variables are automatically and accurately captured very minute. The record is more complete, legible, and accurate than a hand-written record. Both long-term storage and search for the data are much easier. The investment in this system is affordable even for small hospitals. Although there might be some areas in which improvement is needed, we are satisfied with its usefulness and reliability.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

Alpha2-adrenoceptor antagonists have been reported to stimulate colonic motor activity, but the effect on colonic motor dysfunction is unclear. We have investigated the effect of alpha 2-adrenoceptor antagonists on wrap-restraint stress-stimulated and normal colonic propulsion in rats. Colonic propulsion was evaluated by the transit of a charcoal marker along the colon. Faecal pellets output was also measured. A 30-min exposure to wrap-restraint stress starting 120 min after infusion of the charcoal marker significantly stimulated colonic transit with a concomitant increase in faecal pellets. Yohimbine and idazoxan, alpha 2-adrenoceptor antagonists, clonidine, an alpha 2-adrenoceptor agonist, and atropine suppressed wrap-restraint stress-stimulated colonic transit and faecal excretion in a dose-dependent manner. Ondansetron and YM060, 5-hydroxytryptamine3 (5-HT3) receptor antagonists, potently inhibited wrap-restraint stress-stimulated colonic transit, but only weakly inhibited faecal excretion. Neither alpha 2-adrenoceptor antagonists nor atropine had any significant effect on normal colonic transit, whereas clonidine and the 5-HT3 receptor antagonists inhibited it. alpha 2-Adrenoceptor antagonists as well as clonidine, atropine and 5-HT3 receptor antagonists inhibit the stress-induced colonic motor dysfunction in rats.

We studied effects of a novel alpha-2 adrenoceptor antagonist, YNS-15P (N-[(2R,11bS)-9-methoxy-1,3,4,6,7, 11b-hexahydro-2H-benzoquinolizin-2-yl]-N-methylmethanesulfonami de hydrochloride), on colonic propulsion stimulated by wrap-restraint stress (WRS) or bethanechol, on normal colonic propulsion and on diarrhea induced by castor oil in rats. Alpha-2 adrenoceptor antagonists, rauwolscine and RX821002, decreased the increase in the number and weight of fecal pellets induced by WRS. YNS-15P also inhibited WRS-stimulated fecal excretion in a dose-dependent manner. A 5-hydroxytryptamine3 receptor antagonist, granisetron, trimebutine and diazepam, but not a 5-hydroxytryptamine4 receptor antagonist, GR113808, significantly inhibited WRS-stimulated fecal excretion. YNS-15P inhibited WRS-stimulated colonic transit in a dose-dependent manner. However, YNS-15P had no significant effect on normal fecal excretion and colonic transit or on bethanechol-stimulated fecal excretion. YNS-15P also failed to inhibit castor-oil-induced diarrhea. These results indicate that YNS-15P selectively inhibits WRS-stimulated colonic propulsion, and that alpha-2 adrenoceptors may be involved in stress-induced colonic motor dysfunction in fed rats.

We found novel extra-chromosomal telomere repeat (ECTR) DNAs in telomerase-negative immortalized KMST-6 cells, by staining these cells with a (TTAGGG)n probe using both cycling oligonucleotide-primed in situ synthesis and by fluorescence in situ hybridization. Relatively small amounts of ECTR DNAs were also observed in telomerase-negative VA13 and SUSM-1 cells, but not observed in telomerase-positive immortalized HeLa cells. The ECTR DNAs existed mainly in the nucleoplasm with a small amount in the cytoplasm. The nucleoplasm ECTR DNAs were co-stained with an antibody directed to the telomeric-repeat binding factor 1 (TRF1), suggesting that they exist as a complex with TRF1. In consistent with these cytological studies, Southern blot analysis showed the existence of small telomere repeat DNAs. The ECTR DNA may provide an insight into the elucidation of the mechanisms responsible for the maintenance of telomeres in telomerase-negative immortalized cells.

