Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Filamentous brown algae infected by the marine,holocarpic oomycete Eurychasma dicksonii: first results on the organization and the role of cytoskeleton in both host and parasite

The important role of the cytoskeletal scaffold is increasingly recognized in host-pathogen interactions. The cytoskeleton potentially functions as a weapon for both the plants defending themselves against fungal or oomycete parasites, and for the pathogens trying to overcome the resisting barrier of the plants. This concept, however, had not been investigated in marine algae so far. We are opening this scientific chapter with our study on the functional implications of the cytoskeleton in 3 filamentous brown algal species infected by the marine oomycete Eurychasma dicksonii. Our observations suggest that the cytoskeleton is involved in host defense responses and in fundamental developmental stages of E. dicksonii in its algal host.Oomycetes are important plant and animal pathogens and are the cause of significant crop losses every year. Hence, a plethora of studies with different cultivated and model plant species investigate the diversity of parasite infection pathways and host defense responses.¹ However, little information is available on the interactions between algae and marine oomycetes, despite the epidemic outbreaks reported² and the huge impact on intensive algal aquaculture.³Eurychasma dicksonii is a biotrophic, intracellular marine oomycete, capable to infect at least 45 species of brown seaweeds in laboratory cultures.⁴ Molecular data reveal that E. dicksonii has a basal phylogenetic position in the oomycete lineage.^5,6 The basic stages of the infection are known: the attachment of the parasite spore to the host cell wall, the penetration of its cytoplasm into the host cell, the formation of a multinucleated, unwalled thallus, and zoosporogenesis.⁶ Hitherto, though, there was no knowledge about the role of cytoskeleton in the context of infection, which stimulated our research.In land plants, reorganization of the cytoskeleton is part of the reaction to infection by fungal pathogens. The rearrangement of the cytoplasm and the relocation of the nuclei and other organelles are accompanied by rapid rearrangements of the cytoskeletal elements.⁷ The plant cytoskeleton shows an extreme plasticity in order to serve the intracellular realignment.At the same time, this indicates that the plant cytoskeleton could be the parasite’s target by producing anti-cytoskeletal compounds in an effort to overcome plant resistance, a mechanism known in several fungal and oomycete pathogens of higher plants.^8,9Consequently, the changes in microtubule (MT) organization are associated with both the plant defense and/or susceptibility toward oomycetes, respectively.¹⁰ Therefore, our research on the organization and role of cytoskeleton in the host and the parasite sheds some light into the enormous variability in the specificity of the recognition, defense, and infection mechanisms.     

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

Background

The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.

Methods

In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.

Results

Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).

Conclusion

These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

Secretion and degradation of lipoprotein lipase in cultured adipocytes. Binding of lipoprotein lipase to membrane heparan sulfate proteoglycans is necessary for degradation

Equilibrium-binding data of highly purified 125I-labeled avian lipoprotein lipase to cultured avian adipocytes demonstrate the presence of a class of high affinity binding sites. Analysis of the binding function yielded an association constant of 0.62 x 10(8)M-1 and a maximum binding capacity of 2.1 micrograms/60-mm dish. From a time course of dissociation of 125I-lipoprotein lipase from adipocytes at 4 degrees C, a dissociation rate constant of 6.1 x 10(-5)s-1 was obtained. Pretreatment of cells with heparinase and heparitinase resulted in a quantitative suppression of the high affinity binding component, establishing that lipoprotein lipase is bound to cell surface heparan sulfate proteoglycans. At 37 degrees C, cell surface-bound 125I-lipoprotein lipase is internalized and either degraded or recycled to the medium. The degradation rate constant for 125I-lipoprotein lipase was estimated to be 0.78 h-1. The degradation rate constant was reduced 6-fold when cells were exposed to 100 microM chloroquine, indicating that most of the degradation occurs within the lysosomal compartment. By using cells that had been pulsed with Trans35S-label for 1 h, it was demonstrated that acute treatment with endoglycosidases for up to 1 h resulted in a new lipoprotein lipase secretion rate which was 6-fold higher than that of control cells. Degradation of newly synthesized lipoprotein lipase was essentially blocked 30 min after the initiation of the chase. In other studies it was observed that there were no additive effects of chloroquine and either endoglycosidase or heparin treatment on total lipoprotein lipase levels (intracellular, cell surface, and medium) in adipocyte cultures. These experiments support the hypothesis that the release of lipoprotein lipase from its receptor prevents its internalization and degradation and enhances enzyme efflux from the adipocyte. A new model of lipoprotein lipase secretion in cultured adipocytes is proposed: Newly synthesized lipoprotein lipase is transported to the cell surface where it binds to specific heparan sulfate proteoglycan receptors. The enzyme is either released to the medium or internalized via the receptor, in which case the enzyme is degraded or recycled to the cell surface. Major determinants of enzyme efflux from the cell surface include the number and integrity of receptors, the association constant of the enzyme-receptor complex, and the presence in the medium of competing molecules with high affinity for lipoprotein lipase. In this model, modulation of lipoprotein lipase degradation rate may be a significant mechanism for acute regulation of enzyme efflux independent of changes in the rate of enzyme synthesis.     

