Somluk Asuvapongpatana

Solvent extracts of the red seaweed Gracilaria fisheri prevent Vibrio harveyi infections in the black tiger shrimp Penaeus monodon

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

Localization of cathepsin D in mouse reproductive tissues and its acquisition onto sperm surface during epididymal sperm maturation

Sperm maturation in the epididymis involves multiple complex events, that include the adsorption of epididymal secretory proteins, re-organization and removal of sperm surface ligands. In this study, we investigated the existence and distribution of cathepsin D (CAT-D) transcripts and proteins in mouse reproductive tissues and proposed a transfer mechanism of CAT-D to the sperm surface. CAT-D transcripts were highly expressed in cultured Sertoli cells, but not in germ cells. The transcriptional level was relatively higher in the caput epididymis (CP) than in the cauda epididymis (CD). At the translational level, CAT-D was detected in testicular somatic cells and in the principal and basal cells in the CP. The expression of CAT-D was fairly specific to the clear cells in the CD. All forms of CAT-D were detected in ultracentrifuged epididymosomes. In conjunction with the expression levels in epididymal epithelium and epididymosomes, CAT-D expression level on the sperm surface was relatively high in CP sperm, but gradually declined toward the CD. Overall, our results indicated that CAT-D was not inherent to sperm themselves, but rather of epididymal origin and was presumably transported to the sperm surface via epididymosomes.

Cathepsin D in Human Reproductive Tissues: Cellular Localization in Testis and Epididymis and Surface Distribution in Different Sperm Conditions

Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.

Characterization of the thrombospondin (TSP)-II gene in Penaeus monodon and a novel role of TSP-like proteins in an induction of shrimp sperm acrosome reaction.

We have recently shown that water‐soluble materials from the egg extracellular cortical rods (wsCRs) exert the ability to induce the sperm acrosome reaction in Penaeus monodon. In this study, we further demonstrated that the thrombospondin protein family (TSP) existed in wsCRs, and that their mRNA transcripts were detected in developing oocytes as early as stage I. Full sequence analysis revealed that our pmTSP sequence was considerably different from the recently reported pmTSP in the 5′ nonconserved region and in many TSP signature domains, hence, the name pmTSP‐II was given to our variant. The transcripts of pmTSP‐II were detected only in early developing oocytes (stage‐I and ‐II) while TSP‐like proteins were detected in all developing oocytes, particularly at the outer rim of cortical rods situated in the extracellular crypts of the mature, stage‐IV oocytes. In addition, wsCRs contained anti‐TSP‐reactive proteins, suggesting that TSP‐like proteins are dissolved in and are part of the egg water during spawning. The functional importance of TSP‐like proteins was evident by the interference of a wsCR‐induced acrosome reaction response with anti‐TSP in a concentration‐dependent manner. In summary, we found that pmTSP‐II transcripts were present in the developing oocytes and pmTSP‐II protein accumulated in cortical rods, which are partly secreted and thus solubilized to produce dissolved TSP‐like proteins that participate in induction of the sperm acrosome reaction—a novel reproductive role for TSP protein family. Mol. Reprod. Dev. 80: 393–402, 2013.

Isolation of Organic Matrix Nacreous Proteins from Haliotis diversicolor and Their Effect On In Vitro Osteoinduction

ABSTRACT The organic matrix proteins of molluscan nacre are known to engage biocompatible and osteoinductive properties. In this study, we aimed to extract Haliotis diversicolor nacreous proteins and assayed their effect on pre-osteoblastic cell differentiation. We found that acid extracted nacreous proteins (AEP) consisted primarily of a major protein band of 25 kDa and three other minor proteins. AEP enhanced alkaline phosphatase (ALP) activity of MC3T3-E1 cells both in time- and concentration-dependent manners. Transcriptional up-regulation of osteogenic markers, including collagen type I (COL-I), osteopontin (OPN) and osteocalcin (OCN), was also apparent in days 7 and 14 upon AEP treatments. At the translational level, higher protein expression of COL-I was evident in AEP treated cells, and the protein was presumably laid down as extracellular matrix. Further de novo sequencing of an AEP major protein revealed a match with the abalone mantle protein sometsuke. Conclusively, we demonstrated that H. diversicolor AEP contains a factor, potentially the mantle protein sometsuke, which may impart in the osteoblastic cell differentiation.

The development of the cloaca in the human embryo

Subdivision of cloaca into urogenital and anorectal passages has remained controversial because of disagreements about the identity and role of the septum developing between both passages. This study aimed to clarify the development of the cloaca using a quantitative 3D morphological approach in human embryos of 4–10 post‐fertilisation weeks. Embryos were visualised with Amira 3D‐reconstruction and Cinema 4D‐remodelling software. Distances between landmarks were computed with Amira3D software. Our main finding was a pronounced difference in growth between rapidly expanding central and ventral parts, and slowly or non‐growing cranial and dorsal parts. The entrance of the Wolffian duct into the cloaca proved a stable landmark that remained linked to the position of vertebra S3. Suppressed growth in the cranial cloaca resulted in an apparent craniodorsal migration of the entrance of the Wolffian duct, while suppressed growth in the dorsal cloaca changed the entrance of the hindgut from cranial to dorsal on the cloaca. Transformation of this ‘end‐to‐end’ into an ‘end‐to‐side’ junction produced temporary ‘lateral (Rathkes) folds’. The persistent difference in dorsoventral growth straightened the embryonic caudal body axis and concomitantly extended the frontally oriented ‘urorectal (Tourneuxs) septum’ caudally between the ventral urogenital and dorsal anorectal parts of the cloaca. The dorsoventral growth difference also divided the cloacal membrane into a well‐developed ventral urethral plate and a thin dorsal cloacal membrane proper, which ruptured at 6.5 weeks. The expansion of the pericloacal mesenchyme followed the dorsoventral growth difference and produced the genital tubercle. Dysregulation of dorsal cloacal development is probably an important cause of anorectal malformations: too little regressive development may result in anorectal agenesis, and too much regression in stenosis or atresia of the remaining part of the dorsal cloaca.

Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn.

We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm–egg penetration mechanism that is unique in this species.

A sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp

&NA; A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and &bgr;‐1,3‐glucan binding protein (LGBP) and immune related genes including p‐NF‐&kgr;B, IMD, IKK&bgr; and IKK&egr;, antimicrobial peptide PEN‐4, proPO‐I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG‐fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection. HighlightsSG supplemented diet up‐regulated expression of immune genes in shrimp.SG reduced shrimp mortality rate from V. parahaemolyticus infection.SG decreased bacterial colonies and toxA expression.SG maintained a normal histology of hepatopancreas after V. parahaemolyticus infection.

Lectin-Based Profiling of Coelomocytes in Holothuria scabra and Expression of Superoxide Dismutase in Purified Coelomocytes

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some minor staining was also detected in spherulocytes. Our results thus provide a fundamental platform for comparing alterations that may happen to the population and SOD contents of coelomocytes when the sea cucumber is subjected to environmental changes that would activate their immune responses.