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Notes: Recently we developed a new microwave spectroscopy technique in the frequency range up to 40 GHz, and measured the static dielectric constant and the dielectric relaxation time for supercritical water. In the present work we report the dielectric properties of heavy water at temperatures and pressures up to 770 K and 59 MPa, respectively. The static dielectric constant of D2O as well as H2O are well described by the Uematsu–Franck formula when the number density instead of the mass density is used as the input parameter. The dielectric relaxation time decreases rapidly with increasing temperature in liquid H2O and D2O and jumps to a large value at the liquid–gas transition. The relaxation time of D2O is longer than that of H2O in the liquid state, and the difference becomes smaller with decreasing density in the gaseous state. For both H2O and D2O the most relevant parameter determining the relaxation time is the temperature at high densities or at low temperatures, and it is the density at low densities or at high temperatures. Based upon the observation that the dielectric relaxation time becomes fairly long in the dilute limit, we have concluded that the dielectric relaxation in the gaseous state is governed by the binary collision of water molecules and explained the relaxation time quantitatively by the collision time. We have extended the interpretation of the dielectric relaxation to the liquid state by taking into account the contribution of bound water molecules that are incorporated in the hydrogen-bond network. Anomalous relaxation at low temperatures is also discussed. © 1999 American Institute of Physics.

Notes: Microwave spectroscopy that can be applied to study the dielectric relaxation of various fluids under high temperature and pressure has been developed in the frequency range up to 40 GHz. By utilizing this new technique, the dielectric relaxation of water has been measured in the temperature and pressure range up to 750 °C and 120 MPa, which corresponds to a density range between 0.05 and 1 g/cm3. The static dielectric constant cursive-epsilon(0) is deduced from the time required for a microwave signal to travel through the sample by means of the time domain analysis, and is in good agreement with the literature. The dielectric relaxation time τ is obtained by fitting the experimentally observed microwave transmission rate to the value calculated using the S-matrices on the assumption that the dielectric constant obeys the Debye relaxation. The results of τ at ambient pressure agree very well with previous data. Below about 350 °C, τ rapidly decreases with increasing temperature nearly independent of pressure, while above about 350 °C, τ changes little with temperature and increases rapidly with decreasing density. It is concluded that the most relevant parameter determining τ is the temperature at lower temperatures or higher densities, and it is the density d at higher temperatures or lower densities. A possible change in the nature of hydrogen bonding is suggested to explain the observed temperature and density dependence of τ. © 1997 American Institute of Physics.

Notes: Galanin is a modulator of fast transmission in adult brain and recent evidence suggests that it also acts as a trophic factor during neurogenesis and neural injury and repair. Previous studies in our laboratory have identified galanin mRNA in Purkinje cells of adult and developing rat (but not adult mouse) cerebellum; and galanin-binding sites in adult mouse (but not rat) cerebellum. The post-natal development of the cerebellum provides a unique and convenient model for the investigation of developmental processes and to learn more about putative cerebellar galanin systems, the current study examined the presence and distribution of galanin-like-immunoreactivity (– LI), [125I]-galanin binding sites and galanin receptor-1 (GalR1) mRNA in post-natal mouse cerebellum. Using autoradiography and in situ hybridization, [125I]-galanin binding sites and GalR1 mRNA were first detected on post-natal day 10 (P10) in the external germinal layer of all lobes and high levels were maintained until P14. Quantitative real-time PCR assays detected GalR1 mRNA in whole cerebellum across the post-natal period, with a strong induction and peak of expression at P10. Assessment of galanin levels in whole cerebellum by radioimmunoassay revealed relatively similar concentrations from P5 to P20 and in adult mice (80–170 pg/mg protein), with a significantly higher concentration (250 pg/mg, p 〈 0.01) detected at P3. These concentrations were some four- to six-fold lower than those in adult forebrain samples. Using immunohistochemistry, galanin-like-immuno-reactivity was observed in prominent fibrous elements within the white matter tracts of the cerebellum at P3–5 and in more punctate elements in the internal granule cell layer and associated with the Purkinje cell layer at P12 and P20. Increased levels of GalR1 mRNA and galanin binding (attributed to GalR1) in the external granule cell layer at P10–12/(14) coincide with granule cell migration from the external to the inner granule cell layer and together with demonstrated effects of other neuropeptide-receptor systems suggest a role for GalR1 signalling in regulating this or related developmental processes.

