Inhibitory effect of Cinnamomum osmophloeum Kanehira ethanol extracts on melanin synthesis via repression of tyrosinase expression.

Melanin contributes to skin color, and tyrosinase is the enzyme that catalyzes the initial steps of melanin formation. Therefore, tyrosinase inhibitors may contribute to the control of skin hyperpigmentation. The inhibition of tyrosinase activity by Cinnamomum zeylanicum extracts was previously reported. In this report, we test the hypothesis that Cinnamomum osmophloeum Kanehira, an endemic plant to Taiwan, contains compounds that inhibit tyrosinase activity, similar to C. zeylanicum. The cytotoxicity of three sources of C. osmophloeum Kanehira ethanol extracts was measured in B16-F10 cells using a methyl thiazolyl tetrazolium bromide (MTT) assay. At concentrations greater than 21.25 µg/mL, the ethanol extracts were toxic to the cells; therefore, 21.25 µg/mL was selected to test the tyrosinase activities. At this concentration, all three ethanol extracts decreased the melanin content by 50% in IBMX-induced B16-F10 cells. In addition to the melanin content, greater than 20% of the tyrosinase activity was inhibited by these ethanol extracts. The RT-PCR results showed that tyrosinase and transcription factor MITF mRNAs expression were down-regulated. Consistent with the mRNA results, greater than 40% of the human tyrosinase promoter activity was inhibited based on the reporter assay. Furthermore, our results demonstrate that the ethanol extracts protect cells from UV exposure. C. osmophloeum Kanehira neutralized the IBMX-induced increase in melanin content in B16-F10 cells by inhibiting tyrosinase gene expression at the level of transcription. Moreover, the ethanol extracts also partially inhibited UV-induced cell damage and prevented cell death. Taken together, we conclude that C. osmophloeum Kanehira is a potential skin-whitening and protective agent.

The hormonal regulation of color changes in the sexually dichromatic frog Buergeria robusta.

During the breeding season, dynamic changes in body coloration are regularly observed in the male brown tree frog Buergeria robusta. This study investigated the hypothesis that this sexual dichromatism in male B. robusta is mediated through hormonal regulation. Frogs were exogenously injected with testosterone (T) or estradiol (E2). This manipulation revealed that the body coloration (hue, brightness, and saturation) of the male frog increased significantly (i.e., the brilliant yellow color developed) in response to T but not in response to E2. Concurrently, the levels of expression of brain-derived neurotrophic factor (BDNF) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the pituitary gland were reduced in frogs whose coloration was pale brown on a yellow background. In particular, the weakest expressions of BDNF, PACAP, and PACAP type II receptors (VPAC-1R) were found in male frogs with a brilliant yellow body color during the breeding season regardless of background color. These changes may decrease α-melanocyte-stimulating hormone production associated with the PACAP receptors (VPAC-1R), resulting in the aggregation of black pigment in melanophores and the production of a brilliant yellow body color. The effects of hormones on skin coloration were further examined in isolated skin in vitro. The results of this investigation showed that the dispersion of xanthophores was stimulated by T or prolactin (PRL) and that the melanophores were aggregated by melatonin (MEL) but not by E2. Furthermore, yellow pigments in the xanthophores were significantly dispersed following the PRL+T treatment. In the T+MEL, PRL+MEL, and T+PRL+MEL treatments, xanthophores were dispersed, and melanophores were aggregated and subsequently moved to the low spongiosum layer of the dorsal skin, causing the increase in yellow coloration. These results reveal that multiple hormones play major roles in the regulation of the brilliant yellow coloration of male B. robusta by high plasma T during the breeding season.

Characterization and interactome study of white spot syndrome virus envelope protein VP11.

White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570), and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

Penaeus monodon TATA box-binding protein interacts with the white spot syndrome virus transactivator IE1 and promotes its transcriptional activity.

