LY. Ammonium-dependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae. In this study, a scanning ion-selective electrode technique (SIET) was applied to measure H ϩ , Na ϩ , and NH 4 ϩ gradients and apparent fluxes at specific cells on the skin of medaka larvae. Na ϩ uptake and NH3/NH 4 ϩ excretion were detected at most mitochondrion-rich cells (MRCs). H ϩ probing at MRCs revealed two group of MRCs, i.e., acid-secreting and base-secreting MRCs. Treatment with EIPA (100 M) blocked 35% of the NH3/ NH 4 ϩ secretion and 54% of the Na ϩ uptake, suggesting that the Na ϩ /H ϩ exchanger (NHE) is involved in Na ϩ and NH3/NH 4 ϩ transport. Low-Na ϩ water (Ͻ0.001 mM) or high-NH 4 ϩ (5 mM) acclimation simultaneously increased Na ϩ uptake and NH3/NH 4 ϩ excretion but decreased or even reversed the H ϩ gradient at the skin and MRCs. The correlation between NH 4 ϩ production and H ϩ consumption at the skin surface suggests that MRCs excrete nonionic NH3 (base) by an acid-trapping mechanism. Raising the external NH 4 ϩ significantly blocked NH 3/NH4 ϩ excretion and Na ϩ uptake. In contrast, raising the acidity of the water (pH 7 to pH 6) enhanced NH3/NH 4 ϩ excretion and Na ϩ uptake by MRCs. In situ hybridization and real-time PCR showed that the mRNAs of the Na ϩ /H ϩ exchanger (slc9a3) and Rhesus glycoproteins (Rhcg1 and Rhbg) were colocalized in MRCs of medaka, and their expressions were induced by low-Na ϩ acclimation. This study suggests a novel Na ϩ /NH 4 ϩ exchange pathway in apical membranes of MRCs, in which a coupled NHE and Rh glycoprotein is involved and the Rh glycoprotein may drive the NHE by generating H ϩ gradients across apical membranes of MRCs.

In last few years, the use of zebrafish (Danio rerio) in scientific research is growing very rapidly. Initially, it was a popular as a model of vertebrate development because zebrafish embryos are transparent and also develop rapidly. Presently, the research using zebrafish is expanding into other areas such as pharmacology, clinical research as a diseases model and interestingly in drug discovery. The use of zebrafish in pharmaceutical research and discovery and drug development is mainly screening of lead compounds, target identification, target validation, morpholino oligonucleotide screens, assay development for drug discovery, physiology based drug discovery, quantitative structure-activity relationship (QSAR) and structure -activity relationships (SAR) study and drug toxicity study. In this paper, we have described properly all the areas of drug discovery where zebrafish is used as a tool. We are hopeful that the use of these techniques or methods will make the zebrafish a prominent model in drug discovery and development research in the forthcoming years.

Background and purpose: Natural compounds obtained from marine organisms have received considerable attention as potential sources of novel drugs for treatment of human inflammatory diseases. Capnellene, isolated from the marine soft coral Capnella imbricate, 4,4,6a-trimethyl-3-methylene-decahydro-cyclopenta[ə]pentalene-2,3a-diol (GB9) exhibited antiinflammatory actions on activated macrophages in vitro. Here we have assessed the anti-neuroinflammatory properties of GB9 and its acetylated derivative, acetic acid 3a-hydroxy-4,4,6a-trimethyl-3-methylene-decahydro-cyclopenta[ə]pentalen-2-yl ester (GB10). Experimental approach: Effects of GB9 or GB10 on the expression of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in interferon-g (IFN-g)-stimulated mouse microglial BV2 cells were measured by Western blot. The in vivo effects of these compounds were examined in the chronic constriction injury (CCI) rat model of neuropathic pain, measuring thermal hyperalgesia, and microglial activation and COX-2 protein in lumbar spinal cord, by immunohistochemistry. Key results: In BV2 cells, GB9 and GB10 inhibited the expression of iNOS and COX-2, stimulated by IFN-g. Intrathecal administration of GB9 and GB10 inhibited CCI-induced nociceptive sensitization and thermal hyperalgesia in a dose-dependent manner. Intraperitoneal injection of GB9 inhibited CCI-induced thermal hyperalgesia and also inhibited CCI-induced activation of microglial cells and up-regulation of COX-2 in the dorsal horn of the lumbar spinal cord ipsilateral to the injury. Conclusion and implications: Taken together, these data indicate that the marine-derived capnellenes, GB9 and GB10, had anti-neuroinflammatory and anti-nociceptive properties in IFN-g-stimulated microglial cells and in neuropathic rats respectively. Therefore, capnellene may serve as a useful lead compound in the search for new therapeutic agents for treatment of neuroinflammatory diseases.

Tyrosinase is involved in melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders that can be treated with depigmenting agents. A natural product T1, bis(4-hydroxybenzyl)sulfide, isolated from the Chinese herbal plant, Gastrodia elata, is a strong competitive inhibitor against mushroom tyrosinase (IC50 = 0.53 μM, Ki = 58 ± 6 nM), outperforms than kojic acid. The cell viability and melanin quantification assay demonstrate that 50 μM of T1 apparently attenuates 20% melanin content of human normal melanocytes without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that T1 effectively reduces melanogenesis with no adverse side effects. The acute oral toxicity study evidently confirms that T1 molecule is free of discernable cytotoxicity in mice. Furthermore, the molecular modeling demonstrates that the sulfur atom of T1 coordinating with the copper ions in the active site of tyrosinase is essential for mushroom tyrosinase inhibition and the ability of diminishing the human melanin synthesis. These results evident that T1 isolated from Gastrodia elata is a promising candidate in developing pharmacological and cosmetic agents of great potency in skin-whitening.

Five new cembranoids, namely, crassumolides A and B and D-F (1 and 2 and 4-6), along with four known metabolites, 7-10, were isolated from the soft coral Lobophytum crassum. Crassumolide C (3) was isolated for the first time from a natural source. The structures of these compounds were elucidated by extensive spectroscopic analysis and comparison of the NMR data with those of known analogues. The absolute stereochemistry of 1 was determined using the modified Mosher's method. Chemical transformation of 7 into the corresponding methyl ester 3 revealed the absolute stereochemistry of 3. Compounds 1, 3, and 7 were cytotoxic toward Ca9-22 cancer cells, and 10 was broadly cytotoxic toward all six test cancer cell lines used. Compounds 1, 3, 7, and 10 were found to inhibit the accumulation of the pro-inflammatory proteins iNOS and COX-2 at 10 μM.