The ribonucleoprotein enzyme telomerase synthesizes telomeric DNA onto chromosome ends. Telomere length is maintained, by the presence of telomerase activity, in the vast majority of primary tumours and stem cells, suggesting that telomere maintenance is essential for cellular immortalization. Recently, the telomerase RNA component in human and mouse (TERC and Terc, respectively), a telomerase-associated protein TEP1/TLP1 (refs 6,7) and the human catalytic subunit protein TERT (refs 8,9) have been identified. To examine the role of telomerase in telomere maintenance and cellular viability, we established Terc-deficient embryonic stem (ES) cells. It is known that telomerase activity is absent in cells from Terc-knockout mice. Although the study showed that telomere shortening was observed in the Terc-deficient cells from first to six generation animals, whether telomerase-dependent telomere maintenance was essential for cellular viability remained to be elucidated. To address this issue, we examined Terc-deficient ES cells under long-term culture conditions. Accompanying the continual telomere shortening, the growth rate of Terc-deficient ES cells was gradually reduced after more than 300 divisions. An impaired growth rate was maintained to approximately 450 divisions, and then cell growth virtually stopped. These data clearly show that telomerase-dependent telomere maintenance is critical for the growth of mammalian cells.

Lactitol has been reported to decrease the blood ammonia concentration in various experimental hyperammonemia models such as portacaval shunted rats. The mechanism responsible for this lowering of blood ammonia levels was investigated in rats. When lactitol was given orally twice a day for 7 days at doses of 3 and 10 g/kg/day and at half that daily dose on day 8, it significantly decreased the ammonium concentration of the portal blood by 27.3-43.2%, cecal ammonia contents by 49.2-57.6% and the pH of the cecal contents from 6.52 to 5.92-5.54, 4 hr after the final administration. Lactitol also inhibited increases in the portal ammonia concentration induced by the intracecal administration of ammonium acetate (300 mg/kg) 4 hr after the final administration. When lactitol was given orally at bolus doses of 1 and 3 g/kg simultaneously with a charcoal meal, lactitol significantly facilitated small intestinal transit by 12-13%. At a bolus dose of 3 g/kg, given 1 hr before the administration of a charcoal meal into the proximal colon, lactitol significantly facilitated colonic transit by 29.5%. These effects of lactitol were similar to those of lactulose. These findings suggest that lactitol decreases blood ammonia concentration by inhibiting both the production and the absorption of ammonia through reducing intestinal pH and shortening the residence time of intestinal contents in the intestinal tract.

Neovascularization is a prerequisite for glioma growth, so inhibition of angiogenesis may achieve control of glioma growth. We examined whether glioma cells induce angiogenesis and proliferation in microvascular endothelial cells from Fisher 344 rat brains by co-culture in a physical separation system with rat C6 glioma cells or rat T9 gliosarcoma cells. Endothelial cells cultured on type 1 collagen formed capillary-like structures. C6 glioma cells co-cultured with endothelial cells promoted the formation of these capillary-like structures. However, conditioned medium from C6 cells inhibited the proliferation of endothelial cells. T9 cells had little effect on the formation of capillary-like structures and no effect on the proliferation of endothelial cells. We also examined the effects of human tumor necrosis factor (TNF)-alpha on the formation of the capillary-like structures and on the proliferation of endothelial cells. Human TNF-alpha inhibited the formation of capillary-like structures induced by C6 glioma cells at a concentration of 100 U/ml, as well as the proliferation of endothelial cells at a concentration of 1000 U/ml. These results indicate that induction of angiogenesis varies with glioma cell lines and angiogenesis does not correspond with proliferation of endothelial cells. TNF-alpha can inhibit angiogenesis in gliomas in vitro.