Notes: Inflammation is invariably accompanied by angiogenesis; both are important in wound healing. However, it is not known how angiogenic factors that are present during the inflammatory phase of wound healing are involved in inflammation-induced angiogenesis. In this study, we address the contribution of two such factors, interleukin-8 (IL-8/CXCL8) and VEGF, in inflammation-induced angiogenesis. We determined the relative levels of these molecules during the first two weeks of the wound healing process in partial-thickness skin wounds of rabbits. IL-8 levels increase rapidly after wounding, reaching maximal levels after 24 hr; in contrast, VEGF levels peak at 3 days. We also determined the time course of the infiltration of various inflammatory cells and production of new blood vessels after wounding. Neutrophils were markedly increased 4 hr after wounding, and their level peaked at 24 hr. Monocyte infiltration peaked 48 hr after wounding and decreased by day 3. The number of blood vessels was significantly increased by day 3 after wounding and continued to increase through days 5 and 7.In order to determine the contribution of IL-8 and VEGF to the angiogenic process, we performed the wounding experiments in the presence and absence of IL-8 and VEGF antibodies. Treatment with IL-8 antibodies blocked the early stage of blood vessel formation but did not alter the late stage; in contrast, VEGF antibodies blocked the late stage without altering the early stage. Currently, we are performing studies using inhibitors against IL-8 and VEGF receptors to determine whether we observe similar results; we are also beginning to decipher molecular mechanisms involved in this process. This study suggests that IL-8 and VEGF play complementary roles in stimulating angiogenesis during wound healing.

Notes: Permeability of the endothelium occurs early in the angiogenic process, resulting from the initial loss or disruption of endothelial cell-cell adhesions, which is followed by endothelial cell contraction, forming paracellular gaps. We are interested in the angiogenesis and endothelial permeability induced by inflammation; thus, we have chosen to study mechanisms involved in the endothelial permeability stimulated by the angiogenic chemokine Interleukin-8 (IL-8/CXCL8). We have found that IL-8 induces permeability in a receptor-dependent process requiring the activation of Gαi and the regulation of PKA activity. Pertussis toxin abolishes the effect of IL-8 on endothelial permeability, while a PKA inhibitor, H89, results in a similar effect. Thus, regulation of the level of PKA activity appears critical in IL-8-induced permeability, potentially due to the differential regulation of molecules downstream of PKA, such as SREBP and RhoA. PKA and cAMP are thought to inhibit the activation of SREBP, a protein involved in lipid metabolism; we have also found, using inhibitors of SREBP that this molecule is likewise necessary for the permeability stimulated by IL-8. Furthermore, SREBP inhibitors block IL-8-induced activation of RhoA, a GTPase that has been implicated in permeability induced by multiple factors. Taken together, our results indicate a possible signal transduction pathway downstream of the IL-8 receptors, in which the inhibition of cAMP production, and, thus, of PKA activation, by Gαi increases SREBP activity, resulting in increased RhoA activity, leading to endothelial cell contraction and gap formation, increasing endothelial permeability. Because IL-8 plays important roles in the permeability observed in inflammatory disorders, knowledge of the signaling mechanisms induced by this chemokine during permeability increases may be used to identify targets for drug development.This project has been funded by the American Heart Association.

Notes: In mammals, corticotrophin-releasing hormone (CRH) and related peptides are known to play essential roles in the regulation of neuroendocrine, autonomic and behavioural responses to physical and emotional stress. In nonmammalian species, CRH-like peptides are hypothesized to play similar neuroendocrine and neurocrine roles. However, there is relatively little detailed information on the distribution of CRH neurones in the central nervous system (CNS) of nonmammalian vertebrates, and there are currently no comparative data on stress-induced changes in CRH neuronal physiology. We used a specific, affinity-purified antibody raised against synthetic Xenopus laevis CRH to map the distribution of CRH in the CNS of juvenile South African clawed frogs. We then analysed stress-induced changes in CRH immunoreactivity (CRH-ir) throughout the CNS. We found that CRH-positive cell bodies and fibres are widely distributed throughout the brain and rostral spinal cord of juvenile X. laevis. Strong CRH-immunoreactovity (ir) was found in cell bodies and fibres in the anterior preoptic area (POA, an area homologous to the mammalian paraventricular nucleus) and the external zone of the median eminence. Specific CRH-ir cell bodies and fibres were also identified in the septum, pallium and striatum in the telencephalon; the amygdala, bed nucleus of the stria terminalis and various hypothalamic and thalamic nuclei in the diencephalon; the tectum, torus semicircularis and tegmental nuclei of the mesencephalon; the cerebellum and locus coeruleus in the rhombencephalon; and the ventral horn of the rostral spinal cord. To determine if exposure to an acute physical stressor alters CRH neuronal physiology, we exposed juvenile frogs to shaking/handling and conducted morphometric analysis. Plasma corticosterone was significantly elevated by 30 min after exposure to the stressor and continued to increase up to 6 h. Morphometric analysis of CRH-ir after 4 h of stress showed a significant increase in CRH-ir in parvocellular neurones of the anterior preoptic area, the medial amygdala and the bed nucleus of the stria terminalis, but not in other brain regions. The stress-induced increase in CRH-ir in the POA was associated with increased Fos-like immunoreactivity (Fos-LI), and confocal microscopy showed that CRH-ir colocalized with Fos-LI in a subset of Fos-LI-positive neurones. Our results support the view that the basic pattern of CNS CRH expression arose early in vertebrate evolution and lend further support to earlier studies suggesting that amphibians may be a transitional species for descending CRH-ergic pathways. Furthermore, CRH neurones in the frog brain exhibit changes in response to a physical stressor that parallel those seen in mammals, and thus are likely to play an active role in mediating neuroendocrine, behavioural and autonomic stress responses.