Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca 2ϩ -dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffin cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dyn1-KO increased quantal size (QS, but not vesicle size/content) by Ն250% in dyn1-KO mice; (2) the KO-increased QS was rescued by dyn1 (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size (d p ) was increased from Յ9 to Ն9 nm by KO. Therefore, Ca 2ϩ -induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffin cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells. Ca 2ϩ -dependent neurotransmitter release from a single vesicle is the primary event in all neurotransmission, including synaptic/ neuroendocrine forms. To determine whether Ca 2ϩ -dependent vesicular neurotransmitter release is "all-or-none" (quantal), we provide compelling evidence that most Ca 2ϩ -induced secretory events occur via the subquantal mode in native adrenal chromaffin cells. This subquantal release mode is promoted by dynamin 1, which is universally required for most secretory cells, including neurons and neuroendocrine cells. The present work with dyn1-KO mice further confirms that Ca 2ϩ -dependent transmitter release is mainly via subquantal mode, suggesting that subquantal release could be also important in other types of cells.

Mass mortality of cultured white shrimp in southern Taiwan was diagnosed as an outbreak of Taura syndrome (TS). From late 1998 to early 1999, 90% of the shrimp ponds were abandoned at 30 to 45 d after stocking post larvae from Ecuador and elsewhere. The shrimp began to die within 2 to 3 d after they stopped feeding. There were no other gross signs, except that some affected shrimp had reddened tails. Histologically, multifocal necrosis of the cuticular epithelium was the main lesion. The necrotic foci contained pyknotic and karyorrhectic nuclei, and many lightly and densely stained intracytoplasmic and intercellular, spherical inclusions.

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A new cell line (GS-1) was developed from the spleen tissue of the orange-spotted grouper, Epinephelus coioides applied for viral infection studies of fish ranavirus and megalocytivirus. The cells proficiently multiplied in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum at temperatures between 20°C and 32°C. Morphologically, the cell line comprised fibroblast-like cells, and this was confirmed by immunostaining with vimentin, fibronectin, and desmin antibodies. The optimal temperature for grouper iridovirus (GIV) and infectious spleen and kidney necrosis virus (ISKNV) proliferation in GS-1 cells was 25°C, and the highest titer of GIV was 108.4 TCID50/ml, and the highest titer of ISKNV was 105.2 TCID50/ml. Electron micrographs showed that the mean diameter of GIV virions was 180−220 nm, which was larger than ISKNV virions (160−200 nm). Negatively stained GIV particles possessed an envelope structure that was assembled by the three-layered structure with an inner electron-dense core surrounded by a lighter coat (mean diameter, 27 ± 3 nm). The highest GIV-induced mortality of groupers occurred at 25°C, whereas the highest ISKNV-induced mortality occurred at 30°C. In summary, GS-1 cell line is a valuable tool for isolating and investigating fish ranavirus and megalocytivirus in the same host system.

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Orf virus (ORFV), a member of parapoxvirus, is an enveloped virus with genome of double-stranded DNA. ORFV causes contagious pustular dermatitis or contagious ecthyma in sheep and goats worldwide. In general, detection of viral DNA and observing ORFV virion in tissues of afflicted animals are two methods commonly used for diagnosis of orf infection; however, isolation of the ORFV in cell culture using virus-containing tissue as inoculum is known to be difficult. In this work, the ORFV (Hoping strain) isolated in central Taiwan was successfully grown in cell culture. We further examined the biochemical characteristic of our isolate, including viral genotyping, viral mRNA and protein expression. By electron microscopy, one unique form of viral particle from ORFV infected cellular lysate was demonstrated in the negative-stained field. Moreover, immunomodulating and anti-influenza virus properties of this ORFV were investigated. ORFV stimulated human monocytes (THP-1) secreting proinflammatory cytokines IL-8 and TNF-α. And, pre-treatment of ORFV-infected cell medium prevents A549 cells from subsequent type A influenza virus (IAV) infection. Similarly, mice infected with ORFV via both intramuscular and subcutaneous routes at two days prior to IAV infection significantly decreased the replication of IAV. In summary, the results of a current study indicated our Hoping strain harbors the immune modulator property; with such a bio-adjuvanticity, we further proved that pre-exposure of ORFV protects animals from subsequent IAV infection.

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