Effects of urethane on gastric motility and mucosal ulcerogenic responses induced by indomethacin were investigated in the rat in relation to blood glucose levels (BGL) and compared with those of pentobarbital Na. Urethane (1.25 g/kg) given intraperitoneally, caused a progressive and significant rise in BGL, while pentobarbital (30 mg/kg) given intraperitoneally did not affect BGL. Subcutaneous administration of indomethacin (25 mg/kg) caused high-amplitude gastric contractions and induced hemorrhagic lesions in the stomachs of conscious rats. These lesions were significantly inhibited by urethane but not pentobarbital. Administration of urethane abolished basal gastric motility and almost completely suppressed the motility responses induced by indomethacin, while pentobarbital did not have much effect on gastric motility under basal and indomethacin-stimulated conditions. Acid secretion was significantly decreased by urethane and increased by pentobarbital. Pretreatment of the animals with yohimbine (5 mg/kg, subcutaneously) but not prazosin (0.5 mg/kg) inhibited the elevation in BGL seen after administration of urethane and allowed resumption both gastric motility and ulcerogenic responses induced by indomethacin, with less change in acid secretion. These results suggest that intraperitoneal administration of urethane prevented indomethacin-induced gastric lesions, probably by inhibiting the enhanced gastric motility response, and this effect may relate to its hyperglycemic action mediated by alpha 2-adrenoceptors. These findings also provide further evidence to support the importance of gastric motility in the pathogenesis of these lesions.

Intracranial chordomas are rare in childhood. Only 15 cases have been reported in children less than 6 years old. Bone destruction and calcification have been stated to be characteristics of this tumor. We present the case of a 5-year-old boy with clival chordoma without bone involvement.

Angiogenesis induced by rat glioma cells was examined in vitro using a double chamber co-culture system. Cultured microvascular endothelial cells from Fisher 344 rat brain, rat C6 glioma cells and rat T9 gliosarcoma cells were used for this study. Endothelial cells, cultured on type I collagen, formed capillary-like structures. In the co-culture system, C6 glioma cells promoted this formation. On the other hand T9 gliosarcoma cells had no effect on it. The supernatants of C6 glioma cells and T9 gliosarcoma cells suppressed the proliferation of the endothelial cells. C6 glioma cells probably produce and release soluble factors promoting angiogenesis. The proliferation of endothelial cells is thus suppressed while angiogenesis is made more intense. This in-vitro model is useful to elucidate the mechanism of tumor angiogenesis and to evaluate the promoting and inhibiting factors of angiogenesis.

We set up a new system for measuring the gastroduodenal HCO3- responses using pH deflection and the potential difference (PD) in the anesthetized rat. The stomach or the proximal duodenum was perfused at a flow rate of 0.7 ml/min with saline (pH 4.5), the pH of the perfusate and PD were continuously monitored, and HCO3- output was determined as acid-neutralizing capacity by back-titration of the perfusate to pH 4.5. In the case of the stomach, acid secretion was inhibited by omeprazole (60 mg/kg i.p.). Under these conditions, the pH, PD, and HCO3- output were 5.5 +/- 0.03, -3.8 +/- 0.4 mV, and 1.5 +/- 0.3 microEq/10 min in the duodenum and 5.4 +/- 0.04, -52.4 +/- 1.8 mV, and 1.2 +/- 0.3 microEq/10 min in the stomach, respectively. In addition, when various amounts of NaHCO3 were added into the system, a linear relationship was obtained between the area of pH deflection and the amount of added HCO3- (r = 0.999). Both pH and HCO3- output in these tissues were significantly increased by intravenous administration of prostaglandin E2, carbachol, and YM-14673 (a TRH analogue); the net HCO3- output in the duodenum was 8.7 +/- 1.3, 2.3 +/- 0.5, and 5.2 +/- 0.9 microEq, respectively. The values of net HCO3- output measured by back-titration coincided well with those obtained from the area of pH deflection caused by various agents. These results indicate that this system using pH deflection may be useful for quantitative determination of HCO3- response in the gastroduodenal mucosa.

