Koichi Kawanishi

A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.

Immunohistochemical Identification of Galanin and Growth Hormone-Releasing Factor-Containing Neurons Projecting to the Median Eminence of the Rat

Through the combined demonstration of the retrograde transport of True blue and the immunohistochemical staining of galanin (GAL), the GAL neurons that project to the median eminence were identified. Moreover, the distribution of GAL and growth hormone-releasing factor (GRF) was analyzed in the arcuate nucleus (ARC) with an elution-restaining procedure. Following the injection of True blue into the median eminence, GAL-positive cells labeled with True blue were found mainly in the ARC. In addition, a few GAL neurons in the periventricular, the paraventricular and the supraoptic nuclei were labeled with True blue. The elution-restaining results revealed that many retrogradely labeled GAL neurons in the ARC contained GRF. These findings suggest that a subpopulation of GAL neurons in the ARC sending axons to the median eminence produces, stores and releases a GRF-like peptide.

Induction of Fos protein in the rat hypothalamus elicited by insulin-induced hypoglycemia

To evaluate the responses to insulin-induced hypoglycemia of neurons in vivo, we studied Fos protein induction in the brain by means of immunohistochemistry. The induction of Fos protein was maximum after the first injection of insulin for 3 h. This induction was found in the parvocellular division of paraventricular nucleus (PVN), the periventricular, dorsomedial and arcuate nuclei and the lateral hypothalamic area of the hypothalamus. These findings show the activation of specific subsets of neurons in areas of the hypothalamus following hypoglycemic stimulation.

Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.

EFFECT OF CONTINUOUS INTRAVENOUS INJECTION OF INTERLEUKIN-6 AND PRETREATMENT WITH CYCLOOXYGENASE INHIBITOR ON BRAIN C-FOS EXPRESSION IN THE RAT

The aim of the present study was to assess potential brain sites of stimulation by peripheral interleukin (IL)-6 of the hypothalamo-pituitary-adrenal (HPA) axis in the rat, using c-fos protein as a marker of cellular activation. Involvement of prostaglandins in IL-6-induced ACTH secretion and c-fos expression was also investigated. IL-6 was infused continuously (40 ng/min) for 90 min to conscious male rats. Blood samples were taken before the infusion and at 30 and 90 min for measurement of plasma ACTH. Expression of c-fos in the brain was examined by immunohistochemistry. Administration of IL-6 significantly elevated plasma ACTH levels at 30 min (495 +/- 105 vs. 117 +/- 17 pg/ml in controls, p < 0.05). Elevated levels were still present at 90 min (596 +/- 139 vs. 113 +/- 20 pg/ml in controls, p < 0.05). Infusion of IL-6 (3.6 micrograms/rat) markedly triggered c-fos expression in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), as well as in the central amygdaloid nucleus (CeA), the nucleus tractus solitarius and the locus coeruleus. Pretreatment with the cyclooxygenase inhibitor indomethacin (10 mg/kg, i.v.) suppressed the ACTH response induced by IL-6. The number of IL-6-induced immunoreactive cells in the PVN was significantly reduced by indomethacin pretreatment (p < 0.01), but the number of IL-6-induced c-fos-positive cells in the SON and CeA remained unchanged. These findings suggest that circulating IL-6 may exert central actions by acting directly or indirectly on brain neurons. In addition, the ability of IL-6 to activate the HPA axis may depend upon the release of prostaglandins, probably in the brain.

Minimal Hepatitis C Infectivity in Semen

Excerpt To the Editors: Both hepatitis B virus and cytomegalovirus are known to be transmitted sexually. To assess the possibility that hepatitis C virus (HCV), a known blood-based infection, could...

Effect of Excitatory Amino Acid Receptor Agonists on Secretion of Growth Hormone as Assessed by the Reverse Hemolytic Plaque Assay

Recent findings indicate that excitatory amino acids (EAAs) can modulate growth hormone (GH) secretion in several mammalian species in vivo and in vitro. In this study, we examined the effects of EAA receptor antagonists [N-methyl-D,L-aspartate (NMDA), kainic acid, L-glutamate] on GH secretion by the reverse hemolytic plaque assay (RHPA). Anterior pituitary cells of adult male Sprague-Dawley rats were enzymatically dispersed and subjected to RHPA. EAA receptor agonists increased the mean plaque area in a dose-dependent manner: the maximal increase was observed at 10 microM and increased the fraction of somatotrophs forming large plaques. NMDA (10 microM) did not increase the mean plaque area in the presence of the NMDA receptor antagonists 10 microM AP-7 and 10 microM MK-801. Coincubation of kainic acid with the non-NMDA receptor antagonist CNQX blocked the kainic-acid-stimulated increase in GH secretion. The addition of MK-801, AP-7 or CNQX to glutamate caused a partial reduction of the mean plaque area. Ten micromoles per liter glutamate with 10 nM GH-releasing hormone (GHRH) produced an additive effect on GHRH-induced GH release. Somatostatin suppressed the stimulatory action of glutamate. We speculate that glutamate plays a role in the regulation of GH secretion.

Effect of Central and Continuous Intravenous Injection of lnterleukin-1βon Brain c-fosExpression in the Rat: Involvement of Prostaglandins

Utilizing immunohistochemistry for the c-fos protein to detect neuronal activity, we examined the effects of continuous intravenous and intracerebroventricular infusion of interleukin (IL)-1 beta in the rat brain, and the involvement of prostaglandins (PGs) in IL-1 beta-induced c-fos expression. Continuous intravenous infusion of IL-1 beta (10 ng/min) markedly augmented c-fos expression in the paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus as well as in the central amygdaloid nucleus (CeA). The number of IL-1 beta-induced c-fos-positive cells in the PVN and SON was significantly lower in rats pretreated with indomethacin than in vehicle-treated rats. However, the number of IL-1 beta-induced c-fos-positive cells in the CeA remained unchanged. c-fos protein was induced after intracerebroventricular infusion of IL-1 beta (200 ng) in the PVN, SON, and arcuate nuclei of the hypothalamus, and in the CeA. The induction of c-fos immunoreactivity by central administration of IL-1 beta was blocked by indomethacin (500 micrograms/rat), except in the CeA. These findings suggest that PGs are involved in the complex transmission of signals from circulating or central IL-1 beta to hypothalamic neurons.

Neurotensin and growth hormone-releasing factor-containing neurons projecting to the median eminence of the rat: A combined retrograde tracing and immunohistochemical study

The origins of the neurotensin (NT)-containing nerve terminals in the median eminence were determined in rats by a combination of retrograde labeling with True blue and immunofluorescence staining. Moreover, the distribution of NT and growth hormone-releasing factor (GRF) was analyzed in the arcuate nucleus (ARC) with an elution-restaining procedure. The retrogradely labeled NT neurons were found mainly in the ARC. Only a few labeled NT neurons were seen in the periventricular and the paraventricular nuclei. The elution restaining procedure demonstrated that an average of 43% of the labeled NT cells in the ventral part of the ARC restained for GRF. These findings support the hypothesis of co-expression of NT with GRF from the ARC.

Sites of origin of growth hormone-releasing factor-containing neurons projecting to the stalk-median eminence of the rat

The topographical location of neurons containing GRF which project to the median eminence were studied with immunofluorescence for GRF in combination with the retrograde transport of True blue. After the injection of True blue into the median eminence, retrogradely-labeled GRF neurons were identified in the arcuate nucleus and the lateral basal hypothalamus. GRF neurons in the perifornical area contained no positive dye. We concluded that the location of neurons containing hypophysiotrophic GRF are confined within the arcuate nucleus and the lateral basal hypothalamus.