Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.     

Effect of ambient temperatures during oocyte recovery on in vitro production of bovine embryos

Recovery of oocytes from ovaries collected at slaughter was carried out at three ambient temperatures (25 degrees, 30 degrees and 35 degrees C) to assess the effect on subsequent embryonic production in vitro. Oocytes recovered at each temperature were thereafter maintained at temperatures > or =35 degrees C as they were subjected to in vitro maturation, fertilization and culture (IVM/IVF/IVC). The oocytes and resulting embryos within each temperature group were subsequently evaluated for their rates of fertilization, cleavage and development to blastocysts, as well as for the number of cells/blastocyst. The results demonstrate that exposure of cumulus-ocyte-complexes (COCs) to temperatures below 35 degrees C during oocyte recovery is detrimental to optimal embryo production. Although the fertilization and cleavage rates of oocytes recovered at temperatures below 35 degrees C were not significantly lower than that of the controls, the percentage of oocytes recovered at 35 degrees C that developed to the blastocyst stage following fertilization and culture (33.7%) was significantly greater than those from oocytes recovered at either 25 degrees C (22.4%) or 30 degrees C (19.5%). The mean numbers of blastomeres/embryo were significantly lower in embryos derived from oocytes collected at either 25 degrees or 30 degrees compared with those collected at 35 degrees C. The results of this study suggest that exposure of COCs to temperatures below 35 degrees C during oocyte recovery may significantly decrease both the quantity and quality of embryos produced by in vitro methods.     

Incidence of mussel culture on biogeochemical fluxes at the sediment-water interface

Upward nutrient fluxes at the sediment-water interface were studied in a mussel farming zone (Carteau, Gulf of Fos, France) in order to estimate the impact of organic matter input from biodeposition. Nitrate, nitrite, ammonia, silicate, phosphate and oxygen were measured. Fluxes were estimated by means of polyacrylate benthic chambers placed at sites located under (UM) and outside (OM) the rope hanging structures. Transformation of biodeposited organic matter increases phosphate, silicate and ammonia fluxes. No variation in nitrite fluxes could be detected and only minor differences were observed in nitrate and the oxygen production/consumption equilibrium at the two stations. Phosphate and silicate fluxes, which were always higher at the UM than at the OM site, decreased from spring to winter. Ammonia fluxes were very high under mussel cultures in May and September and lower in November. The fact that ammonia flux was always higher at the UM than at the OM sites might be explained by degradation of mussel biodeposit, as well as by benthic macrafauna excretion. Discrepancies between fluxes of the nutrients studied at the UM and OM sites increased as organic particulate matter in the water column decreased. Variations of oxygen flux followed a different pattern, since they were correlated with presence and abundance of photosynthetic microphytes on the bottom and in the water. Bottom respiration exceeded production of oxygen only in May 1988 at the UM station.As it now stands, biodeposit input into the sediment under mussel ropes does not affect the ecosystem, although the flow of nutrients towards the water column is higher than in other areas.     

3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

How mechanisms of habitat preference evolve and promote divergence with gene flow

Habitat preference may promote adaptive divergence and speciation, yet the conditions under which this is likely are insufficiently explored. We use individual‐based simulations to study the evolution and consequence of habitat preference during divergence with gene flow, considering four different underlying genetically based behavioural mechanisms: natal habitat imprinting, phenotype‐dependent, competition‐dependent and direct genetic habitat preference. We find that the evolution of habitat preference generally requires initially high dispersal, is facilitated by asymmetry in population sizes between habitats, and is hindered by an increasing number of underlying genetic loci. Moreover, the probability of habitat preference to emerge and promote divergence differs greatly among the underlying mechanisms. Natal habitat imprinting evolves most easily and can allow full divergence in parameter ranges where no divergence is possible in the absence of habitat preference. The reason is that imprinting represents a one‐allele mechanism of assortative mating linking dispersal behaviour very effectively to local selection. At the other extreme, direct genetic habitat preference, a two‐allele mechanism, evolves under restricted conditions only, and even then facilitates divergence weakly. Overall, our results indicate that habitat preference can be a strong reproductive barrier promoting divergence with gene flow, but that this is highly contingent on the underlying preference mechanism.     

Adjustment of juvenile tuatara to a cooler,southern climate: operative temperatures,emergence behaviour and growth rate

Translocations for conservation often involve species limited to relict distributions. However, uncertainty can exist regarding the ability of source individuals to acclimatise following a shift to a distant location. We investigated the ability of captive-reared juvenile tuatara (Sphenodon punctatus) of Cook Strait stock (41°S) to adjust to outdoor, predator-protected pens within Orokonui Ecosanctuary (45 °S). We examined potential basking and within burrow temperatures, the influence of temperature on emergence, and growth rates in comparison with other locations. Tuatara at Orokonui reached their preferred temperature when basking over summer, and burrows provided protection from freezing over winter. Emergence was temperature-dependent and essentially ceased during winter. Growth rates of Orokonui-held juveniles were within the range for four other captive-rearing facilities and faster than for wild juveniles from a Cook Strait population. As all Orokonui-held juveniles have survived and grown we conclude that the climate at this southern location is suitable to consider a free-release.     

