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Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. Within a low range of LMWH concentration (<0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin-bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.

A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS-containing solutions were evaluated as extractants. Several preelectrophoretic operations--such as treatment with RNase/DNase, ultrafiltration and desalting--and up to ten types of gels and three SDS-PAGE systems were considered. The SDS-containing solution allowed a higher protein extractability than urea. Unlike urea extraction, SDS extraction seemed not to be influenced so much by the state of the sample (raw, cooked at 60 degrees C, cooked at 85 degrees C). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under the optimized technical conditions described above, all the fish species tested, either raw and cooked, yielded reproducible and discriminant species-specific protein patterns.

By using single-strand conformation polymorphism (SSCP) analysis of three amplicons of the cytochrome b gene obtained by the polymerase chain reaction (PCR) it was possible to differentiate between various species of tunas and bonitos processed as canned fish. Four different techniques were used to produce single-strand DNA (ssDNA): (i) Denaturation of double-strand DNA (dsDNA) by formamide and alkali, (ii) two-step asymmetrical PCR, (iii) one-step asymmetrical PCR, and (iv) exonuclease digestion of the phosphorylated strand of dsDNA. The technique rendering optimal results depended on the type of amplicon (i.e. the sequence).

1. Electrophoretic separation patterns of water-soluble proteins of five species of hake, Merluccius merluccius; M. australis; M. productus; M. bilinearis and M. angustimanus, have been examined. 2. The patterns were reproducible within each species and distinguishable from one another. 3. The frequency distribution of numbers of vertebrae and of gillrakers were obtained for the same fish. 4. Only the electrophoretic patterns gave an unequivocal identification of the species.

Two incidents of toxin-type food poisoning in N.E. Scotland associated with the consumption of red whelks (Neptunea antiqua) are described. Four patients developed symptoms within 1 h of consuming whole whelks. These included visual disturbances--double vision and difficulty in focusing--tingling of the fingers, prostration and in one subject nausea, vomiting, diarrhoea and ataxia. In all cases recovery was complete in 24 h. Using a newly developed analytical technique the concentration of the causative toxin, tetramine, in the salivary glands of the whelks consumed was estimated at 0.07%, equivalent to a content of 3.75 mg/100 g of the shellfish.