The Immunocytochemical Detection of P53 Protein In Cytological Specimens: Technical Considerations

The p53 gene encodes a 393 amino acid nuclear phosphoprotein that appears to act as a cell cycle checkpoint, possibly by transactivating other target genes. Abnormalities of the p53 gene are common in a wide range of human tumours and are associated in many cases with immunologically detectable p53 protein. Detection of p53 immunoreactivity is uncommon in normal cells, but is frequently seen in neoplasia. Here we define the optimum conditions for the detection of p53 immunoreactivity in cytological material, including fixation and storage. Immersion in acetone-methanol for 10 min is optimal, and after air drying, smears or cytospin preparations can be stored at - 70°C for at least 6 months. We describe the range of controls necessary, including the use of positive control cell lines with known mutations of the p53 gene and defined abnormalities of p53 protein. Negative controls should include cell lines (or strains) with no p53 abnormality as well as the conventional negative immunological controls. It is only with these technical caveats and controls that p53 immunoreactivity can be performed reliably on cytological specimens. Le géne p53 code pour une phosphoprotéine nucléaire de 393 acides aminés qui semble jouer en rôle dans la régulation du cycle cellulaire, probablement par transactivation d'autres gènes cibles. Les anomalies du gène p53 sont présentes dans un large éventail de tumeurs humaines et sont associées a la présence d'une protéine p53 détectable immunologiquement. La détection d'une immunoréactivité anti p53 est rare dans les cellules normales alors qu'elle est fréquente dans les tumeurs. Nous avons défini dans ce travail les conditions optimales pour la détection de l'immunoréactivité anti p53 sur matériel cytologique, y compris les conditions de fixation et de conservation. L'immersion dans l'acétone-méthanol pendant 10 minutes est optimale. Aprés séchage à l'air, les frottis ou les préparations par cytocentrifugation peuvent être stockés à—70°C pendant au moins 6 mois. Nous décrivons aussi l'éventail des contrôles nécessaires incluant I'utilisation, comme contrôle positif, de lignées cellulaires avec des mutations connues du gène p53 et des anomalies définies de la protéine p53. Les contrôles négatifs doivent comporter des lignées cellulaires (ou des espèces) sans anomalie de p53 ainsi que les contrôles immunologiques négatifs convrentionnels. C'est seulement lorsque ces précautions techniques et ces contrôles sont respectés que l'immunoréactivité anti p53 peut être étudiée valablement sur les prélèvements cytologiques. Das p53 kodiert ein aus 393 Aminosären bestehendes Phosphoprotein, das offensichtlich den Zellzyklus blockiert, möglicherweise durch Aktivierung anderer Gene. Veränderungen des p53-Gens wurden in zahlreichen menschlischen Tumoren nachgewiesen, sodaß eine positive Reaktion in Neoplasien häufig, in Normalzellen jedoch ungewöhnlich ist. Die optimalen Bedingungen für den p53-Nachweis in cytologischem Material werden hinsichtlich Fixation und Lagerung untersucht. Eine 10minütige Aceton-Methanol-Fixierung mit anschlies-sender Lufttrocknung erlaubt die Lagerung von Ausstrichen und Cytozentrifugen-präparaten bei - 70°C für mindestens 6 Monate. Die erforderlichen Kontrollen einschließlich positiver Zellinien mit bekannten Mutationen des p53-Gens und definierten Anomalien des Proteins werden beschrieben. Negativkontrollen sollten Zellinien ohne p53-Anomalie ebenso umfassen wie die üblichen negativen immunologischen Kontrollen. Nur unter diesen Bedingungen ist ein zuverlässiger Nachweis von p53 mögloch.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

Relationships within theRugopharynx delta species complex (Nematoda: Strongyloidea) from Australian marsupials inferred from allozyme electrophoretic data

Relationships between the strongyloid nematodesRugopharynx delta, R. zeta, R. omega, R. longibursaris, R. mawsonae andR. sigma, all from macropodid marsupials, were investigated using allozyme data. The phylogenetic trees derived from the electrophoretic data set were congruent with those of the hosts and were consistent with the hypothesis that the species complex originated in pademelons of the genusThylogale and diversified in rock-wallabies (Petrogale spp.) and scrub wallabies of the subgenusNotamacropus. Host switching is evident only between closely related macropodid taxa.     

The xanthophyll cycle and sustained thermal energy dissipation activity in Vinca minor and Euonymus kiautschovicus in winter

The influence of low temperature on the operation of the xanthophyll cycle and energy dissipation activity, as ascertained through measurements of chlorophyll fluorescence, was examined in two broad-leaved evergreen species, Vinca minor L. and Euonymus kiautschovicus Loessner. In leaves examined under laboratory conditions, energy dissipation activity developed more slowly at lower leaf temperatures, but the final, steady-state level of such activity was greater at lower temperatures where the rate of energy utilization (through photosynthetic electron transport) was much lower. The rate at which energy dissipation activity increased was similar to that of the de-epoxidation of violaxanthin to antheraxanthin and zea-xanthin at different temperatures. However, leaves in the field examined prior to sunrise on mornings following cold days and nights exhibited a retention of antheraxanthin and zeaxanthin that was associated with sustained decreases in photosystem II efficiency. We therefore suggest that this phenomenon of ‘photoinhibition’ in response to light and cold temperatures during the winter results from sustained photoprotective thermal energy dissipation associated with the xanthophyll cycle. Such retention of the de-epoxidized components of the xanthophyll cycle responded to day-to-day changes in temperature, being greatest on the coldest mornings (when photoprotective energy dissipation might be most required) and less on warmer mornings when photosynthesis could presumably proceed at higher rates.     

Exposure of Gambian children to Anopheles gambiae malaria vectors in an irrigated rice production area

Abstract. Variation in exposure of children to malaria vectors of the Anopheles gambiae complex was recorded in a Gambian village situated near an irrigated area of rice cultivation. Observations were made in 1987 and 1988 during two dry seasons, when pumped water was used to grow rice, and two rainy seasons, when rice was produced using a combination of irrigated and rainfed paddies. Routine collections of mosquitoes were made from under bednets. Most of these specimens were assumed to have fed on the occupants of the net and thus represented a crude measure of exposure to malaria. Most nets in the village were in good condition, but even these were a poor defence against blood-seeking mosquitoes. Two annual peaks in the numbers of An. gambiae s.l. corresponded with the irrigation of rice paddies in the dry and wet seasons. When there were few vectors in the village the frequency distribution of mosquitoes caught under nets was described best by a Poisson process. When high numbers were present the daily distributions were over-dispersed and fitted a negative binomial model. The spatial distribution of mosquitoes varied between dry and wet seasons and was related to the predominant wind direction at night, suggesting that wind assisted the dispersal of mosquitoes from their breeding sites. For individual children in the rainy season, increased exposure to malaria vectors was associated with living adjacent to a mosquito breeding site, being resident in larger compounds, having open eaves in the house, a store-room adjacent to the bedroom, the absence of a ceiling in the bedroom, the absence of wood smoke indoors and leaving the bednet untucked at night. In the dry season a high level of exposure was associated with living close to a mosquito breeding site, having an unfenced compound, sleeping in a room without a ceiling and using insecticide aerosols. These observations demonstrate that within a village there are systematic and persistent differences in the level of exposure to malaria parasites experienced by individual children.