The effects of YM-14673, an analogue of thyrotropin-releasing hormone, on duodenal HCO3- secretion were characterized in comparison with those of prostaglandin E2 and carbachol in anesthetized rats. The proximal duodenum was perfused with saline (pH 4.5), the pH of the perfusate and of the transmucosal potential difference were continuously monitored, and HCO3- output was determined by back-titration of the perfusate and by pH change. YM-14673 (0.1, 0.3 and 1 mg/kg) increased these parameters in a dose-related manner; the total HCO3- output at 1 mg/kg (5.8 +/- 0.7 mumol) was about 50% of that induced by prostaglandin E2 (1 mg/kg, i.v.) and 2 times greater than that induced by carbachol (4 micrograms/kg, i.v.). These responses, induced by YM-14673, were almost completely blocked by bilateral vagotomy and significantly inhibited by atropine (0.3 mg/kg, i.v.) or cyclooxygenase inhibitors, such as indomethacin (5 mg/kg, s.c.) and acetylsalicylic acid (40 mg/kg, s.c.), but not affected by pirenzepine (1 mg/kg, i.v.), a selective M1 antagonist. None of the latter agents had any effect on the HCO3(-)-stimulating action of prostaglandin E2, while the carbachol-induced HCO3- output was significantly reduced by atropine. These results suggest that YM-14673 induces the vagal-dependent HCO3- secretion in the rat duodenum and that this mechanism is mediated by muscarinic cholinoceptors, excluding the M1-subtype, and may involve endogenous prostaglandins.

Pathogenesis of duodenal ulcers induced by diethyldithiocarbamate (DDC), a superoxide dismutase (SOD) inhibitor, was investigated in the rat. Repeated s.c. administration of DDC (750 mg/kg) every 12 hr induced duodenal ulcers in the fed rats, and the severity of the ulcers reached the maximum after three injections. DDC not only reduced basal acid output but also impaired duodenal alkaline secretion. These ulcers were significantly prevented by antioxidative agents such as SOD (50000 units/kg, s.c.), allopurinol (50 mg/kg, s.c.) or glutathione (200 mg/kg, s.c.) as well as the antisecretory agent cimetidine (100 mg/kg, s.c.). The impaired HCO3- response caused by DDC was partially but significantly reversed by either SOD (15000 units/kg/hr, i.v.), allopurinol or glutathione; and SOD by itself significantly elevated the rate of basal alkaline secretion. 16,16-Dimethyl prostaglandin E2 (10 micrograms/kg, s.c.) increased duodenal HCO3- output in the presence of DDC and significantly prevented the development of duodenal ulcers in response to DDC. These results suggest that the mucosal antioxidative system including SOD may play a role in the regulatory process of alkaline secretion and contribute to the mucosal defensive ability in the duodenum. The insufficiency of this system may be involved in the pathogenesis of DDC-induced duodenal ulcers.

The effects of N-ethylmaleimide (NEM), a sulfhydryl (SH) blocker, on ethanol-induced gastric lesions were investigated in rats by varying the route of administration. Oral administration of acidified ethanol (60% ethanol in 150 mM HCl, 1 ml) produced hemorrhagic bandlike lesions in the gastric mucosa. Pretreatment of the animals with orally administered NEM (0.1-10 mg/kg) dose-dependently inhibited these lesions (the inhibition was over 80% at 1 mg/kg or greater), and the effects were partially reversed by indomethacin (5 mg/kg, subcutaneous). However, when NEM (10 mg/kg) was given subcutaneously, this agent significantly worsened the lesions. Intragastrically applied NEM produced a dose-dependent reduction of the transmucosal potential difference (PD) and the mucosal nonprotein SH levels, an increase of the volume of gastric contents, and an inhibition of gastric motility, while these parameters remained unaltered after subcutaneous administration of the agent. The microvascular permeability in the mucosa was significantly increased by both oral and subcutaneous administration of NEM (10 mg/kg) but remained unchanged in response to lower doses of orally administered (less than 3 mg/kg). These results suggest that NEM given orally is cytoprotective to the stomach against ethanol, probably by acting as a mild irritant and due to dilution of an irritant and inhibition of gastric motility (muscle relaxation), but when given subcutaneously it aggravates the lesions by unknown mechanisms.

Acid secretory and mucosal ulcerogenic responses to hypothermia (36-24 degrees C) were examined in anesthetized rats, and the role of thyrotropin-releasing hormone (TRH) in these responses was investigated. Lowering of body temperature (less than 32 degrees C) induced acid hypersecretion and damage in the gastric mucosa. These responses reached a maximum at a body temperature of 28 degrees C and were completely abolished by bilateral cervical vagotomy and significantly inhibited by intracerebroventricular (i.c.v.) administration of TRH antiserum (10 microliters/rat). TRH (10 micrograms/rat) given i.c.v. to the normothermia rat, caused an increase of acid secretion with a pattern similar to those observed during hypothermia. The blood levels of thyroid-stimulating hormone rose significantly during exposure of cold, and this response preceded the onset of acid hypersecretion and lesion formation. Thus, lowering of body temperature induces vagal-dependent gastric acid secretion, probably mediated by TRH released in response to cold exposure, and may be an important element in the etiology of stress ulceration.

