Piyachat Chansela

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography-tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.

Characterization of red pigment concentrating hormone (RPCH) in the female mud crab (Scylla olivacea) and the effect of 5-HT on its expression.

Red pigment concentrating hormone (RPCH) is a member of the chromatophorotropic hormones and, in crustaceans, it is synthesized in the eyestalk. We have isolated a full-length cDNA for a RPCH preprohormone gene (Scyol-RPCH) from the eyestalks of female mud crabs, Scylla olivacea. The open reading frame consists of 642 nucleotides, and encodes a deduced 108 amino acid precursor protein, which includes a signal peptide, the RPCH (pQLNFSPGWamide), and an associated peptide. We show that the mud crab RPCH peptide exhibits 100% identity with 15 other decapods. Expression of Scyol-RPCH within adult mud crab takes place in the eyestalk, brain, and ventral nerve cord, comprising subesophageal ganglion, thoracic ganglion, and abdominal ganglion. In situ hybridization demonstrates specific expression within neuronal clusters 1, 2, 3, and 4 of the eyestalk X-organ, clusters 6, 8, 9, 10, and 17 of the brain, and in neuronal clusters of the ventral nerve cord. We found that administration of 5-HT up-regulates RPCH gene expression in the eyestalk, suggesting that RPCH may play a role as a downstream hormone of 5-HT.

Composition and Localization of Lipids in Penaeus merguiensis Ovaries during the Ovarian Maturation Cycle as Revealed by Imaging Mass Spectrometry

Ovary maturation, oocyte differentiation, and embryonic development in shrimp are highly dependent on nutritional lipids taken up by female broodstocks. These lipids are important as energy sources as well as for cell signaling. In this study, we report on the compositions of major lipids, i.e. phosphatidylcholines (PCs), triacylglycerols (TAGs), and fatty acids (FAs), in the ovaries of the banana shrimp, Penaeus merguiensis, during ovarian maturation. Thin-layer chromatography analysis showed that the total PC and TAG signal intensities increased during ovarian maturation. Further, by using gas chromatography, we found that (1) FAs 14∶0, 16∶1, 18∶1, 18∶2, 20∶1, and 22∶6 proportionally increased as ovarian development progressed to more mature stages; (2) FAs 16∶0, 18∶0, 20∶4, and 20∶5 proportionally decreased; and (3) FAs 15∶0, 17∶0, and 20∶2 remained unchanged. By using imaging mass spectrometry, we found that PC 16∶0/16∶1 and TAG 18∶1/18∶2/22∶6 were detected in oocytes stages 1 and 2. PCs 16∶1/20∶4, 16∶0/22∶6, 18∶3/22∶6, 18∶1/22∶6, 20∶5/22∶6, and 22∶6/22∶6 and TAGs 16∶0/16∶1/18∶3, 16∶0/18∶1/18∶3, 16∶0/18∶1/18∶1, and 16∶0/18∶2/22∶6 were present in all stages of oocytes. In contrast, the PC- and TAG-associated FAs 20∶4, 20∶5, and 22∶6 showed high signal intensities in stage 3 and 4 oocytes. These FAs may act as nutrition sources as well as signaling molecules for developing embryos and the hatching process. Knowledge of lipid compositions and localization could be helpful for formulating the diet for female broodstocks to promote fecundity and larval production.

Gonadotropin-releasing hormone and adipokinetic hormone/corazonin-related peptide in the female prawn.

Crustacean neuropeptides (NPs) play important roles in the regulation of most physiological activities, including growth, molting and reproduction. In this study, we have performed an in silico analysis of female prawn (Macrobrachium rosenbergii) neural transcriptomes to identify NPs not previously identified. We predict that approximately 1309 proteins are destined for the secretory pathway, many of which are likely post-translationally processed to generate active peptides. Within this neural secretome, we identified a gene transcript that encoded a precursor protein with striking similarity to a gonadotropin-releasing hormone (GnRH). We additionally identified another GnRH NP superfamily member, the adipokinetic hormone/corazonin-related peptide (ACP). M. rosenbergii GnRH and ACP were widespread throughout the nervous tissues, implicating them as potential neuromodulators. Furthermore, GnRH was found in non-neural tissues, including the stomach, gut, heart, testis and ovary, in the latter most prominently within secondary oocytes. The GnRH/corazonin receptor-like gene is specific to the ovary, whereas the receptor-like gene expression is more widespread. Administration of GnRH had no effect on ovarian development and maturation, nor any effect on total hemolymph lipid levels, while ACP administration decreased oocyte proliferation (at high dose) and stimulated a significant increase in total hemolymph lipids. In conclusion, our targeted analysis of the M. rosenbergii neural secretome has revealed the decapod GnRH and ACP genes. We propose that ACP in crustaceans plays a role in the lipid metabolism and the inhibition of oocyte proliferation, while the role of the GnRH remains to be clearly defined, possibly through experiments involving gene silencing.

Ultrastructure, Composition, and Possible Roles of the Egg Coats in Haliotis asinina

ABSTRACT Spawned eggs of a tropical abalone Haliotis asinina have 2 protective barriers: the egg jelly coat and the vitelline envelope. At the electron microscopic level, the egg jelly is composed of a network of large fibers (40–50 nm thick) cross-linked by smaller fibers (15–20 nm thick), whereas the vitelline envelope is a thin, tough sheet containing pores that might be channels for sperm contact and entry. Electrophoretically, the egg jelly contains 2 major glycoproteins at 107 kDa and 178 kDa, whereas the vitelline envelope contains a broad spectrum of protein bands ranging from 15–200 kDa, which also includes the corresponding egg jelly protein bands. Glycoproteins of egg jelly and vitelline envelope exhibit strong cross-reactivities, and they appear in late oocytes (Oc4, Oc5). Glucose is the major sugar composition of both egg jelly and vitelline envelope glycoproteins, whereas minor proportions of arabinose, fructose, galactose, and fucose are present in both the egg jelly and vitelline envelope. Our findings suggest that a sperm acrosome reaction could be induced by isolated vitelline envelope glycoproteins, whereas acceleration of sperm motility could be stimulated by egg jelly glycoproteins.

Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes

BACKGROUND & AIMS Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3. METHODS The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases. RESULTS We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders. CONCLUSIONS The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.

Effects of andrographolide on intrahepatic cholestasis induced by alpha-naphthylisothiocyanate in rats

Cholestasis is a cardinal manifestation of liver diseases but effective therapeutic approaches are limited. Therefore, alternative therapy for treating and preventing cholestatic liver diseases is necessary. Andrographolide, a promising anticancer drug derived from the medicinal plant Andrographis paniculata, has diverse pharmacological properties and multi-spectrum therapeutic applications. However, it is unknown whether andrographolide has a hepatoprotective effect on intrahepatic cholestasis. The aims of this study were to investigate the protective effect and possible mechanisms of andrographolide in a rat model of acute intrahepatic cholestasis induced by alpha-naphthylisothiocyanate (ANIT). Andrographolide was administered intragastrically for four consecutive days, with a single intraperitoneal injection of ANIT on the second day. Liver injury was evaluated biochemically and histologically together with hepatic gene and protein expression analysis. Rats pretreated with andrographolide prior to ANIT injection demonstrated lower levels of serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, as well as bilirubin and bile acids as compared to rats treated with ANIT alone. Andrographolide also decreased the incidence and extent of periductular fibrosis and bile duct proliferation. Analysis of protein expression in livers from andrographolide-treated cholestatic rats revealed markedly decreased expression of alpha-smooth muscle actin and nuclear factor kappa-B (NF-κB). In conclusion, andrographolide has a potent protective property against ANIT-induced cholestatic liver injury. The mechanisms that underlie this protective effect are mediated through down-regulation of NF-κB expression and inhibition of hepatic stellate cell activation. These findings suggest that andrographolide could be a promising therapeutic option in prevention and slowing down the progression of cholestatic liver diseases.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100μg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1μg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10μg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10μg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100μg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100μg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100μg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

The Tetrapeptide Apgw-Amide Induces Somatic Growth in Haliotis asinina Linnaeus

ABSTRACT APGW-amide is a well-known neurohormone modulator in several molluscs, and is involved in motor activities, feeding, and sexual behavior. In this report we show that injections of APGW-amide into 4-mo-old juvenile Haliotis asinina stimulate growth of body weight and, to a lesser degree, shell length. The injections were given at 0 (control), 20, and 200 ng/g body weight (BW), at 1-wk intervals for 14 wk. BW and shell length (SL) were measured every week, and growth rates were calculated. When compared with control animals, there was an approximate 2-fold increase in body growth rates of animals given 20 ng/g BW and 200 ng/g BW APGW-amide (P ≤ 0.05), whereas only 20 ng/g BW APGW-amide produced significantly greater SL than controls (P ≤ 0.05), with an approximate 1.2-fold increase. Using an immunoperoxidase technique, we showed the presence of APGW-amide in neuronal cells of the cerebral ganglia and nerve fibers. Overall, these data indicate that APGW-amide is an important neurohormone/neuromodulator in the nervous system of H. asinina and plays a role in controlling the body growth of H. asinina.

Characterization and tissue distribution of neuropeptide F in the eyestalk and brain of the male giant freshwater prawn, Macrobrachium rosenbergii

We previously analyzed the central nervous system (CNS) transcriptome and found three isotypes of long neuropeptide F (MrNPF-I, −II, −III) and four isoforms of short NPF (sMrNPF) in the giant freshwater prawn, Macrobrachium rosenbergii. We now validate the complete sequences of the MrNPF-I and −II precursor proteins, which show high similarity (91–95 %) to NPFs of the penaeus shrimp (PsNPF). MrNPF-I and -II precursors share 71 % amino acid identity, whereas the mature 32-amino-acid MrNPF-I and 69-amino-acid MrNPF-II are identical, except for a 37-amino-acid insert within the middle part of the latter. Both mature MrNPFs are almost identical to PsNPF-I and −II except for four amino acids at the mid-region of the peptides. Reverse transcription plus the polymerase chain reaction revealed that transripts of MrNPF-I and -II were expressed in various parts of CNS including the eyestalk, brain and thoracic and abdominal ganglia, with the highest expression occurring in the brain and thoracic ganglia and with MrNPF-I showing five- to seven-fold higher expression than MrNPF-II. These peptides were also expressed in the midgut hindgut, and hepatopancreas, with MrNPF-I expression in the former two organs being at the same level as that in the brain and thoracic ganglia and about 4-fold higher than NPF-II. The expression of NPFs was also detected in the testes and spermatic duct but appeared much weaker in the latter. Other tissues that also expressed a considerable amount of NPF-I included the hematopoeitic tissue, heart and muscle. By immunohistochemistry, we detected MrNPFs in neurons of clusters 2, 3 and 4 and neuropils ME, MT and SG of the optic ganglia, neurons in cluster 6 and neuropils AMPN, PMPN, PT, PB and CB of the medial protocerebrum, neurons in clusters 9 and 11 and neurophils ON and OGTN of the deutocerebrum and neurons in clusters 14, 15 and 16 and neuropils TN and AnN of the tritocerebrum. Because of their high degree of conservation and strong and wide-spread expression in tissues other than CNS, we believe that, in addition to being a neuromodulator in controlling feeding, MrNPFs also play critical roles in tissue homeostasis. This should be further explored.