Identification of coiled bodies inBrassica napus nuclei during embryogenesis and early germination

Summary Nucleolus-associated bodies characterize interphase nuclei of many plant species. The recent demonstration that such bodies contain small nuclear ribonucleoproteins as well as coilin clearly indicates that they belong to a larger family of nuclear structures, known as coiled bodies, that have been intensively studied in a variety of animal cell types. In a previous work, we have shown that coiled bodies were present in close association with the nucleolus inZea mays dry seeds as well as during subsequent stages of germination. This study reveals that similar nuclear structures were also present duringBrassica napus embryogenesis starting at the torpedo stage and that they were, likewise, generally located on the nucleolar surface. As in the case ofZ. mays, coiled bodies were observed in cells of dry seeds as well as in those of early germinating tissues. These bodies were labelled with monoclonal antibody K121, an antibody reacting with the unique 5-terminal cap structure containing 2,2,7-trimethylguanosine that characterizes small nuclear RNAs. Owing to their intimate association with the nucleolus in all stages studied, the possibility is considered that, in these plant cells, coiled bodies are assembled on an organizer element located within this organelle.Abbreviations BSA bovine serum albumin - IgM immunoglobulin M - NAB nucleolus-associated body - NAC nucleolus-associated chromatin - PBS phosphate-buffered saline - snRNA small nuclear ribonucleic acid - snRNP small nuclear ribonucleoprotein     

Participation of alkaline phosphatase in the active transport of phosphates in brush border membrane vesicles

This paper reports the separation and preliminary characterization of the products formed by the reaction of the antitumor compound cis-Pt(NH₃)₂Cl₂ with DNA. Electrophoresis of the acid hydrolysed platinum-DNA complex gave a profile of platinum concentration which contained 5 peaks whose relative intensities varied with the amount of cis-Pt(NH₃)₂Cl₂ fixed on the DNA. Similar analysis of the products formed between DNA and trans-Pt(NH₃)₂Cl₂ or [Pt(dien)Cl]Cl, which are not active antitumor agents, indicated that these compounds bound to DNA in a different manner than cis-Pt(NH₃)₂Cl₂. DNA isolated from Escherichia coli which had been treated with cis-Pt(NH₃)₂Cl₂ or [Pt(dien)Cl]Cl did not give the same electrophoresis profiles as the corresponding platinum-DNA complexes formed in vitro.     

“Animals Like Us”: Identifying with Nonhuman Animals and Support for Nonhuman Animal Rights

While anthropomorphizing nonhuman animals has been shown to increase identification with them and, by extension, concern for their wellbeing, little research has directly tested whether identifying with nonhuman animals is similarly associated with concern for their wellbeing. We tested hypotheses related to this premise across three cross-sectional studies. In study 1 (n = 224), we tested the hypothesis that therians—a group of people who self-identify with nonhuman animals, show greater concern for nonhuman animal rights than non-therian furries—people with a fan-like interest in media featuring anthropomorphized animal characters. In study 2 (n = 206), we further tested this hypothesis using implicit and explicit measures of identification with nonhuman animals to predict behavioral intentions to support nonhuman animal rights. In study 3 (n = 182), we tested the generalizability of our findings in a sample of undergraduate students. Taken together, the studies show that explicit, but not implicit, identification with nonhuman animals predicts greater support for their rights. The implications of these findings for research on anthropomorphism and animal rights activism are discussed, as well as the limitations of these findings and possible avenues for future research.     

Human placental steroid sulfatase: purification and properties

Steroid sulfatase is recovered quantitatively from the 105,000 g h supernatant of human placental microsomes extracted with Triton X-100. The solubilized enzyme has been purified using conventional techniques. Throughout the purification procedure, steroid sulfatase appears to be heterogeneous as evidenced by certain, but not all, criteria. Following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the final preparation exhibits a major component and varying amounts of two minor ones. Antibodies raised in rabbits with the heterogeneous immunogen give rise to a single precipitation line when the native enzyme is analyzed by double immunodiffusion or by immunoelectrophoresis. In addition, using aged preparations of microsomes and immunoaffinity techniques, steroid sulfatase activity was found to be associated with the fastest migrating minor component. This finding would suggest that the apparent heterogeneity of purified steroid sulfatase is linked to degradation processes occurring within the microsomal preparations. Steroid sulfatase has a Stokes radius of 56 A, a sedimentation coefficient of 4.85 +/- 0.15S (in Triton-containing buffers) and binds 1.3 g of Triton X-100-per g of protein. The molecular weight of the Triton-protein complex was calculated to be 166,000 in which the glycoprotein portion contribution is about 43% (72,000). In contrast, the apparent molecular weight of the major polypeptide determined on calibrated SDS-gels is 62,000. The purified enzyme exhibits two pH optima with cholesterol sulfate as substrate, an acidic one at pH 5.0 and a second one at pH 7.5. The Km values for cholesterol sulfate, dehydroandrosterone sulfate and p-nitrophenylsulfate were 5.26, 14 and 1,320 microM, respectively.