Study of drug effects of calcium channel blockers on retinal degeneration of rd mouse

In the present study, we studied drug effects of Ca(2+) antagonists on the retinal degeneration of rd mouse to evaluate their efficacy. Several kinds of Ca(2+) antagonists, diltiazem, nicardipine, nilvadipine or nifedipine were administrated intraperitoneally and thereafter retinal morphology and functions were analyzed. In addition, we performed DNA microarray analysis both in nilvadipine treated and control retinas to understand their drug effects at molecular levels. We found that nilvadipine caused significant preservation of retinal thickness in rd mouse during the initial stage of the retinal degeneration, and nicardipine showed also significant but lesser preservation than nilvadipine. However, we recognized no preservation effects of diltiazem and nifedipine. In the total 3774 genes, the expressions of 27 genes were altered upon administration of nilvadipine, including several genes related to the apoptotic pathway, neuro-survival factor, Ca(2+) metabolisms, and other mechanisms. It is suggested that some types of Ca(2+) channel blockers, such as nilvadipine and nicardipine, are able to preserve photoreceptor cells in rd mouse and can potentially be used to treat some RP patients.     

Full-term development of hamster embryos produced by injection of round spermatids into oocytes

The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.     

Endogenous levels of abscisic acid and gibberellins in leaf protoplasts competent for plant regeneration in Betula platyphylla and Populus alba

Differences in endogenous levels of abscisic acid and gibberellinsbetween Betula platyphylla and Populusalba leaf protoplasts were determined using micro-scale extractionand purification steps, including thin layer chromatography ormicro-high-performance-liquid-chromatography and quantification by enzymelinkedimmunosorbent assay or micro-bioassay. The content of abscisic acid was tentimes higher in B. platyphylla than in P.alba on the basis of both cell number and dry weight; in contrast,levels of gibberellins were lower in the former. Leaf protoplasts of bothspecies are competent for plant regeneration through the exogenous supply ofauxins and cytokinins. The function of abscisic acid in these protoplastcultures is discussed in relation to the need for a strong cytokinin,N-(2-chloro-4-pyridyl)-N-phenylurea (4-CPPU) for colony proliferation inB. platyphylla, in contrast to a weak cytokininrequirementin P. alba.     

Down-Regulation of the Noradrenaline Transporter by Interferon-α in Cultured Bovine Adrenal Medullary Cells

Abstract: The aim of this study was to investigate the effect of long-term treatment with interferon (IFN)-α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN-α caused a decrease in uptake of [³H]noradrenaline by the cells in time (4–48 h)- and concentration (300–1,000 U/ml)-dependent manners. IFN-β also inhibited [³H]noradrenaline uptake to a lesser extent than did IFN-α, whereas IFN-γ had little effect. An anti-IFN-α antibody reduced the effect of IFN-α on [³H]noradrenaline uptake. Saturation analysis of [³H]noradrenaline uptake showed that the inhibitory effect of IFN-α was due to a reduction in the maximal uptake velocity ( V _max) values without altering apparent Michaelis constant ( K _m) values. Incubation of cells with IFN-α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN-α on [³H]noradrenaline uptake was diminished in protein kinase C-down-regulated cells. Incubation of cells with IFN-α for 48 h significantly reduced the specific binding of [³H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [³H]desipramine binding revealed that IFN-α decreased the maximal binding ( B _max) values without any change in the dissociation constant ( K _D) values. These findings suggest that IFN-α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Expression of the Dan gene during chicken embryonic development.

The Dan gene was first identified as the putative rat tumor suppressor gene and encodes a protein structurally related to Cerberus and Gremlin in vertebrates. Xenopus DAN, as with Cerberus and Gremlin, was demonstrated to block bone morphogenetic protein (BMP) signaling by binding BMPs, and to be capable of inducing additional anterior structures by ectopic overexpression in Xenopus embryos. DAN, thus, is suggested to play pivotal roles in early patterning and subsequent organ development, as in the case of other BMP antagonists. In this report, we isolated the chicken counterpart of Dan. Chicken Dan is mainly expressed in the cephalic and somitic mesoderm and several placodes during organ development.     

Transient local overexpression of human vascular endothelial growth factor (VEGF) in mouse feto-maternal interface during mid-term pregnancy lowers systemic maternal blood pressure.

The effect on maternal circulation of transient human vascular endothelial growth factor (VEGF) (165) cDNA transfection into the mouse feto-maternal interface at day 14.5 post coitus (p.c.) using a hemagglutinating virus of Japan-envelope (HVJ-E) vector system is reported. On day 15.5 p.c., Western blotting clearly showed overexpression of 18 kD VEGF protein in the uterus. After VEGF transfection, the blood pressure was significantly lowered for 48 hours. On day 17.5 p.c., the blood pressure returned to the control level. Proteinuria was not observed after VEGF transfection. No preterm birth was observed during the course of pregnancy after the transfection procedure. After 24 hours of transfection, human VEGF was not detectable and the mouse VEGF level was similar to that in peripheral blood. However, the soluble fms-like tyrosine kinase (Flt)-1 concentration was significantly lower in VEGF-transfected mice. These results suggest that extraamniotic VEGF overexpression lowered the systemic blood pressure without altering the VEGF concentration in the peripheral blood. Local overexpression of VEGF may become a novel treatment for pregnancy-related disorders such as hypertension complicated-pregnancy and preeclampsia.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.