Transfer function analysis of cerebral autoregulation dynamics during jaw movements.

Mastication as a mild but life-long exercise has been examined as a possibility for maintaining brain function in the elderly. Little is known, however, about the cerebral circulatory response during masticatory movement. The aim of this study was to develop a monitoring system for circulation dynamics during masticatory movement and to apply this system to the study of cerebral autoregulation. Cerebral blood flow, heart rate, and arterial blood pressure were simultaneously recorded, and changes in these circulatory systems were quantitatively evaluated in 38 young healthy volunteers. Transfer function analysis was also performed on blood pressure and cerebral blood flow for investigation of cerebral autoregulation during gum chewing. Although increases in cerebral blood flow, mean blood pressure, and heart rate suggested the activation of cerebral and systemic circulation during gum chewing, increased cerebral circulation was independent of systemic circulation. Our results suggest that cerebral autoregulation is well maintained during jaw movements.

Interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice.

Cytokines may be crucially involved in the pathogenesis of inflammatory bowel diseases (IBD), but it remains controversial whether interferon (IFN)-gamma, a typical proinflammatory cytokine, is an essential mediator to cause the disorders. In the present study, IFN-gamma(-/-) and wild-type (WT) C57BL/6 mice were fed 2.5% dextran sodium sulphate (DSS) in drinking water for 7 days, in order to investigate DSS-induced intestinal inflammation. The DSS-treated WT mice exhibited a robust production of IFN-gamma in the gut, a remarkable loss of body weight, as well as high rate of mortality (60%). In striking contrast, IFN-gamma deficient mice did not develop DSS-induced colitis, as indicated by the maintenance of body weight and survival rate of 100%. Severe intestinal inflammation was demonstrated exclusively in WT animals in terms of the shortening of the bowel as well as the elevation of the disease activity index, myeloperoxidase (MPO) activity and serum haptoglobin level. Histological study of DSS-treated WT intestine revealed disruption of mucosal epithelium and massive infiltration of inflammatory cells, while the organ from IFN-gamma(-/-) mice remained virtually normal in appearance. Enzyme-linked immunosorbent assay (ELISA) analyses indicated abundant production of three chemokines, i.e. monokine induced by interferon-gamma (MIG), interferon-inducible protein 10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), in the DSS-irritated intestine of WT but not of IFN-gamma(-/-) mice. The present results demonstrate clearly that IFN-gamma plays indispensable roles in the initiation of DSS colitis, and some chemokines are produced in an IFN-gamma-dependent fashion.

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart.

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

Six4, a putative myogenin gene regulator, is not essential for mouse embryonal development.

Six4 is a member of the Six family genes, homologues of Drosophila melanogaster sine oculis. The gene is thought to be involved in neurogenesis, myogenesis, and development of other organs, based on its specific expression in certain neuronal cells of the developing embryo and in adult skeletal muscles. To elucidate the biological roles of Six4, we generated Six4-deficient mice by replacing the Six homologous region and homeobox by the beta-galactosidase gene. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of the heterozygous mutant embryos revealed expression of Six4 in cranial and dorsal root ganglia, somites, otic and nasal placodes, branchial arches, Rathke's pouch, apical ectodermal ridges of limb buds, and mesonephros. The expression pattern was similar to that of Six1 except at the early stage of embryonic day 8.5. Six4-deficient mice were born according to the Mendelian rule with normal gross appearance and were fertile. No hearing defects were detected. Six4-deficient embryos showed no morphological abnormalities, and the expression patterns of several molecular markers, e.g., myogenin and NeuroD3 (neurogenin1), were normal. Our results indicate that Six4 is not essential for mouse embryogenesis and suggest that other members of the Six family seem to compensate for the loss of Six4.

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

The gamete fusion process is defective in eggs of Cd9-deficient mice.

The cell-surface molecule Cd9, a member of the transmembrane-4 superfamily, interacts with the integrin family and other membrane proteins. and is postulated to participate in cell migration and adhesion. Expression of Cd9 enhances membrane fusion between muscle cells and promotes viral infection in some cells. Fertilization also involves membrane fusion, between gametes. In mammals, the sperm binds to microvilli on the egg surface, and sperm-egg membrane fusion first occurs around the equatorial region of the sperm head12. The fused membrane is then disrupted, and the sperm nucleus as well as the cytoplasm is incorporated into the egg. Cd9 is expressed on the plasma membrane of the mouse egg, and an anti-Cd9 monoclonal antibody inhibits sperm-egg surface interactions. We generated Cd9 mice and found that homozygous mutant females were infertile. Sperm-egg binding was normal, but sperm-egg fusion was almost entirely inhibited in eggs from Cd9 females. Intracellular Ca2 oscillations, which signal fertilization, were absent in almost all mutant eggs; in rare cases, a response occurred after a long time period. In normal animals, Cd9 molecules were expressed on the egg microvilli and became densely concentrated at the sperm attachment site. Thus, our results show that Cd9 is important in the gamete fusion process at fertilization.