The pathophysiological changes associated with hypothermia were investigated in the rat stomach under anesthetized conditions. The animal was placed in a styrene foam box and the core body temperature was kept between 24 and 36 degrees C using a heat lamp and refrigerant pack. Lowering of body temperature (less than 30 degrees C) produced acid hypersecretion and induced hemorrhagic lesions in the gastric mucosa; these responses reached the maximum at 28 degrees C, and a significant relationship was found between acid output and lesion score. Hypothermia (28 degrees C) also caused a marked increase of gastric contractile activity and mucosal blood flow (MBF), but the ratio of acid output to MBF became greater when compared to that obtained under normothermic conditions. These changes induced by hypothermia (28 degrees C) were completely blocked by vagotomy and were significantly inhibited by atropine, hexamethonium, clonidine, or TRH antiserum. However, lowering body temperature did not significantly affect acid secretory, motility, and ulcerogenic responses induced by carbachol in the vagotomized rat, excluding local mechanisms (suppression of the inhibitory nerves) in the hypothermia-induced changes. We conclude that hypothermia alone stimulates vagally dependent acid secretion and motility, resulting in damage in the gastric mucosa. These changes may be centrally mediated by TRH, which is released in association with the thermogenic response to hypothermia.

We have examined the effect of orally administered capsaicin on gastric motility in the rat to investigate a possible relationship between motility change and cytoprotection induced by this agent. Capsaicin, given orally (1-30 mg/kg), dose-dependently inhibited hemorrhagic band-like lesions induced by ethanol (60% in 150 mM HCl). This protection was significantly mitigated by desensitization of afferent neurons following capsaicin pretreatment 2 weeks before the experiment, and it was also significantly attenuated by prior administration of indomethacin, but not by spantide. Intragastric administration of capsaicin (30 mg/kg) significantly inhibited gastric motility and increased the mucosal blood flow, but had no effect on the transmucosal potential difference of the stomach. These functional changes induced by capsaicin were also less marked in the afferent neuronal desensitized rat, and they were significantly attenuated by indomethacin but not by spantide. These results suggest that the mucosal protection by intragastric capsaicin may be associated with the inhibition of gastric motility and the increase of mucosal blood flow. These responses may be induced by activation of primary afferent neurons which are probably sensitized by endogenous prostaglandins.

We set up a new system for measuring the gastroduodenal HCO3- responses using pH change and potential difference (PD) in the anesthetized rat. The stomach or the proximal duodenum was perfused at the flow rate of 0.7 mL/min with saline (pH 4.5), the pH of the perfusate and PD were continuously monitored, and HCO3- output was determined by back-titrating the perfusate and by measuring the area of pH change. In the case of the stomach, acid secretion was inhibited by omeprazole (60 mg/kg, i.p.). Output of both pH and HCO3- in these tissues was significantly increased by intravenous administration of prostaglandin (PGE2), 16, 16-dimethyl PGE2, carbachol, and YM-14673 (a thyrotropin-releasing hormone (TRH) analog), whereas the PD responded to these agents by a significant rise in the duodenum and decrease in the stomach. These parameters also responded to physiological stimulation such as mucosal acidification. When the area of pH change caused by various agents was plotted against the net amount of HCO3- output determined from back-titration, a significant relationship was found between these two factors (r = 0.98). These results indicate that this system using pH change may be useful for quantitative determination of HCO3- response in the gastroduodenal mucosa.