We show here that the white spot syndrome virus (WSSV) immediate-early protein IE1 interacts with the Penaeus monodon TATA box-binding protein (PmTBP) and that this protein-protein interaction occurs in the absence of any other viral or cellular proteins or nucleic acids, both in vitro and in vivo. Mapping studies using enhanced green fluorescent protein (EGFP) fusion proteins containing truncations of IE1 and PmTBP delimited the interacting regions to amino acids (aa) 81 to 180 in IE1 and, except for aa 171 to 230, to aa 111 to 300 in PmTBP. A WSSV IE1 transactivation assay showed that large quantities (>800 ng) of the GAL4-IE1 plasmid caused "squelching" of the GAL4-IE1 activity and that this squelching effect was alleviated by the overexpression of PmTBP. Gene silencing of WSSV ie1 and PmTBP by pretreatment with double-stranded RNAs (dsRNAs) prior to WSSV challenge showed that the expression of these two target genes was specifically inhibited by their corresponding dsRNAs 72 and 96 h after dsRNA treatment. dsRNA silencing of ie1 and PmTBP expression also significantly reduced WSSV replication and the expression of the viral early gene dnapol (DNA polymerase gene). These results suggest that WSSV IE1 and PmTBP work cooperatively with each other during transcription initiation and, furthermore, that PmTBP is an important target for WSSV IE1's transactivation activity that can enhance viral gene expression and help in virus replication.

A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.

BACKGROUND: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus), is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. PRINCIPAL FINDINGS: In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument) proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. CONCLUSIONS: From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

Ovine thyroid stimulating hormone (TSH) heterologously stimulates production of thyroid hormones from Chinese soft-shell turtle (Pelodiscus sinensis) and bullfrog (Rana catesbeiana and Rana rugulosa) thyroids in vitro.

Thyroid hormones are important for regulating a variety of developmental processes in vertebrates, including growth, differentiation, metamorphosis, and oxidative metabolism. In particular, this study focused on the in vitro production of thyroxine (T(4)) and triiodothyronine (T(3)) from thyroids in American bullfrogs (Rana catesbeiana), Chinese bullfrogs (Rana rugulosa Wiegmann), and Chinese soft-shell turtles (Pelodiscus sinensis) treated with ovine thyroid stimulating hormone (TSH) at different culture intervals (2, 4, 8, and 12 h) and dosages (1, 10, 50 or 100 ng). The levels of T(4) and T(3) in the tested animals were elevated upon stimulation in a time- and dose-dependent manner, indicating de novo synthesis of T(4) and T(3). Significantly higher hormone levels were observed in the Chinese bullfrog compared to the other two species, for both the time-course and dose-response experiments. Although the bullfrog secreted significantly higher levels of T(4) and T(3), a higher T(4)-conversion capacity was found in the Chinese soft-shell turtle. The highest ratios of T(3) to T(4) were observed in the American bullfrog and Chinese soft-shell turtle for the time-course and dose-response experiments, respectively. These findings suggest that the Chinese soft-shell turtle and bullfrog thyroids can accept ovine TSH for T(4)- and T(3)-formation in a time- and dose-dependent manner, supporting the hypothesis that the binding interactions between TSHs and thyroidal receptors are conserved in vertebrates.

Expression and in vitro regulation of pituitary adenylate cyclase-activating polypeptide (pacap38) and its type I receptor (pac1-r) in the gonads of tilapia (Oreochromis mossambicus).

