Glucose, a carbohydrate metabolite, plays a major role in the energy supply for fish iono- and osmoregulation, and the way that glucose is transported in ionocytes is a critical process related to the functional operations of ionocytes. Eighteen members of glucose transporters (GLUTs, SLC2A) were cloned and identified from zebrafish. Previously, Na(+),K(+)-ATPase-rich (NaR), Na(+)-Cl(-) cotransporter-expressing (NCC), H(+)-ATPase-rich (HR), and glycogen-rich (GR) cells have been identified to be responsible for Ca(2+) uptake, Cl(-) uptake, Na(+) uptake, and the energy deposition, respectively, in zebrafish skin/gills. The purpose of the present study was to test the hypothesis of whether GLUT isoforms are specifically expressed and function in ionocytes to supply energy for ion regulatory mechanisms. On the basis of translational knockdown of foxi3a/3b (2 transcriptional factors related to the ionocytes' differentiation) and triple in situ hybridization/immunocytochemistry, 3 GLUT isoforms, zglut1a, -6, and -13.1, were specifically localized in NaR/NCC cells, GR cells, and HR cells, respectively. mRNA expression of zglut1a in embryos and adult gills were stimulated by the low Ca(2+) or low Cl(-) freshwater, which has been previously reported to upregulate the functions (monitored by epithelial Ca(2+) channel, NCC mRNA) of NaR/NCC cells, respectively while that of zglut13.1 was stimulated only by low Na(+), a situation to upregulate the function (monitored by carbonic anhydrase 15a mRNA) of HR cells. On the other hand, ambient ion compositions did not affect the zglut6 mRNA expression. Taken together, zGLUT1a, -6, and 13.1, the specific transporters in NaR/NCC cells, GR cells, and HR cells, may absorb glucose into the respective cells to fulfill different physiological demands.

We report the encapsulation of quantum dots with biologically important beta-N-acetylglucosamine (GlcNAc) in different ratios, together with studies of their specific/sensitive multivalent interactions with lectins and sperm by fluorimetry, transmission electron microscopy, dynamic light scattering microscopy, confocal imaging techniques, and flow cytometry. These GlcNAc-encapsulated quantum dots (QDGLNs) specifically bind to wheat germ agglutinin, and cause fluorescence quenching and aggregation. Further studies of QDGLNs and the mannose-encapsulated QDs (QDMANs) with sperm revealed site-specific interactions, in which QDGLNs bind to the head of the sperm, while QDMANs spread over the whole sperm body.

The production of inositol 1,4,5-trisphosphate (InsP3) has been reported to mediate the transient rise in intracellular Ca2+ activity ([Ca2+]i) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP3 during fertilization is still lacking. We investigated this question by blocking the InsP3 synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca2+ release in a dose-dependent manner, although above 15 microM U73122 eggs showed an elevated resting [Ca2+]i and a lower fertilization rate. The inhibition of Ca2+ transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca2+ response. U73122 also prevented the accompanying rise in intracellular pH (pHi), which is mediated by the activation of the Na+-H+ antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol, which is the other hydrolytic product of phosphatidylinositol 4,5-bisphosphate by PLC. Further evidence of the specificity of U73122 action was inhibition of the increase in InsP3 mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTPgammaS-induced Ca2+ release and pHi rise in unfertilized eggs. These results suggested that the transient rise in Ca2+ in sea urchin during fertilization requires the production of InsP3.

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BackgroundZebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.Methodology/Principal FindingsThis study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling.Conclusion/SignificanceThe killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo.