Sen-Shyong Jeng

Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata

Shellfish (Niotha clathrata) were collected in both July and November from three locations in Taiwan (Pingtung, Kaohsiung and Chiai Prefecture) and assayed for anatomical distribution of tetrodotoxin (TTX) and aerobic heterotrophic bacteria. Pingtung specimens showed higher toxicity than those from Kaohsiung and Chiai, and did not show much seasonal variation. At each site, the total aerobic bacterial counts in November samples were higher than in July. The predominant genera were Vibrio, Pseudomonas, Pasteurella, Aeromonas and Plesiomonas. Vibrio comprised more than 35% of the genera, with V. alginolyticus as the major species. The viable counts of Vibrio species were higher in November than in July. However, the results did not suggest any relationship between the total count or viable count and the toxicity of the shellfish. HPLC, UV and gas chromatographic-mass spectrometric analyses demonstrated that some of the bacteria isolated, such as V. alginolyticus, V. parahaemolyticus, Pseudomonas spp. Plesiomonas sp. and Aeromonas sp., produced TTX and/or related substances.

Toxicity and toxic components of two xanthid crabs, atergatis floridus and demania reynaudi, in taiwan

Paralytic toxicity was detected by tetrodotoxin bioassay in eight specimens of Atergatis floridus and seven specimens of Demania reynaudi, collected from Taiwan in 1994. The toxicity of crab specimens was 161 +/- 115 (mean +/- S.D.) mouse units (MU) for A. floridus and 640 +/- 273 MU for D. reynaudi. The respective toxins were partially purified from specimens of A. floridus and D. reynaudi by ultrafiltration using a Diaflo YM-1 membrane, followed by chromatography on a Bio-Gel P-2 column. Electrophoresis, thin-layer chromatography, high-performance liquid chromatography, ultraviolet spectrum and gas chromatography-mass spectrometry indicated that the toxin of A. floridus was mainly composed of tetrodotoxin (85%), along with minor gonyautoxin 1-4 (15%), and the toxin of D. reynaudi was mainly composed of tetrodotoxin (88%), along with minor gonyautoxin 2-4 and neosaxitoxin (12%).

Association of zinc with connective tissue in the digestive tract of common carp

Zinc (Zn) concentration in the digestive tract of common carp is always > 10 times higher than most animal tissues. In a previous paper, it was reported that this high Zn came from a 43 kDa Zn-binding membrane protein. In this present study it was further found that in the digestive tract of common carp, Zn content was closely associated with the amounts of extracellular macromolecules. The higher the Zn content, the more there is of collagen and glycosaminoglycans. An indirect immunoperoxidase staining method using antibody against the 43 kDa Zn-binding protein, or the fibroblast marker (Thy 1.1 protein) was applied to the sections of digestive tract of fish. It was found that the Zn-binding protein in the digestive tract of common carp is mainly located in the connective tissue of its lamina propria and submucosal layer. Connective tissue cells, probably fibroblasts, hold the 43 kDa Zn-binding protein. In the common carp Zn might be bound to the external side of the fibroblast. The present finding may have a significant meaning on extending the studies of Zn in biology to the field of Zn with extracellular matrix.

High zinc in the erythrocyte plasma membranes of common carp Cyprinus carpio

The distribution of zinc (Zn) concentration in the blood and erythrocytes of common carp, grass carp, silver carp and tilapia was studied. It was found that in whole blood, the average Zn concentrations in these four species of fish (approx. 6–14 μg/[ml whole blood]) were relatively similar to those in other species of fish and mammals. However, the mean Zn concentration in the erythrocytes of common carp is approximately two times higher than the other three species of fish (approx. 5 vs approx. 2 μg/[ml whole blood]). It was found that approximately 70% of the Zn in the common carp whole blood came from its erythrocytes. In addition, approximately 43% of the Zn in the erythrocytes of common carp was found to be located on its outer plasma membranes. When an antibody against a 43 kDa Zn-binding protein, isolated from the digestive tract tissue of common carp, was used, significant quantities of the protein were shown to be present on the erythrocyte plasma membranes of common carp by an indirect immunofluorescent staining. High Zn on the outer plasma membrane of the common carp erythrocyte most probably comes from the 43 kDa Zn-binding protein.

Binding characteristics of the Zn-binding membrane protein from common carp

This study incorporated the 43 kDa Zn-binding membrane protein isolated from common carp into liposome. The specificity and strength of the binding of 65Zn to the 43 kDa protein-liposomes, and the binding of the 65Zn-labeled 43 kDa protein-liposomes to laminin were studied. It was found that 65Zn was bound to the external side of the 43 kDa protein-liposomes. Specific binding of 65Zn to the protein-liposomes was detected. The binding parameter of Zn to the protein was found to be: maximum binding site (Nmax), 76.7 pmole/μg protein (approx. 3 mole of Zn2+/mole); and equilibrium dissociation constant (Kd), 0.19 μM. Of the cations introduced (Ca2+, Cd2+, Co2+, Cr2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+), only Co2+ competed significantly with Zn. The protein-liposomes were also found to bind specifically to laminin with a Nmax of 1.1 pmole/μg laminin, and Kd of 4.79 μM. No significant protein-liposome binding occurred to other extracellular matrix proteins (fibronectin, fibrinogen or vitronectin). Furthermore, the binding was specifically inhibited by the Arg-Gly-Asp (RGD) peptide or GRGDSPG, while two other analogs (GRGESPG and GRADSPG) were without effect.