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, has diverse functions in mammals. However, studies of the expression and function of PACAP and its receptor in fish, particularly in the reproductive system, are still limited. In this report, semi-quantitative RT-PCR and immunohistochemical staining were performed to identify expression domains of commercially important tilapia (Oreochromis mossambicus). PACAP (tpacap(38)) and its type I receptor (tpac(1)-r). Transcripts were detected in the brain, gallbladder, gill, heart, intestine, kidney, muscles, pancreas, spleen, stomach, testes, and ovaries, but not in the liver. Expression of tpacap(38) and tpac(1)-r mRNA in brain tissue was significantly higher in both sexes compared with other tissues. Addition of exogenous ovine PACAP(38) (0.25-5 nM), cAMP analog (dibutyryl-cAMP, 0.25-1.5 mM) or forskolin (adenylate cyclase activator, 1-10 microM) significantly upregulated tpacap(38) in the gonads via a dose- and time-dependent fashion. This effect reached a maximal level at 2 h after induction, and then decreased with prolonged culture for up to 4 or 8 h. Additionally, the expression levels of tpac(1)-r were not significantly affected by ovine PACAP(38) or dibutyryl-cAMP in either sex. Forskolin had a slightly inductive effect and its function could be suppressed with the addition of protein kinase A (PKA) inhibitor, H89 (10 microM), indicating involvement of the cAMP-PKA signaling pathway in the regulation of tpacap(38). Expression of tpacap(38) and tpac(1)-r in the gonads of tilapia suggests that PACAP may mediate gonadotropin action via paracrine/autocrine mechanisms in this bony fish.

Characterization of white spot syndrome virus envelope protein VP51A and its interaction with viral tegument protein VP26.

In this study, we characterize a novel white spot syndrome virus (WSSV) structural protein, VP51A (WSSV-TW open reading frame 294), identified from a previous mass spectrometry study. Temporal-transcription analysis showed that vp51A is expressed in the late stage of WSSV infection. Gene structure analysis showed that the transcription initiation site of vp51A was 135 bp upstream of the translation start codon. The poly(A) addition signal overlapped with the translation stop codon, TAA, and the poly(A) tail was 23 bp downstream of the TAA. Western blot analysis of viral protein fractions and immunoelectron microscopy both suggested that VP51A is a viral envelope protein. Western blotting of the total proteins extracted from WSSV virions detected a band that was close to the predicted 51-kDa mass, but the strongest signal was around 72 kDa. We concluded that this 72-kDa band was in fact the full-length VP51A protein. Membrane topology assays demonstrated that the VP51A 72-kDa protein is a type II transmembrane protein with a highly hydrophobic transmembrane domain on its N terminus and a C terminus that is exposed on the surface of the virion. Coimmunoprecipitation, colocalization, and yeast two-hybrid assays revealed that VP51A associated directly with VP26 and indirectly with VP28, with VP26 acting as a linker protein in the formation of a VP51A-VP26-VP28 complex.

Application of RNAi technology to the inhibition of zebrafish GtHalpha, FSHbeta, and LHbeta expression and to functional analyses.

Zebrafish (Danio rerio) were used as a model fish, and the technique of RNA interference (RNAi) was employed to knockdown three subunits of the gonadotropin alpha (GtHalpha, common alpha), follicle-stimulating hormone beta (FSHbeta), and luteinizing hormone beta (LHbeta) genes. Three short-hairpin RNA (shRNA) expression vectors and three mismatched shRNA expression vectors as controls for each subunit gene were constructed, and the depression efficiency was tested in vivo by microinjection; the RNA or protein expression levels of the GtH genes were monitored by RT-PCR, Southern blotting, and green fluorescent protein (GFP) analyses. Expression of GtH mRNA was obviously and more efficiently depressed by GtHalpha RNAi expression compared with the other two subunits. A GtHalpha morpholino analysis showed that the GtHalpha morpholino led to suppression of embryonic development and the production of embryonic mutants as a result of an injection of GtHalpha -shRNA. Taken together, these results show that GtHalpha-shRNA, which more efficiently targets RNAi, may have an essential role in the further development of sterility technology of transgenic fish for biosafety purposes.

Steroid hormones (17beta-estradiol and hydrocortisone) upregulate hepatocyte nuclear factor (HNF)-3beta and insulin-like growth factors I and II expression in the gonads of tilapia (Oreochromis mossambicus) in vitro.

