Effects of Osmotic Stress on the mRNA Expression of prl, prlr, gr, gh, and ghr in the Pituitary and Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii.

In euryhaline teleost black porgy, Acanthopagrus schlegelii, the glucocorticoid receptor (gr), growth hormone receptor (ghr), prolactin (prl)-receptor (prlr), and sodium-potassium ATPase alpha subunit (α-nka) play essential physiological roles in the osmoregulatory organs, including the gill, kidney, and intestine, during osmotic stress. The present study aimed to investigate the impact of pituitary hormones and hormone receptors in the osmoregulatory organs during the transfer from freshwater (FW) to 4 ppt and seawater (SW) and vice versa in black porgy. Quantitative real-time PCR (Q-PCR) was carried out to analyze the transcript levels during salinity and osmoregulatory stress. Increased salinity resulted in decreased transcripts of prl in the pituitary, α-nka and prlr in the gill, and α-nka and prlr in the kidney. Increased salinity caused the increased transcripts of gr in the gill and α-nka in the intestine. Decreased salinity resulted in increased pituitary prl, and increases in α-nka and prlr in the gill, and α-nka, prlr, and ghr in the kidney. Taken together, the present results highlight the involvement of prl, prlr, gh, and ghr in the osmoregulation and osmotic stress in the osmoregulatory organs (gill, intestine, and kidney). Pituitary prl, and gill and intestine prlr are consistently downregulated during the increased salinity stress and vice versa. It is suggested that prl plays a more significant role in osmoregulation than gh in the euryhaline black porgy. Furthermore, the present results highlighted that the gill gr transcript's role was solely to balance the homeostasis in the black porgy during salinity stress.

QTRAP LC/MS/MS of Garlic Nanoparticles and Improving Sunflower Oil Stabilization during Accelerated Shelf Life Storage.

The purpose of this research was to assess and utilize the bioactive compounds of garlic nanoparticles (Ga-NPs) as a natural antioxidant in sunflower oil (SFO) stored at 65 ± 1 °C for 24 days. The garlic nanoparticles (Ga-NPs) from the Balady cultivar were prepared, characterized, and added to SFO at three concentrations: 200, 600, and 1000 ppm (w/v), and they were compared with 600 ppm garlic lyophilized powder extract (Ga-LPE), 200 ppm BHT, 200 ppm α-tocopherol, and SFO without Ga-NPs (control). The QTRAP LC/MS/MS profile of Ga-NPs revealed the presence of four organosulfur compounds. Ga-NPs exhibited the highest capacity for phenolic, flavonoid, and antioxidant compounds. In Ga-NP SFO samples, the values of peroxide, p-anisidine, totox, conjugated dienes, and conjugated trienes were significantly lower than the control. The antioxidant indices of SFO samples containing Ga-NPs were higher than the control. The Ga-NPs enhanced the sensory acceptability of SFO treatments up to day 24 of storage. The shelf life of SFO treated with Ga-NPs was substantially increased (presuming a Q10 amount). The results show that Ga-NPs are a powerful antioxidant that improves SFO stability and extends the shelf life (~384 days at 25 °C).

Localization of the Neuropeptide Arginine Vasotocin and Its Receptor in the Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii: Gills, Kidneys, and Intestines.

The neurohypophysial hormone arginine vasotocin (avt) and its receptor (avtr) regulates ions in the osmoregulatory organs of euryhaline black porgy (Acanthopagrus schlegelii). The localization of avt and avtr transcripts in the osmoregulatory organs has yet to be demonstrated. Thus, in the present study, we performed an in situ hybridization analysis to determine the localization of avt and avtr in the gills, kidneys, and intestines of the black porgy. The avt and avtr transcripts were identified in the filament and lamellae region of the gills in the black porgy. However, the basal membrane of the filament contained more avt and avtr transcripts. Fluorescence double tagging analysis revealed that avt and avtr mRNAs were partially co-localized with α-Nka-ir cells in the gill filament. The proximal tubules, distal tubules, and collecting duct of the kidney all had positive hybridization signals for the avt and avtr transcripts. Unlike the α-Nka immunoreactive cells, the avt and avtr transcripts were found on the basolateral surface of the distal convoluted tubule and in the entire cells of the proximal convoluted tubules of the black porgy kidney. In the intestine, the avt and avtr transcripts were found in the basolateral membrane of the enterocytes. Collectively, this study provides a summary of evidence suggesting that the neuropeptides avt and avtr with α-Nka-ir cells may have functions in the gills, kidneys, and intestines via ionocytes.

