Cytochemical and Biochemical Analysis of Development of Urechis Oocytes

The echiuroid marine worm Urechis caupo is uniquely suited forthe study of oogenesis. A relatively large quantity of oocytesat various developmental stages can be obtained and subjectedto coordinated cytochemical and biochemical analysis Oocytesat the cluster, early diplotene, mid-diplotene, and diffusediplotene or lampbrush stages are active in the synthesis andaccumulation of ribosomal RNA, several proteins, carbohydrates,lipids, and also, perhaps, yolk constituents. Only corticalgranule formation, which occurs during later stages of oogenesis,appears to be stage specific. Ribosomal RNA genes are also transcribedin the nucleolus of the mature oocytes or unfertilized eggs.However, the rate of production in these eggs appears to beregulated at the level of maturation of rRNA precursor molecules.     

Cytohistological correlation as a measure of quality assurance of a cytology laboratory

Cytohistological correlation as a measure of quality assurance of a cytology laboratory In a hospital-based cytology screening programme for the early detection of preinvasive lesions of the uterine cervix, 166 women with abnormal smears (human papillomavirus (HPV) changes, cervical intraepithelial neoplasia (CIN) and invasive carcinoma) were referred to the central colposcopy clinic between January 1989 and December 1991. The colposcopist (V.S.) was able to take a direct biopsy in 156 cases. In the remaining 10 cases, biopsy could not be taken because of unsatisfactory colposcopy. A cytohistological correlation was obtained in 121/156 (77.5%) cases, and the remaining 35 cases showed a disparity in diagnosis. These were reviewed by one of us (P.S.) and the reasons for underdiagnosis/false negatives and overdiagnosis/false-positive results were analysed. It was found that sampling error was the cause of false negativity and underdiagnosis in most cases, while interpretative errors resulted in the overdiagnosis and false-positive smears. The reasons for interpretative errors were studied in detail. Les corrélations cyto-histologiques comme mesure de l'assurance qualité dans un laboratoire de cytologie Dans le cadre d'un programme hospitalier de dépistage précoce des lésions précurseurs et des lésions invasives du col utérin, 166 femmes ayant un frottis anormal (lésions virales à HPV, CIN, carcinome infiltrant) ont été vues dans le service central de colposcopie entre janvier 1989 et décembre 1991. Dans 156 cas, le colposcopiste (VS) a pu réaliser une biopsie. Dans les 10 cas restants, la colposcopie n'était pas satisfaisante et la biopsie n'a pas été pratiquée. Une corrélation cyto-histologique a pu être établie dans 121 cas sur 156 (77, 5%) avec, dans 35 cas, constatation d'une discordance diagnostique. Ces cas ont été revus par l'un de nous (PS) et les raisons de cette sous-évaluation diagnostique/faux-négatifs et de surévaluation diagnostique/faux-positifs, ont été analysées. Cette analyse montre que le prélèvement est à l'origine des faux-négatifs et de la sous-évaluation diagnostique dans la majorité des cas, alors que l'erreur d'interprétation est responsable de la surévaluation diagnostique et des faux-positifs. Les sources d'erreur d'interprétation ont été étudiées dans le détail. Die Korrelation zwischen Zytologie und Histologie als Massnahme der Qualitatssicherung eines zytologischen Labors Im Rahmen des Screeningprogramms eines Krankenhauses wurden 166 Frauen mit auffälligen Befunden (HPV, CIN oder invasive Karzinome) zwischen Januar 1989 und Dezember 1991 einer zentralen Kolposkopieklinik überwiesen. Biopsien konnten in 156 Fällen gewonnen werden, während das kolposkopische Bild dies in 10 Fällen nicht rechtfertigte. Übereinstimmung zwischen Zytologie und Histologie wurde in 121/156 (77,5%) erzielt. Abweichungen ergaben sich in 35 Fällen, die genau analysiert wurden. Entnahmefehler verursachten die meisten falsch negativen und unterbewerteten Fälle, während falsch positive und zu hoch bewertete Fälle auf Interpretationsfehlern beruhten. Diese wurden genau studiert.     