Hepatocyte nuclear factors (HNF-1alpha, -1beta and -3beta) and insulin-like growth factors (IGF-I and -II), which are involved in liver-specific gene expression, metabolism, development and cell growth, have been found in the gonads of tilapia (Oreochromis mossambicus). However, the functions of these factors and how they interact within the gonads of bony fish are not understood. In the present study, we provided experimental evidence that the expression of HNF-3beta in the gonads of tilapia, but not HNF-1alpha and -1beta, was affected in vitro by 17beta-estradiol and hydrocortisone. Immunohistochemical staining confirmed that tilapia HNF-3beta was mainly found in the nuclei of hepatocytes, the follicular granulosa cells of the ovaries, and the interstitial cells of the testes of adult tilapia. Further data were gathered at various steroid concentrations (0.1, 1, 10, 100, and 1000 nM) over various culture intervals (6, 12, 18, 24, 30, and 36 h) and subjected to semi-quantitative RT-PCR analysis. The expression of downstream genes (IGF-I and -II) followed the same temporal patterns as HNF-3beta, albeit at decreased levels for 30 and 36 h culture intervals. Both hormones upregulated HNF-3beta mRNA expression at concentrations of 0.1-10 nM, and reached optimal physiological concentrations for induction of IGFs at 1-10 nM. The identity of the PCR fragments was concurrently verified by sequencing and PCR-Southern hybridization. We inferred that HNF-3beta and IGFs may play a regulatory role in tilapia gonads during oocyte maturation and spermatogenesis.

Temperature and humidity alter prolactin receptor expression in the skin of toad (Bufo bankorensis and Bufo melanostictus).

Bufo bankorensis and Bufo melanostictus, the only two species of Bufonidae genus in Taiwan, live in habitats that differ in altitude and humidity. This study tested the hypothesis that prolactin receptor (PRLR) expression responds to environmental change. Western blot analysis showed that the PRLR protein was widely distributed in brain, lung, liver, kidney, dorsal skin and ventral skin of toads. The level PRLR protein was elevated in the dorsal skin of the two toad species treated with dry or wet conditions for 14 days. The increase in PRLR of dorsal skin in B. bankorensis was higher than that in B. melanostictus. This experimental result suggests that B. bankorensis secretes more mucus to reduce water evaporation from its thinner cuticle than B. melanostictus. The expression of PRLR protein was increased in the lung of B. bankorensis and decreased in the lung of B. melanostictus. Moreover, PRLR protein levels were increased in the kidneys in the two species toad, likely due to reduction in water lost through lung and urine. The two toad species were subjected to varying temperatures (25 degrees C, 15 degrees C and 10 degrees C) for 14 days. The lowest PRLR protein expression was observed at 10 degrees C. Comparison of the decreasing trend in PRLR protein levels demonstrated that the variation in B. bankorensis was significantly higher than that in B. melanostictus. Comparisons of variation in PRLR protein expression in the two species under different environments suggest that B. bankorensis is more adaptable to different environments than B. melanostictus.

Response to acute changes in salinity of two different muscle type creatine kinase isoforms, from euryhaline teleost (Oreochromis mossambicus) gills.

Two CKM isoforms (CKM1 and CKM2) from the gills of tilapia (Oreochromis mossambicus) were obtained after transfer from freshwater (FW) to seawater (SW, 25 ppt). Based on the 5' and 3' RACE, the identity of CKM1 and CKM2 was determined to be 59% in the 5'-untranslated region (5'-UTR) and 41.9% in the 3'-UTR. Using Northern blot hybridization with the CKM1 and CKM2 3'-UTR probes, CKM1 and CKM2 were found to be expressed in muscle, heart and gill. The levels of these two different CK isoforms (CKM1 and CKM2) were shown to be different in FW than after acute SW transfer, showing that CKM isoforms respond to changes in salinity.

Molecular cloning and tissue-specific, developmental-stage-specific, and hormonal regulation of IGFBP3 gene in zebrafish.