Pathogenesis of indomethacin-induced gastric lesions was investigated in the rat by measuring lesions, gastric motility, and terminal blood glucose levels and correlating them with each other. Subcutaneously administered indomethacin (3-25 mg/kg) dose-dependently produced lesions in the stomach with concomitant gastric hypermotility and reduction of blood glucose levels. When the lesion score and the motility were plotted against terminal glucose levels, a highly significant relationship was found among these three factors (P less than 0.01). Gastric lesions and hypermotility induced by indomethacin (25 mg/kg) were suppressed significantly by 16,16-dmPGE2 (10 micrograms/kg) with no effect on the glucose levels, while intravenous infusion of glucose (25% w/w, 1.4 ml/hr) prevented these responses and restored the reduced glucose levels above the basal values. In addition, both 16,16-dmPGE2 and glucose infusion afforded a significant protection against gastric lesions induced by indomethacin even in the acid-perfused stomach (150 mM HCl). These results confirmed gastric hypermotility as a key element in the pathogenesis of indomethacin-induced lesions and further suggested that indomethacin may sensitive gastric contractility through glycoprivic receptors by inducing hypoglycemia and PG deficiency.

Effects of a newly synthesized compound, KB-5492, on gastric lesions and gastric secretion were studied in rats. Oral KB-5492 inhibited the lesions induced by HCl.ethanol, HCl.aspirin, water-immersion stress, indomethacin, histamine and prednisolone at 30-300 mg/kg. The ED50 values varied from about 35 to 98 mg/kg. KB-5492 had no effect on gastric acid secretion even at 300 mg/kg. KB-5492 appeared to have a much more potent protective effect than a known anti-ulcer drug, sofalcone, against acute gastric lesions.

Possible involvement of increased mucosal permeability in the stimulation by prostaglandin E2 (PGE2) of duodenal HCO3- secretion was investigated in rats. PGE2 (0.3, 1 mg/kg, s.c.) dose-dependently increased HCO3- secretion in the duodenum with a significant elevation of transmucosal potential difference (PD); the PD was increased from -4.5 +/- 0.3 mV to -10.0 +/- 1.5 mV (mucosa negative) at 1 mg/kg. These responses caused by PGE2 were abolished by sacrificing the animals with saturated KCl (i.v.). Although a significant increase of HCO3- output was observed after exposure of the mucosa to 1 M NaCl (0.5 ml), this response was accompanied by a significant reduction of PD and was not abolished after KCl injection. The mucosal permeability determined by Evans blue (1%, i.v.) was not affected by PGE2, while 1 M NaCl markedly elevated the amount of extravasated dye in both the luminal content and the mucosa. Stimulation of HCO3- output by PGE2 was significantly mitigated by ouabain (3 mg/kg, s.c.) or prior exposure of the mucosa to 1 M NaCl. These results suggest that stimulation by PGE2 of duodenal HCO3- secretion is not simply due to the increased mucosal permeability, but depends rather on both the Na/K ATPase activity and the intact perfusion of the organ. The HCO3- response as induced by 1 M NaCl may result from the increased permeability and is accompanied by a marked reduction of PD.

The effect of baclofen (PCPGABA) on acid secretion, motility, and mucosa was investigated in the anesthetized rat stomach under various body temperatures (BT: 28-38 degrees C), and they were compared with those of 2-deoxy-D-glucose (2DG), an acid stimulant through cytoglycopenia. Under these conditions PCPGABA induces lesions dose-dependently (greater than 1 mg/kg, subcutaneously) in both the stomach and duodenum, and this action was dependent on BT; lowering of BT enhanced the ulcerogenicity. PCPGABA (3 mg/kg) had no effect on acid secretion at higher BT (36-38 degrees C) but produced a marked increase of acid output at lower BT (30-32 degrees C). 2DG caused a stimulation of acid output and gastric lesions without BT dependency, but the duodenal ulcerogenicity enhanced at lower BT. Gastric motility was enhanced significantly by these two agents to similar degrees, at either high or low BT. Neither PCPGABA nor 2DG affected alkaline secretion in the duodenum, while lowering of BT by itself reduced alkaline secretory responses. The above changes caused by PCPGABA and 2DG were blocked by both atropine and vagotomy. These results suggest that (1) acid stimulatory and ulcerogenic action of PCPGABA may involve a temperature-dependent process but does not relate to a cytoglycopenia, and (2) the vagus nerve mediating acid secretion and motility may be different in the temperature dependency.

