Differential distribution of3H dihydrotestosterone and3H estradiol nuclear binding sites in mouse male accessory sex organs

Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with³H dihydrotestosterone (³H DHT) and³H estradiol (³H E₂). With³H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With³H E₂, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with³H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With³H E₂, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with³H E₂. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with³H DHT, but not with³H E₂. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of³H DHT and³H E₂ nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens. Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

Comparing ethylene glycol with glycerol and with or without dithiothreitol and sucrose for cryopreservation of bull semen in egg-yolk containing extenders

There are few studies performed for investigating the roles of different ratio and cryoprotectants with dithiothreitol or sucrose on sperm motility characteristics and antioxidant capacities of post-thawed bull spermatozoa. The objectives of this study were to compare glycerol (G) and ethylene glycol (EG) at different concentrations as cryoprotectants and dithiothreitol (D) or sucrose (S) (with/without) as antioxidants in Tris extender for cryopreservation of bull semen. Twenty-four ejaculates obtained from three bulls were included in the study. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and diluted using both of the dithiothreitol 5 mM or sucrose 25 mM, and control (without additives) was cooled to 4 °C and frozen in 0.25-ml French straws. when compared to control, different doses cryoprotectants and antioxidants addition no significantly increased the percentages of post-thaw sperm progressive and motitilities, acrosome abnormality and plasma membrane integrity (P > 0.05). However, EG3 + S yielded the greatest percentages of the total abnormality (P < 0.05). As regard to antioxidant activities G7 and EG5 led to lowest MDA activity with or without D or S but, these results were not supported to the GPx activity (P < 0.01). The sperm motion characteristics such as VAP, VCL, ALH and BCF gave significantly different results (P < 0.05). When compared the DNA integrity, different doses cryoprotectants without antioxidants addition significantly increased the percentages of the tail intensity and tail moment (P < 0.05). There were no significant differences observed in non-return rates among all treatment groups (P > 0.05).     

Interspecific variation in arrangement and morphology of micrasters of Tethya species (Porifera,Demospongiae)

Summary A new distinctive feature between the two Mediterranean species of Tethya, T. aurantium and T. citrina has been found in the body arrangement of different types of micrasters. Contrary to the previous assumptions, T. aurantium has two clearly distinct categories of micrasters: the chiaster-tylaster in the cortex and the larger, slender oxyaster in the choanosome. T. citrina has only slightly differentiated micraster sets in the cortex and choanosome; in the latter the shape of micrasters is close to that of oxyasters. SEM analysis shows that differences in micraster shape depend on the cylindrical or conical form of rays and on the distribution, density and strength of the microspines along their axis. The relationship between the degree of micraster differentiation and the development of the cortex in the two species is discussed.     

Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

Climate determinants of breeding and wintering ranges of lesser kestrels in Italy and predicted impacts of climate change

Climate warming would theoretically create conditions for the breeding range expansion of pseudo‐steppe Mediterranean and long‐distance migrant species and provide the possibility for these to overwinter in the same breeding areas. However, contemporary changes in rainfall regimes might have negative effects on the climate suitability and in turn, shrink species potential range. The lesser kestrel Falco naumanni is highly sensitive to rainfall oscillations and has recently extended its Italian breeding range towards northern latitudes and increasing its wintering records. We modelled the effects of temperature and rainfall on current and future climate suitability for lesser kestrels in both the breeding and wintering periods by using MaxEnt. Models were based on the distribution of 298 colonies and 40 wintering records. Future climate suitability was assessed under eight different scenarios. Spring rainfall amount resulted as the main determinant of breeding climate suitability, so its predicted reduction will determine a shrinkage in suitable areas (–42.10% in 2050; –32.07% in 2070). Specifically, the 66.05% of Italian colonies will be outside the climatically suitable area by 2050. However wide areas, suitable under current climate conditions, are still not occupied by lesser kestrel and allow the potential expansion of its Italian breeding range in the short term. Temperature seasonality mainly determined the species’ winter climate suitability, which is overall predicted to boost in the next decades (+145.03% in 2050; and +123.91% in 2070). All but one future scenarios predicted a northward shift of about 40 km for both breeding and wintering climate suitability. Despite its recent expansion, we have found that climate change will pose conservation concerns for the Italian breeding population of lesser kestrels. Indeed, changes in non‐climate factors will also outline the future suitability of the Italian range for lesser kestrels in both seasons with effects that might both strengthen or mitigate climate effects.