The role of ribonucleoproteins in the production of mitotic abnormalities

Summary Immersion of growing roots of onion and lily in aerated solutions of ribonuclease affected their pattern of growth and altered the structure and mitotic distribution of the chromosomes. Action of the enzyme on meristematic cells caused enlargement of nucleoli, excessive contraction, stickiness, adhesion, and clumping of chromosomes, and production of aneuploid and polyploid chromosome complexes, tripolar and multipolar spindles, binucleate and multinucleate cells. Very few cases of chromosome fragmentation were observed.Accumulation of abnormalities accompanied the passage of ribonuclease across the root as determined by alterations in stainability of the cells with pyronin and fast green. There was no visible modification of stainability of the chromosomes with methyl green or the Feulgen reagent.These results, when compared with those produced by control solutions, indicate that ribonuclease enters the living cell and degrades ribonucleoproteins essential for maintenance of structural and functional integrity. The implications of these results, with respect to the production of aberrations by other agents, are discussed.This study was supported by a research grant (RG-149) from the National Institutes of Health, U.S. Public Health Service.     

Intrachloroplast Localization of 65Zn and 63Ni in a Zn-tolerant Plant, Ocimum basilicum Benth

Intrachloroplast localization studies of ⁶⁵Zn and ⁶³Ni weremade in a Zn-tolerant plant Ocimum basilicum var. purpurascensBenth. in order to investigate the mechanism and specificityof metal tolerance. The isotopes were supplied in solution tothe roots 14 d before fractionation. It was observed that ⁶⁵Znactivity was comparatively greater in the chloroplast envelopemembranes and stroma than the ⁶³Ni; and ⁶³Ni was largely foundin the lamellar and stroma fractions. Further analysis of lamellaerevealed that photosystem II (PS II) particles were richer inradioactivity than photosystem I (PS I) particles. The photochemicalevents of photosynthesis were less affected in Zn-treated plantsthan in the Ni-treated plants. The changed levels of the electrontransport chain intermediates including cytochromes, plastocyaninand ferredoxin provide supporting evidence for the localizationstudies. The activity of carbonic anhydrase, a zinc metalloprotein,was increased in Zn-treated plants with increase in nutrientZn concentration, indicating the binding of zinc to a proteinmoiety in the chloroplasts.     

Size-spectra as indicators of the effects of fishing on coral reef fish assemblages

The data requirements and resources needed to develop multispecies indicators of fishing impacts are often lacking and this is particularly true for coral reef fisheries. Size-spectra, relationships between abundance and body-size class, regardless of taxonomy, can be calculated from simple sizeabundance data. Both the slope and the mid-point height of the relationship can be compared at different fishing intensities. Here, we develop size-spectra for reef fish assemblages using body size- abundance data collected by underwater visual census in each of ten fishing grounds across a known gradient of fishing intensity in the Kadavu Island group, Fiji. Slopes of the size-spectra became steeper (F_9,69=3.20, p<0.01) and the height declined (F_9,69=15.78, p<0.001) with increasing fishing intensity. Regressions of numbers of individuals per size class across grounds were negative for all size classes, although the slope was almost zero for the smallest size class. Response to exploitation of each size class category was greatest for larger fish. Steepening of the slope with increasing fishing intensity largely resulted from reductions in the relative abundance of large fish and not from the ecological release of small fish following depletion of their predators. The slope and height of the size-spectrum appear to be good indicators of fishing effects on reef fish assemblages.     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Leishmania donovani Attachment Stimulates PKC-Mediated Oxidative Events in Bone Marrow-Derived Macrophages

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O₂^- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O₂^- and NO production and stimulation of O₂ consumption. Optimal NO and O₂^- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O₂^- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment. Activation of O₂^- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.