Extraction and purification of prostaglandins and thromboxane from biological samples for gas chromatographic analysis

An efficient extraction procedure for the isolation of prostaglandins (PGs) from biological samples for their subsequent quantification by gas chromatography-electron capture detection (GC-ECD) is described. PGs were extracted from lung, kidney, spleen and stomach fundus into ethyl acetate at different pHs. The highest recovery and least extraction of contaminating pigments was obtained at pH 4.5. Pigments and other contaminants are removed by thin-layer chromatography using a solvent system chloroform-isopropyl alcohol-ethanol-formic acid (45:5:0.5:0.3). The isolated PGs were determined by GC-ECD after appropriate derivatization. The overall recovery of PGs using this procedure is 60%.     

Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.     

Self-incompatibility in passion fruit: evidence of two locus genetic control

 The self-incompatibility in yellow passion fruit was previously described as homomorphic sporophytic with monofactorial inheritance. Five progenies were obtained by bud-selfing. The plants of these progenies were selfed, reciprocally crossed within each progeny and crossed with known incompatible phenotypes to identify their phenotypic group. Fruit set was evaluated at the 7th day after pollination. Two progenies consisted of two self-incompatible groups, the other three formed three suck groups. The groups were identified as S₁, S₂, S₃, S₄, S₅ and S₆. The results provide evidence that the self-incompatibility of passion fruit is controlled by two loci, the S-gene and another, whose expression needs to be investigated. Received: 20 June 1998 / Accepted: 13 July 1998     

Human cerebral cysticercosis: immunolocalization of a sodium-dependent glucose cotransporter (SGLT) in larval and adult tapeworms.

Light microscopic immunocytochemistry was used to examine human brain cysticerci resected from the fourth ventricles of patients who had not been treated with anthelminthic drugs. Tissues were examined from 3 different patients undergoing surgery for treatment of hydrocephalus. A rabbit polyclonal antiserum to the peptide corresponding to amino acids 564-575 unique to the rabbit sodium-dependent, SGLT1 glucose cotransporter labeled with immunoperoxidase, localized immunoreactive SGLT epitopes. This antibody localizes SGLT1 in the apical brush borders of human enterocytes, but is negative in cytoplasm, as well as lateral and basal enterocyte membranes. Taenia solium neurocysticerci were SGLT positive; transporter protein was highly expressed on the surface microvilli of the external cyst wall. The well-developed network of small and larger osmoregulatory ducts within racemose larval cystcerci displayed high expression of SGLT cotransporter, consistent with a resorptive function for this system of tubules. Because water is cotransported with glucose molecules by the SGLT protein, its high expression in neurocysticerci may contribute to the expansive growth of these larvae in subarachnoid and intraventricular sites. The SGLT epitopes were also immunolocalized in gravid proglottids of Taenia saginata, indicating that cotransporter expression persisted in intestinal-dwelling, adult tapeworms. Cotransporter antibody was abundantly localized at the proglottid tegumentary surface and in the lateral osmoregulatory ducts, analogous to the SGLT localization in cysticerci. Furthermore, high expression of this cotransporter was seen in the branches of the uterus, suggesting that SGLT-mediated absorption of glucose and water has an important functional role within the reproductive system of adult tapeworms.     

The importance of the marine ornamental reef fish trade in the wider Caribbean

The marine ornamental fish trade began in the 1930s in Sri Lanka, spread to Hawaii and the Philippines in the 1950s, and expanded to a multi-million dollar industry in the 1970s with fisheries established throughout the tropical Pacific, Indian and Atlantic Oceans. Currently, 45 countries supply global markets an estimated 14-30 million fish annually, with an import value of US$28-44 million. The largest suppliers are Indonesia and the Philippines, followed by Brazil, Maldives, Vietnam, Sri Lanka and Hawaii. In the tropical Western Atlantic, 16 countries have export fisheries, including the U.S. (Florida and Puerto Rico). The U.S. is the world's largest buyer, followed by the European Union and Japan. The global trade consists of over 1400 species of reef fishes, of which only about 25 are captive bred on a commercial scale. Damselfish, anemonefish, and angelfish constitute over 50% of the global volume; butterflyfish, wrasses, blennies, gobies, triggerfish, filcfish, hawkfishes, groupers and basselets account for 31% of the trade, and the remaining 16% is represented by 33 families. The most important fishes from the Caribbean are angelfish (six species), seahorses (two species), royal gramma, jawfish, queen triggerfish, redlip blenny, puddingwife, bluehead wrasse, and blue chromis. The Caribbean currently supplies a small percentage of the global trade in marine ornamental species, but ornamental fisheries in this region represent important emerging industries. It is critical that effective ornamental fishery management plans and regulations are developed and enforced, and fishery-dependent and fishery-independent data are collected and utilized in decision making processes to ensure sustainable ornamental fisheries throughout the region.     

Control of vesicle fusion by a tyrosine phosphatase

The tyrosine phosphatase PTP-MEG2 is targeted by its amino-terminal Sec14p homology domain to the membrane of secretory vesicles. There it regulates vesicle size by promoting homotypic vesicle fusion by a mechanism that requires its catalytic activity. Here, we identify N-ethylmaleimide-sensitive factor (NSF), a key regulator of vesicle fusion, as a substrate for PTP-MEG2. PTP-MEG2 reduced the phosphotyrosine content of NSF and co-localized with NSF and syntaxin 6 in intact cells. Furthermore, endogenous PTP-MEG2 co-immunoprecipitated with endogenous NSF. Phosphorylation of NSF at Tyr 83, as well as an acidic substitution at the same site, increased its ATPase activity and prevented alphaSNAP binding. Conversely, expression of a Y83F mutant of NSF caused spontaneous fusion events. Our results suggest that the molecular mechanism by which PTP-MEG2 promotes secretory vesicle fusion involves the local release of NSF from a tyrosine-phosphorylated, inactive state. This represents a novel mechanism for localized regulation of NSF and the first demonstrated role for a protein tyrosine phosphatase in the regulated secretory pathway.     

Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections

Proteoglycans are among the major extracellular matrix components of the central nervous system. In the cerebral cortex and many subcortical regions, chondroitin sulphate proteoglycans, which are related to the aggrecan-versican- neurocan family, have been detected immunocytochemically in perineuronal nets that surround various types of neurons. This indicates that, in the brain, there is a nonhomogeneous but defined distribution of extracellular matrix components. The present study is a further attempt to characterize the perineuronal nets in the cerebral cortex. Sections obtained from fixed and unfixed rat brains were subjected to different enzymatic treatments prior to the visualization of perineuronal nets using N-acetylgal actosamine-binding Wisteria floribunda agglutinin, antibodies against chondroitin sulphate proteoglycans or hyaluronectin, and biotinylated hyaluronectin which detects hyaluronan. In all perineuronal nets the binding of the Wisteria floribunda agglutinin was abolished after the incubation of sections with chondroitinase ABC. The protein components of the proteoglycan complexes became easier to digest after removal of chondroitin sulphate chains or hyaluronan. Since only quantitative, and not qualitative, differences in the labelling properties and the structural appearance of cortical perineuronal nets were observed after the various treatments, it is concluded that, with regard to their proteoglycan composition, these structures have common basic properties     

International regulatory requirements for Leptospira vaccine potency testing. Roundtable: Current requirements and opportunity for harmonization

Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests.