Occurrence of tetrodotoxin and paralytic shellfish poisons in a gastropod implicated in food poisoning in southern Taiwan.

The toxicity of the gastropod Nassarius papillosus implicated in a food paralytic poisoning incident in Liuchiu Island, Taiwan, in October 2005 is reported. The symptoms of a victim (67 years old) were featured by general paresthesia, paralysis of phalanges and extremities, paralysis, coma, and aphasia. The remaining specimens of shell were assayed for toxicity. The range of specimen toxicity was found to be 63-474 mouse units (MU) per specimen for N. papillosus by a tetrodotoxin (TTX) bioassay. The mean (SD) toxicity of the digestive gland and other portions were 296 +/- 120 and 382 +/- 156 MU in N. papillosus. The toxin was partially purified from the acidic methanol extract of the gastropod by using a C18 solid-phase extraction column. The eluate was then filtered through a 3000 MW cut-off ultrafree microcentrifuge filter. It was shown that the toxin purified from gastropods analysed by high-performance liquid chromatography and liquid chromatography/mass spectrometry contained TTX 42-60 microg g(-1) (about 90%), whereas along with minor paralytic shellfish poisons (PSP) it was 3-6 microg g(-1) (about 10%).

Aligned core-shell nanofibers delivering bioactive proteins.

AIMS: Continuous nanostructures embedded with proteins may synergistically present topographical and biochemical signals to cells for tissue engineering applications. This study presents the co-axial electrospinning of aligned poly(epsilon-caprolactone) nanofibers encapsulated with bovine serum albumin and platelet-derived growth factor-bb for demonstration of controlled release and bioactivity retention, respectively. MATERIALS & METHODS: Controllable release kinetics is achieved by incorporation of poly(ethylene glycol) as a porogen in the shell of the nanofibers. RESULTS & DISCUSSION: Poly(ethylene glycol) leaches out in a concentration- and molecular weight-dependent fashion, leading to bovine serum albumin release half-lives that range from 1 to 20 days. Optimized platelet-derived growth factor-bb-encapsulated nanofibers can completely release the protein with near zero-order kinetics and preserved bioactivity. CONCLUSION: Co-axial electrospinning is shown to be a versatile technique in achieving the delivery of biochemical signals in a controlled manner for regenerative medicine applications.

Cloning of the cDNA for thyroid stimulating hormone beta subunit and changes in activity of the pituitary-thyroid axis during silvering of the Japanese eel, Anguilla japonica.

The purposes of this study were: (1) to clone the cDNA encoding pituitary thyroid-stimulating hormone beta subunit (TSH beta) of the Japanese eel, Anguilla japonica, together with its genomic DNA sequence, for phylogenetic analysis, and to study the regulation of the TSH beta gene expression in cultured pituitaries; and (2) to investigate the transcript levels of pituitary TSH beta mRNA and the serum thyroxine profiles at different stages of ovarian development before and during silvering in the wild female eels. The maturity of female eels was divided into four stages, juvenile, sub-adult, pre-silver, and silver, based on skin color and oocyte diameter. The genomic DNA of the TSH beta subunit contains two introns and three exons, and the TSH beta protein possesses a putative signal peptide of 20 amino acids and a mature peptide of 127 amino acids. The amino acid sequence identities of TSH beta mature peptide of Japanese eel compared with those of teleosts and other vertebrates are: European eel (98.4%), salmonids (60.6-61.3%), carps (52.0-56.7%), sturgeon (48.4%), and tetrapods (42.9-45.2%). In in vitro studies of the regulation of TSH beta mRNA it was found that thyrotropin-releasing hormone increased while thyroxine decreased its expression. RT-PCR and real-time quantitative PCR analysis showed that the transcript levels of TSH beta subunit increased during eel silvering. The serum thyroxine levels also increased in parallel with TSH beta mRNA expression during silvering, supporting the hypothesis that the hypothalamus-pituitary-thyroid axis is correlated to silvering in the wild female Japanese eels.

Immunomodulation by dietary beta-1, 3-glucan in the brooders of the black tiger shrimp Penaeus monodon.

The present study evaluated the effectiveness of beta-1,3-glucan derived from Schizophyllum commune in enhancing shrimp survival as well as haemocyte phagocytosis and superoxide anion production in brooder Penaeus monodon. Pond-reared P. monodon adults (135 +/- 25 g) stocked in outdoor or indoor tanks were fed either a test diet containing beta-1,3-glucan (2.0 g kg(-1) or a glucan-free control diet for 40 days. Their survival was compared. The brooders reared in indoor tanks were analysed at days 0, 1, 3, 6, 12, 24, 30 and 40 for their haemocyte phagocytic activity and superoxide anion production. The results showed that regardless of indoor or outdoor rearing the survival rate of shrimp fed the glucan diet was significantly higher (P<0.001) than that of the control group. The brooders showed enhanced haemocyte phagocytic activity, cell adhesion and superoxide anion production when glucan was administered in their diets. The immunostimulatory enhancement peaked at day 24 after starting the dietary exposure and subsequently decreased to the pre-feeding level at the end of the 40 days feeding trial.

Introducing foreign DNA into tiger shrimp (Penaeus monodon) by electroporation.

Electroporation was used to introduce pFLAG-CMV-1-BAP, a DNA fragment that includes a bacterial alkaline phosphatase gene driven by a human cytomegalovirus (CMV) promoter, into Penaeus monodon zygotes. The transgenic tiger shrimp was achievedby using 10kV, 28 pulses, 120 g sec pulse time, 10 cycles, and a DNA concentration of 37.5 microg/mL. The hatching rate of electroporated zygotes (46%) was significantly lower than that of zygotes in the untreated group (89%). The survival rate of postlarvae in the electroporated group using a DNA concentration of 37.5 microg/mL decreased from 0.6% for postlarva 45 to 0.4% for postlarva 120. Based on dot blot analysis, the rate of gene transfer was 37% in mysis-stage, 23% postlarva 15(PL15), 19% postlarva 45(PL45), and 21% 4-month-old (about PL120). Genomic Southern blotting demonstrated that DNA from transgenic tiger shrimp contained fragments of exogenous DNA that were smaller, larger and of the same molecular size as pFLAG-CMV-1-BAP. Transferred DNA fragments were integrated into the genomes of 31% of the transgenic tiger shrimp. The exogenous DNA was mosaically distributed in a wide variety of tissues. Immunohistochemical staining revealed that the FLAG-BAP fused-protein encoded by pFLAG-CMV-1-BAP was present in the ovaries of some transgenic tiger shrimp.

[The development of shrimp blood agar for testing the hemolysis of shrimp's haemocyte by bacteria].

A new plating medium, shrimp blood agar, was developed by using the haemolymph of shrimp plus 200 ppm rose bengal as the substrate. The colorless shrimp haemocytes were dyed by rose bengal to red. On the present agar, the hemolytic bacteria strain may show a clear zone surrounding the bacterial colony. The result on hemolysis of 45 bacteria representing 12 genera isolated from aquaculture environments against shrimp blood agar and sheep blood agar was also evaluated. There are 11 strains with different results. The study showed that, with the aim of screening the hemolytic bacteria to shrimp, shrimp blood agar might reveal a relatively quick and accurate results than that of sheep blood agar.

The biological properties of black porgy (Acanthopagrus schlegeli) sperm and its cryopreservation.

This paper describes the general biology of the testes, milt and spermatozoa of the black porgy, Acanthopagrus schlegeli and reports some preliminary results in which the techniques for cryopreservation of spermatozoa were investigated. During the spawning season from December to February, the gonadosomatic index ranged from 2.0 to 3.5. The milt had an average pH value of 7.4 and osmotic pressure of 385 mOsm/kg. The head of the spermatozoon was apple-shaped and averaged at 1.6 microns in diameter. The best quality of milt was obtained only in the early spawning season. Good motility of spermatozoa could be maintained for up to 10 days in vials hanging in a water bath at 4 degrees C. For cryopreservation, an extender containing 5% glucose mixed with glycerol, serving as the cryoprotective agent (CPA), at a 4:1 ratio was used and the black porgy milt was diluted with the extender at a 1:1 ratio. After an equilibration period no longer than 10 minutes, straws containing this mixture were submerged in isopropanol at -10 degrees C and then frozen at a rate of 2 degrees C/min until the temperature reached -80 degrees C or were held in liquid nitrogen (LN) vapor (-90 to -100 degrees C) for 10 to 20 minutes. A total 720 of 0.5 ml straws were stored in LN at -196 degrees C for long term preservation. Between 50 and 90% of the post-thawed sperm were motile. After being cryopreserved for 1, 7, 7 and 342 days, sperm showed fertilities of 99.0, 93.2, 91.9 and 91.5% respectively.