The biological functions of insulin-like growth factor (IGF) I and II are modulated by a family of IGF-binding proteins (IGFBPs) in complex IGF-dependent and IGF-independent pathways. For further understanding of the actions of IGFs, some of these binding proteins have been cloned and characterized. We report the molecular cloning of IGFBP-3 cDNA for zebrafish. The tissue-specific and developmental stage-specific expression of IGFBP-3 and the hormonal regulation of its expression have also been determined by comparative reverse transcription polymerase chain reaction. Zebrafish IGFBP-3 cDNA contains an open reading frame of 879 bp, encoding a polypeptide of 293 amino acid residues. Results of this analysis revealed high levels of IGFBP-3 messenger RNA in ovary and fin tissue. Expression of IGFBP-3 mRNA was throughout the entire embryonic development, with the highest level of expression observed at 36 hours after the onset of development. Elevated levels of expression of IGFBP-3 were observed 24 hours after injection with IGF-I and 48 hours with IGF-II or insulin. These results suggest that the expression of IGFBP3 gene might be modulated by IGF-I, IGF-II, and insulin.

Transfer of porcine embryos through mini-laparotomy.

The aim of this experiment was to examine the suitability of mini-laparotomy for transferring embryos in pigs. Expanded blastocysts collected from estrus-induced prepuberal gilts were transferred to the uterus of synchronous recipients. Each recipient received 18 embryos transferred unilaterally either by conventional laparotomy (n = 20), mini-laparotomy (n = 15) or laparoscopy (n = 14). The mini-laparotomy consisted of a midventral incision of 4 cm enabling the surgeon to grasp a uterine horn with two fingers and exteriorize about 3 cm of it. To close the suture wound, only three or four interrupted skin sutures are required. Pregnancy rates after conventional surgery, mini-laparotomy and laparoscopy were 60.67 and 21%, respectively. Corresponding litter size was 7.4, 6.2 and 6.0 and total embryo survival 25, 23 and 7%. The differences in pregnancy rate and total embryo survival between conventional and mini-surgery were negligible, whereas between laparoscopy and the other two techniques it was significant. It may be concluded that, with a little practice, the time saving and less traumatic mini-laparotomy is a practicable alternative to conventional surgery.

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus).

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.

Bioenergetics of adaptation to a salinity transition in euryhaline teleost (Oreochromis mossambicus) brain.

Freshwater (FW) teleosts are capable of acclimating to seawater (SW) following such a transfer from FW. However, their osmoregulating mechanisms are still unclear, particularly those in the brain. The present study was conducted to examine acute changes that occur in brain Na(+)-K(+)-ATPase activity, creatine kinase (CK) activity, creatine, creatinine contents, and ATP levels of tilapia (Oreochromis mossambicus) in response to this transition. After transfer to SW (25 ppt), the Na(+)-K(+)-ATPase activity was maintained for 8 hr at higher levels than that in FW. In contrast, in 35 ppt SW, Na(+)-K(+)-ATPase was maintained at a even higher level than in FW for the first 2 hr. Brain Na(+)-K(+)-ATPase contents in both the 25 and 35 ppt SW groups were significantly elevated within 1 and 0.5 hr after transfer from FW, respectively. Interestingly, brain CK activities and content (homodimer of the B subunit [BB] form) in both the 25 and 35 ppt SW groups were significantly elevated within 1 hr after transfer from FW. The ATP contents in 35 ppt SW increased abruptly within 0.5 hr, and then gradually decreased during the next 2 hr. Unlike the 35 ppt group that declined in ATP contents, the 25 ppt group leveled off within 24 hr. The elevations in CK activity and creatine levels after transfer from FW to SW imply that abrupt salinity changes alter phosphocreatine/CK ratio. Such changes are needed to satisfy the increases in the energetic requirement of the cotransport mechanisms mediating osmoregulation.