In anesthetized rats oral administration (2 ml) of both ethanol (50% in 150 mM HCl) and aspirin (80 mM in 150 mM HCl) produced bandlike lesions in the stomach, while more generalized lesions occurred in the pylorus-ligated stomach when the irritant was given intragastrically through the fistula prepared in the rumen and the mucosal folds were removed by stomach distension. The bandlike lesions induced in the intact stomach by both irritants were significantly and dose-dependently prevented by 16,16-dimethyl PGE2 (dmPGE2: 3 and 10 micrograms/kg, subcutaneously), cysteamine (30 and 100 mg/kg, subcutaneously) or timoprazole (10 and 30 mg/kg, per os) at the doses which significantly inhibited gastric motility. In the pylorus-ligated stomach, however, neither of these agents showed any protection against the generalized lesions induced by ethanol, but such lesions caused by aspirin were significantly prevented only by dmPGE2. These agents also showed similar effects against the reduction of transmucosal PD in the pylorus-ligated stomach exposed to ethanol and aspirin. These results suggest that (1) the formation of bandlike lesions caused by ethanol and aspirin depends on the presence of mucosal folds and may be prevented by the agents that inhibit gastric motility, (2) the pathogenesis of the lesions induced by aspirin and ethanol may be different in the pylorus-ligated stomach, and (3) dmPGE2 has a unique protective ability that is not shared by usual cytoprotective agents.

We set up a system to measure the luminal pH, potential difference (PD) and bicarbonate output in the anesthetized rat duodenum, and investigated these responses caused by prostaglandins (PGs) and cholinergic agents. When the proximal duodenum (1.7 cm) was perfused at a flow rate of 0.7 ml/min with saline adjusted to pH 4.5, the duodenal pH, PD and HCO3- output were 5.5 to 6.0, -4 to -6 mV and 1.2 to 1.6 muEq/10 min, respectively; they were markedly reduced by i.v. injection of saturated KCl. Both natural (PGE1, PGE2) and synthetic (PGE2, PGl2) PGs, given either s.c. or i.v., significantly elevated all these parameters, while indomethacin (s.c.) decreased the pH as well as the PD. Small but significant increases of the pH were observed after i.v. administration of cholinergic agents (carbachol, bethanechol), a GABAergic agent (baclofen) and an analogue of thyrotropin releasing hormone (YM-14673), with a temporal elevation of the PD; the degree of net HCO3- output caused by these agents was 20-50% of the values obtained with PGE2 (100 micrograms/kg, i.v.), and they were significantly reduced in the presence of atropine. These results suggest that (a) the system using pH deflections can be used to sensitively detect HCO3- output in the rat duodenum, and (b) duodenal acid neutralizing capacity may be regulated by central and peripheral cholinergic systems as well as endogenous PGs.

A case of recurrent meningioma with malignant change and extracranial multiple metastases is reported. A 51-year-old female was operated on and left parasagittal meningioma was extirpated by Simpson grade II. Histological diagnosis was fibroblastic and transitional meningioma with slight atypism. Six years later, however, the tumor (transitional meningioma with slight mitosis) recurred in the same portion and was removed again by Simpson grade II. Further more, four years after the second operation, bilateral parasagittal meningioma (atypical meningioma; transitional type) was extirpated by Simpson grade I including superior sagittal sinus and falx. Only eight months after the last operation, a few tumors with central necrosis were demonstrated in the bilateral parasagittal area on a computerized tomography scan and she received radiation therapy. But the tumor had metastasized to the extracranial multiple organs including lungs, liver, pancreas, adrenal gland, muscles, multiple bones and lymph nodes. Post mortem diagnosis was malignant meningioma. We reviewed and discussed the characteristics of metastasizing meningioma, the effectiveness of radiation therapy on the prevention of recurrence of meningioma and the curative effect of radiation therapy for recurrent or metastasized meningioma.