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners.

Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer's disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aß42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression.

Expression and Transcript Localization of star, sf-1, and dax-1 in the Early Brain of the Orange-Spotted Grouper Epinephelus coioides.

We investigated the developmental expression and localization of sf-1 and dax-1 transcripts in the brain of the juvenile orange-spotted grouper in response to steroidogenic enzyme gene at various developmental ages in relation to gonadal sex differentiation. The sf-1 transcripts were significantly higher from 110-dah (day after hatching) and gradually increased up to 150-dah. The dax-1 mRNA, on the other hand, showed a decreased expression during this period, in contrast to sf-1 expression. At the same time, the early brain had increased levels of steroidogenic gene (star). sf-1 and star hybridization signals were found to be increased in the ventromedial hypothalamus at 110-dah; however, dax-1 mRNA signals decreased in the early brain toward 150-dah. Furthermore, the exogenous estradiol upregulated star and sf-1 transcripts in the early brain of the grouper. These findings suggest that sf-1 and dax-1 may have an antagonistic expression pattern in the early brain during gonadal sex differentiation. Increased expression of steroidogenic gene together with sf-1 during gonadal differentiation strongly suggests that sf-1 may play an important role in the juvenile grouper brain steroidogenesis and brain development.

Differential Expression of Hypothalamic and Gill-crh System With Osmotic Stress in the Euryhaline Black Porgy, Acanthopagrus schlegelii.

The local gill production of corticotropin releasing hormone (crh) and crh-receptor (crhr) is hypothesized to play important roles during seawater (SW) and freshwater (FW) acclimation in euryhaline black porgy (Acanthopagrus schlegelii). The mRNA expression of crh, crhr, and Na +/K + -ATPase (a-nka) was examined in SW and FW diencephalon (Dien) and in the gills at different exposure time by Q-PCR analysis. The in situ hybridization results indicate that crh mRNA hybridization signals were more abundant in FW fish in the gigantocellular (PMgc) and parvocellular (PMpc) part of the magnocellular preoptic nucleus versus SW fish. The crh and crhr-expressing cells were located in basal cells of gill filament. Furthermore, in vitro dexamethasone (DEX) treatment could increase the crh-system in the gill. Increased transcripts of the crh-system in the gill via in vitro and in vivo CRH treatments suggest that CRH may regulate the system in a local manner. The a-Nka cells were localized in the filament and secondary lamellae mitochondria rich cells (MRCs) of FW fish at 8 h and 1 day. a-Nka cells were seen in both filament and lamellae in the FW but much less in SW fish indicating that gills play key roles in black porgy osmoregulation. Gill crh and crhr play important roles in the response to salinity stress.

Neurohypophysial Hormones Associated with Osmotic Challenges in the Brain and Pituitary of the Euryhaline Black Porgy, Acanthopagrus schlegelii.

Our study showed differential expression of the arginine vasotocin (avt)/isotocin (it) in the brain and pituitary gland of the euryhaline black porgy (Acanthopagrus schlegelii) during osmotic stress. A decrease in serum osmolality and increased cortisol levels were observed after acute transfer from seawater (SW) to freshwater (FW). The increased expressions of avt, avt receptor (avtr: v1a), and isotocin receptor (itr: itr1) transcripts on day 1 and it and itr transcripts on days 7 and 30 were found in the brains and pituitary glands of FW fish. Increased levels of avt mRNA in the diencephalon and avtr mRNA in the pituitary together with serum cortisol on day 1 of FW exposure indicated activation of the hypothalamic-pituitary-interrenal (HPI) axis. The expression levels of avtr and itr after FW transfer were increased in the pituitary on days 7 and 30. Furthermore, in situ hybridization demonstrated spatially differential expression of avt and itr transcripts in nucleus preopticus parvocellularis of pars gigantocellularis (PMgc), magnocellularis (PMmc), and parvocellularis (PMpc) of the preoptic area (POA). Positive signals for avt and it were highly abundant in PMpc after FW exposure. The data suggest involvement of neurohypophysial hormones in the brain (telencephalon and diencephalon) and pituitary for osmotic stress.

WWOX Possesses N-Terminal Cell Surface-Exposed Epitopes WWOX7-21 and WWOX7-11 for Signaling Cancer Growth Suppression and Prevention In Vivo.