Assessment of exocrine pancreatic dysfunction in chronic pancreatitis.

The clinical diagnosis of chronic pancreatitis is usually based on imaging studies, pancreatic function tests, and the presence of characteristic clinical features. In Japan, diagnostic criteria for chronic pancreatitis were established in 1995. The secretin test (a duodenal intubation test) and the combination of noninvasive tests, N-benzoyl-L-tyrosyl-p-aminobenzoic acid (BT-PABA) and fecal chymotrypsin (FCT), have been recommended for evaluating exocrine pancreatic function in patients with chronic pancreatitis. In the present study, the diagnostic value of these two noninvasive tests was compared to the secretin test. Although noninvasive tests are less sensitive and specific for determining exocrine pancreatic dysfunction than the secretin test, greater reliability for diagnosing chronic pancreatitis can be obtained by performing the BT-PABA and FCT simultaneously. Assessment of exocrine pancreatic function is important not only to diagnose chronic pancreatitis but also to decide a treatment method with pancreatic enzyme preparation.

Pathophysiologic studies of experimental chronic pancreatitis in rats induced by injection of zein-oleic acid-linoleic acid solution into the pancreatic duct.

An experimental model of chronic pancreatitis was induced by a retrograde injection of a viscous solution consisting of zein-oleic acid-linoleic acid (0.05 ml/100 g body weight) into the rat pancreatic duct. Histologic and biochemical changes were investigated over a period of 6 months after induction of this model. The treated rats gained weight, but pancreatic weight decreased with time. Histologically, the widening of acinar lumen and cellular vacuolization occurred within 24 h at the parenchyma neighboring the small ducts filled with the injected solution. Degenerative parenchyma, interstitial edema, and inflammatory cell infiltration were pronounced 1 week later. Thereafter, duct-like tubular complex formation progressed, and the exocrine tissue exhibited marked atrophy of the gland with irregular fibrosis and fat replacement over a period of 6 months. Pancreatic contents of protein, amylase, DNA, and RNA markedly decreased, as did pancreatic weight, whereas hydroxyproline content increased. Oral administration of camostat did not affect pancreatic weight and contents of enzyme in this model. Urinary para-aminobenzoic acid (PABA) excretion in the BT-PABA test decreased to 54% at 6 weeks and 22% at 6 months. Although three quarters of pancreatic immunoreactive insulin (IRI) content was lost after 6 months, overt diabetes did not occur. The results suggest that an obstructive mechanism in the small ducts plays an important role in the genesis and development of chronic pancreatitis.

Relationship of plasma CCK to acinar cell regeneration in acute pancreatitis as studied by proliferating cell nuclear antigen.

In order to elucidate the relationship of cholecystokinin to acinar cell regeneration, the current study examined the changes in plasma cholecystokinin and immunostaining of proliferating cell nuclear antigen in the pancreas of rats with acute necrotizing pancreatitis. Proliferating cell nuclear antigen immunohistochemistry has been used to examine the proliferation of cells in several types of tissues. We compared the usefulness of proliferating cell nuclear antigen immunostaining and the incorporation of 5-bromodeoxyuridine to demonstrate acinar cell proliferation in the pancreas of rats with acute necrotizing pancreatitis. We also examined the relationship between these labeling indices and plasma cholecystokinin concentrations. The labeling index of paraformaldehyde-fixed specimens stained with proliferating cell nuclear antigen showed biphasic peaks at 12 hr and day 7. On the other hand, the methanol-fixed specimens stained with proliferating cell nuclear antigen and specimens stained with bromodeoxyuridine showed monophasic peaks in their labeling indices on day 5. There was a linear correlation (r = 0.808, P < 0.001) between the labeling index of bromodeoxyuridine and that of methanol-fixed proliferating cell nuclear antigen during the entire experimental period. During the regenerating phase, plasma cholecystokinin bioactivity showed positive correlations with the labeling index of bromodeoxyuridine and that of methanol-fixed proliferating cell nuclear antigen, r = 0.555 and 0.566, respectively (P < 0.001). Immunostaining of methanol-fixed proliferating cell nuclear antigen may be a useful tool for analyzing proliferating acinar cells. Acinar cell proliferation correlates with the bioactivity of plasma cholecystokinin during the regenerating phase of acute pancreatitis.