The mechanism by which bilateral adrenalectomy worsens indomethacin-induced gastric lesions was investigated in rats. In sham-operated rats subcutaneously administered indomethacin produced gastric lesions at doses of 10 mg/kg body wt or greater, in association with lowering of blood glucose levels. In a parallel study, indomethacin induced gastric hypermotility at the same dose levels but had no effect on acid output or mucosal blood flow even at 25 mg/kg body wt. Adrenalectomy (2 wk) itself significantly reduced the blood glucose levels (approximately 50%) and markedly potentiated the ulcerogenic and motility responses caused by indomethacin; the ED50 values dropped to approximately 10 times lower than those in sham-operated rats. Both acid output and mucosal blood flow were significantly reduced by adrenalectomy, but these values were increased after indomethacin treatment (3 mg/kg body wt). The ulcerogenic and motility responses caused by indomethacin were significantly reduced by acute infusion of glucose (25% wt/wt, 1.2 ml/h) intravenously in both sham-operated and adrenalectomized rats, and by subcutaneous administration of hydrocortisone acetate (10 mg/kg body wt for 2 wk) in the latter group. When the motility and the ulcer score were determined in the same animals, a highly significant relationship was found between these two factors in both sham-operated and adrenalectomized rats. These results suggest that (a) the increased gastric motility may be a key element in the pathogenesis of indomethacin-induced lesions and in the mechanism for aggravation of the lesions and in the mechanism for aggravation of the lesions by adrenalectomy, and (b) abrasion of adrenal glands by inducing hypoglycemia may sensitize the system to indomethacin and increase gastric motility.

We assembled a new system using a lucite chamber and rat stomach for simultaneous measurement of transmucosal potential difference (PD) and luminal pH as indicators of the mucosal integrity and the secretory activity, respectively. The biological preparation involved only the glandular mucosa and responded to a variety of mucosal damaging agents by different degrees of PD reduction, pH increases and histological damages. When the mucosa was exposed for 10 min to 1 M NaCl, the reduced PD was restored with time, reaching the baseline values within 2 hr with histological restitution. Titration of gastric effluent showed that after the exposure, acid secretion ceased and a considerable amount of HCO3- was evident in the lumen, followed by re-secretion of acid. These secretory changes corresponded with those of luminal pH; this remained elevated for 1 hr after the exposure and returned to the basal values 2 hr later. The histological restitution as well as the PD recovery after damage were significantly interfered with by indomethacin (5 mg/kg, s.c.) or vasopressin (10 unit/kg/hr, i.v.), respectively, at the dose which inhibited the increased pH responses caused by 1 M NaCl or reduced the mucosal blood flow. These results suggest that this system may be useful for studying physiological changes of gastric mucosa after acute injury and for screening drugs that may have an effect on the repair process.

The present study was undertaken in rats using 2-deoxy-D-glucose (2DG) as a stimulator of gastric motility and a low dose of indomethacin as a prostaglandin (PG) synthesis inhibitor to investigate the roles of gastric motility and PG deficiency in the pathogenesis of indomethacin-induced gastric lesions. Subcutaneously administered indomethacin at 5 mg/kg did not induce any visible damage in the mucosa within 4 hr, but at 25 mg/kg produced linear hemorrhagic lesions along the long axis of the stomach. 2DG (100 mg/kg/hr), given intravenously, produced linear nonhemorrhagic lesions along the mucosal folds and, in the presence of 5 mg/kg of indomethacin, caused severe hemorrhagic lesions in the same areas of the stomach. Gastric motility was markedly enhanced by both indomethacin (25 mg/kg) and 2DG, while acid output and mucosal blood flow were increased only by the latter. Mucosal PGE2 levels were significantly reduced by indomethacin (25 mg/kg) but not by 2DG. Indomethacin at 5 mg/kg alone had no or little effect on any parameter except PG levels, which were reduced to similar degrees as caused by 25 mg/kg of the agent. Time-course development of the lesions was closely associated with those changes in gastric motility after administration of indomethacin (25 mg/kg) and 2DG. These results suggest that the enhanced gastric motility is, by itself, sufficient to induce damage (nonhemorrhagic) in the mucosa and that a PG deficiency alone does not induce any damage but is required for further extension to hemorrhagic lesions of nonhemorrhagic ones that are initially induced by enhanced gastric motility.(ABSTRACT TRUNCATED AT 250 WORDS)