Membrane hyaluronidase Hyal-2 supports cancer cell growth. Inhibition of Hyal-2 by specific antibody against Hyal-2 or pY216-Hyal-2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal-2. Alternatively, WWOX undergoes self-polymerization and localizes in the cell membrane. Proapoptotic pY33-WWOX binds Hyal-2, and TGF-ß induces internalization of the pY33-WWOX/Hyal-2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14-WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface-exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3-dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7-21 peptide, or truncation to 5-amino acid WWOX7-11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time-lapse microscopy revealed that WWOX7-21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33-WWOX, downregulation of prosurvival pERK, prompt increases in Ca2+ influx, and disruption of the IkBα/WWOX/ERK prosurvival signaling. In contrast, pS14-WWOX7-21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib-mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14-WWOX significantly enhanced the ceritinib-induced apoptosis. Together, the N-terminal epitopes WWOX7-21 and WWOX7-11 are potent in blocking cancer growth in vivo. WWOX7-21 and WWOX7-11 peptides and pS14-WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

L-Theanine alleviates the neuropathological changes induced by PCB (Aroclor 1254) via inhibiting upregulation of inflammatory cytokines and oxidative stress in rat brain.

The present study is aimed at evaluating the protective role of L-theanine on aroclor 1254-induced oxidative stress in rat brain. Intraperitoneal administration of Aroclor 1254 (2 mg/kg b.wt. for 30 days) caused oxidative stress in rat brain and also caused neurobehavioral changes. Oxidative stress was assessed by determining the levels of lipid peroxide (LPO), protein carbonyl content, and changes in activities of creatine kinase (CK), acetylcholinesterase (AchE), and ATPases in the hippocampus, cerebellum and cerebral cortex of control and experimental rats. Histopathological results showed that PCB caused neuronal loss in all three regions. PCB upregulated the mRNA expressions of inflammatory cytokines. Oral administration of L-theanine (200 mg/kg b.wt.) increased the status of antioxidants, decreased the levels of LPO, nitric oxide (NO) and increased the activities of CK, AchE and ATPases. L-Theanine restored normal architecture of brain regions and downregulated the expression of inflammatory cytokines. In conclusion, L-theanine shows a protective role against PCBs-induced oxidative damage in rat brain.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia.

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic-pituitary-interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes.

Increase in estrogen signaling in the early brain of orange-spotted grouper Epinephelus coioides: a mini-review.

Despite neurosteroidogenic enzymes are playing important roles in the regulation of brain development and function, the potential link between brain and gonad by the action of steroid hormones during gonadal sex differentiation is still not clear in teleosts. In this mini-review, we summarized our understanding on the early brain development related to the synthesis of neurosteroids and receptor signaling during gonadal sex differentiation in protogynous orange-spotted grouper, Epinephelus coioides (functional females for the first 6 years of life and start to sex change around the age of 7 years) and protandrous black porgy (functional males for the first 2 years of life but begin to change sex during the third year). We found a similar profile in the increased expression of brain aromatase gene (aromatatse B or cyp19a1b), aromatase activity, estradiol (E(2)), and estrogen signaling in the brain of both grouper and black porgy fish during gonadal sex differentiation. In contrast to mammals, teleost fish Cyp19a1b expressed in a unique cell type, a radial glial cell, which is acted as progenitors in the brain of developing and adult fish. In agreement with these pioneer studies, we demonstrated that the grouper cyp19a1b/Cyp19a1b was expressed in radial glial cells. Further, in vivo data in the grouper brain showed that exogenous E(2) upregulated Cyp19a1b immunoreactivity (ir) in radial glial cells. These data suggest the possible roles of Cyp19a1b and E(2) in early brain development which is presumably related to gonadal sex differentiation.

Neurosteroidogenic enzymes and their regulation in the early brain of the protogynous grouper Epinephelus coioides during gonadal sex differentiation.