Renal Carcinogenesis After Uninephrectomy

Nephrectomized rats have widely been used to study chronic renal failure. Interestingly, renal cell carcinoma occurred in the remnant kidney after uninephrectomy (UNX). In this study, we probed insulin-like growth factor (IGF)-1 signaling pathway in UNX-induced renal cancer. Adult male Sprague-Dawley rats were randomized into two groups: UNX rats (n = 22) and sham-operated rats (n = 12). Rats were killed at 3, 7, and 10 months. After 7 months after nephrectomy, the UNX rats developed renal cell carcinoma with increased expression of proliferating cell nuclear antigen, and 68.2% (15/22) of the animals exhibited invasive carcinoma. Western blot demonstrated significant down-regulation of IGF binding protein 3 contrasting with the up-regulation of protein kinase Cζ and Akt/protein kinase B in the renal cancer tissues. These findings indicate a unique rat model of UNX-induced renal cancer associated with enhanced IGF-1 signaling pathway.     

Multilamellar liposomes of triamcinolone acetonide: preparation,stability, and characterization

The aim of this study was to assess and characterize the stability of multilamellar liposomes as a delivery vehicle for triamcinolone acetonide. A standardized preparation method for a liposomal delivery vehicle was developed, after varying composition and storage conditions, and assessing encapsulation efficiency and loss of active principle. The assessment of temperature as a factor in formula stability during storage showed that stability improved under refrigeration (4–6°C) (less early diffusion of active principle through the liposomal wall), in comparison with samples stored at room temperature. To improve stability, cholesterol was added to some formulae, which although resulting in a decrease in average encapsulation efficiency, mitigated subsequent losses of retained active principle (formulae 4, 5, and 6), in comparison with those without cholesterol (formulae 1, 2, and 3). This was evident both under refrigerated and room-temperature conditions. Finally, after testing the effects of adding an antioxidant and/or preservative to the formulae, a liposomal design was achieved with acceptable stability, vesicle dimensions, and encapsulation efficiency.     

The effect of nutrients on carbon and nitrogen fixation by the UCYN-A–haptophyte symbiosis

Symbiotic relationships between phytoplankton and N₂-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N₂) fixation and primary production by the UCYN-A–haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N₂ fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO₂ fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO₂ fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N₂ fixation and the transfer of N₂-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.     

Role for rodent Smc6 in pericentromeric heterochromatin domains during spermatogonial differentiation and meiosis

Chromatin structure and function are for a large part determined by the six members of the structural maintenance of chromosomes (SMC) protein family, which form three heterodimeric complexes: Smc1/3 (cohesin), Smc2/4 (condensin) and Smc5/6. Each complex has distinct and important roles in chromatin dynamics, gene expression and differentiation. In yeast and Drosophila, Smc6 is involved in recombinational repair, restarting collapsed replication forks and prevention of recombination in repetitive sequences such as rDNA and pericentromeric heterochromatin. Although such DNA damage control mechanisms, as well as highly dynamic changes in chromatin composition and function, are essential for gametogenesis, knowledge on Smc6 function in mammalian systems is limited. We therefore have investigated the role of Smc6 during mammalian spermatogonial differentiation, meiosis and subsequent spermiogenesis. We found that, during mouse spermatogenesis, Smc6 functions as part of meiotic pericentromeric heterochromatin domains that are initiated when differentiating spermatogonia become irreversibly committed toward meiosis. To our knowledge, we are the first to provide insight into how commitment toward meiosis alters chromatin structure and dynamics, thereby setting apart differentiating spermatogonia from the undifferentiated spermatogonia, including the spermatogonial stem cells. Interestingly, Smc6 is not essential for spermatogonial mitosis, whereas Smc6-negative meiotic cells appear unable to finish their first meiotic division. Importantly, during meiosis, we find that DNA repair or recombination sites, marked by γH2AX or Rad51 respectively, do not co-localize with the pericentromeric heterochromatin domains where Smc6 is located. Considering the repetitive nature of these domains and that Smc6 has been previously shown to prevent recombination in repetitive sequences, we hypothesize that Smc6 has a role in the prevention of aberrant recombination events between pericentromeric regions during the first meiotic prophase that would otherwise cause chromosomal aberrations leading to apoptosis, meiotic arrest or aneuploidies.     

SPINDLES, SPINDLE PLAQUES, AND MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE (HANSEN)

The intranuclear spindle of yeast has an electron-opaque body at each pole. These spindle plaques lie on the nuclear envelope. During mitosis the spindle elongates while the nuclear membranes remain intact. After equatorial constriction there are two daughted nuclei, each with one spindle plaque. The spindle plaque then duplicates so that two side-by-side plaques are produced. These move rapidly apart and rotate so that they bracket a stable 0.8 µm spindle. Later, during mitosis, this spindle elongates, etc. Yeast cells placed on sporulation medium soon enter meiosis. After 4 hr the spindle plaques of the more mature cells duplicate, producing a stable side-by-side arrangement. Subsequently the plaques move apart to bracket a 0.8 µm spindle which immediately starts to elongate. When this meiosis I spindle reaches its maximum length of 3–5 µm, each of the plaques at the poles of the spindle duplicates and the resulting side-by-side plaques increase in size. The nucleus does not divide. The large side-by-side plaques separate and bracket a short spindle of about 1 µm which elongates gradually to 2 or 3 µm. Thus there are two spindles within one nucleus at meiosis II. To the side of each of the four plaques a bulge forms on the nucleus. The four bulges enlarge while the original nucleus shrinks. These four developing ascospore nuclei are partially surrounded by cytoplasm and by a prospore wall which originates from the cytoplasmic side of the spindle plaque. Eventually the spore nuclei pinch off and the spore wall closes. In some of the larger yeast cells this development is completed after 8 hr on sporulation medium.