The regulatory role of neurosteroids in the early brain during gonadal sex differentiation is unclear. The aim of this study was to investigate the expression and cellular localization of key steroidogenic enzymes in the early brain of the protogynous orange-spotted grouper Epinephelus coioides and the temporal expressions has been correlated with gonadal sex differentiation. In this study, we showed that peak neurosteroidogenesis occurs in the early brain during gonadal sex differentiation. The temporal expressions of key enzymes, cyp11a1 (cytochrome P450 side chain cleavage), hsd3b1 (3ß-hydroxysteroid dehydrogenase) and cyp17a1 (cytochrome P450c17) were studied at different developmental ages (from 90- to 150-dah: days after hatching) using quantitative real-time PCR (q-PCR). q-PCR analysis indicated that the transcript expressions of cyp11a1, hsd3b1 and cyp17a1 were increased in the brain around the period of gonadal sex differentiation. Further, in situ hybridization (ISH) analysis showed that cyp11a1, hsd3b1 and cyp17a1 transcripts were widely expressed in several discrete brain regions, especially the intense expression in the forebrain, with an overall similar expression pattern. High density in the cyp19a1b/Cyp19a1b expression was detected in radial glial cells. Thus, the expression of grouper cyp19a1b/Cyp19a1b is restricted to radial glial cells, suggesting estrogens can modulate their activity. Next, by combining Cyp19a1b immunohistochemistry (IHC) with florescence ISH (FISH) of cyp11a1, hsd3b1 and cyp17a1, we showed that sub-cellular localization of cyp11a1, hsd3b1 and cyp17a1 transcripts, in partial, appeared to be in Cyp19a1b radial glial cell soma. Further, exogenous estradiol (E(2)) increased the expression of cyp17a1 and cyp19a1b/Cyp19a1b in the brain of grouper. Consequently, our results illustrated that the locally synthesized E(2) upregulated neurosteroidogenic enzymes in the early brain and suggest a role for these enzymes in the neurogenic process during gonadal sex differentiation.

Differential expression patterns and localization of glucocorticoid and mineralocorticoid receptor transcripts in the osmoregulatory organs of tilapia during salinity stress.

The glucocorticoid receptor (GR) plays an essential role during seawater (SW) acclimation. However, the regulation of GR isoforms 1 and 2 (GR1 and GR2) and the mineralocorticoid receptor (MR) during SW acclimation is poorly understood. To address this, we localized and examined the GR1, GR2 and MR transcripts in the tilapia gill, kidney and intestine. Our results indicated that the GR1, GR2 and MR levels were increased in the kidney and intestine on day 1 in seawater (SW) fish, which is in agreement with the recognized osmoregulatory role of the corticosteroid receptors. The SW transfer increased the GR2 and MR transcripts in the gill on day 1 and 4, respectively. Surprisingly, no significant difference was obtained for the GR1 mRNA level. Analysis of the plasma parameters in freshwater (FW) and SW tilapia showed that the plasma cortisol levels were significantly increased at day 1 in the SW fish compared to the FW fish. This is the first study that focused on the spatial distribution of GR1, GR2 and MR in the osmoregulatory organs of freshwater (FW)- and SW-acclimated tilapia by in situ hybridization. Consistent with the Q-PCR results, the expression levels of the GR1, GR2 and MR transcripts were increased or decreased in the SW-acclimated tilapia's gill, kidney and intestine compared to the FW fish. We observed that GR1, GR2 and MR were localized in the branchial epithelial cells and chloride cells of the gill, proximal tubules of the kidney and columnar cells of the intestine. Together, these results indicate that the mobilization of corticosteroid receptors is dependent on the target tissue, salinity and exposure time.

Developmental expression of genes involved in neural estrogen biosynthesis and signaling in the brain of the orange-spotted grouper Epinephelus coioides during gonadal sex differentiation.

In the brain, the synthesis of neurosteroids and receptor activation during gonadal sex differentiation in teleosts are poorly understood. In the present study, the protogynous orange-spotted grouper (Epinephelus coioides) was selected as a model fish, and we hypothesized that de novo synthesis of neural estrogen may play a role in the female grouper brain during gonadal sex differentiation. We investigated the temporal expression of the genes StAR, cyp19a1b and pcna and the sex steroid nuclear receptors for estrogen (ERα, ERß1 and ERß2), androgen (AR) and the plasma membrane-associated estrogen receptor (GPR30) in the brain during early developmental ages from 90 days after hatching (dah) to 180dah after gonadal sex differentiation. Our results revealed that mRNA for ERs and GPR30 but not AR was significantly increased at 110dah (a time close to gonadal sex differentiation) in the forebrain and midbrain and for cyp19a1b at 110dah in the forebrain. Brain aromatase activity and estradiol (E2) levels, but not testosterone (T), were increased in the forebrain at 110 and 120dah, respectively. Furthermore, exogenous E2 stimulated cyp19a1b transcripts in the forebrain and hypothalamus and immunoreactive (ir)Cyp19a1b (aromatase enzyme) in the forebrain. irCyp19a1b localized in the glial cells of the forebrain regions. Therefore, we identified a peak of functional aromatase activity and estrogen signaling in the early grouper brain during gonadal sex differentiation. Moreover, pcna transcripts (a marker for cell proliferation activity) were higher in the early brain at 110-150dah. Thus, a peak time of development in the brain is suggested to occur during gonadal sex differentiation in the grouper.