Expression, localization and functions in acrosome reaction and sperm motility of Ca(V)3.1 and Ca(V)3.2 channels in sperm cells: an evaluation from Ca(V)3.1 and Ca(V)3.2 deficient mice

In spermatozoa, voltage-dependent calcium channels (VDCC) have been involved in different cellular functions like acrosome reaction (AR) and sperm motility. Multiple types of VDCC are present and their relative contribution is still a matter of debate. Based mostly on pharmacological studies, low-voltage-activated calcium channels (LVA-CC), responsible of the inward current in spermatocytes, were described as essential for AR in sperm. The development of Ca(V)3.1 or Ca(V)3.2 null mice provided the opportunity to evaluate the involvement of such LVA-CC in AR and sperm motility, independently of pharmacological tools. The inward current was fully abolished in spermatogenic cells from Ca(V)3.2 deficient mice. This current is thus only due to Ca(V)3.2 channels. We showed that Ca(V)3.2 channels were maintained in sperm by Western-blot and immunohistochemistry experiments. Calcium imaging experiments revealed that calcium influx in response to KCl was reduced in Ca(V)3.2 null sperm in comparison to control cells, demonstrating that Ca(V)3.2 channels were functional. On the other hand, no difference was noticed in calcium signaling induced by zona pellucida. Moreover, neither biochemical nor functional experiments, suggested the presence of Ca(V)3.1 channels in sperm. Despite the Ca(V)3.2 channels contribution in KCl-induced calcium influx, the reproduction parameters remained intact in Ca(V)3.2 deficient mice. These data demonstrate that in sperm, besides Ca(V)3.2 channels, other types of VDCC are activated during the voltage-dependent calcium influx of AR, these channels likely belonging to high-voltage activated Ca(2+) channels family. The conclusion is that voltage-dependent calcium influx during AR is due to the opening of redundant families of calcium channels.     

Development and characterization of a mouse floxed <Emphasis Type="Italic">Bmp2</Emphasis> osteoblast cell line that retains osteoblast genotype and phenotype

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.     

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

Background  

Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.     

Microbial reductive dechlorination of aroclor 1260 in Baltimore harbor sediment microcosms is catalyzed by three phylotypes within the phylum Chloroflexi

The specific dechlorination pathways for Aroclor 1260 were determined in Baltimore Harbor sediment microcosms developed with the 11 most predominant congeners from this commercial mixture and their resulting dechlorination intermediates. Most of the polychlorinated biphenyl (PCB) congeners were dechlorinated in the meta position, and the major products were tetrachlorobiphenyls with unflanked chlorines. Using PCR primers specific for the 16S rRNA genes of known PCB-dehalogenating bacteria, we detected three phylotypes within the microbial community that had the capability to dechlorinate PCB congeners present in Aroclor 1260 and identified their selective activities. Phylotype DEH10, which has a high level of sequence identity to Dehalococcoides spp., removed the double-flanked chlorine in 234-substituted congeners and exhibited a preference for para-flanked meta-chlorines when no double-flanked chlorines were available. Phylotype SF1 had similarity to the o-17/DF-1 group of PCB-dechlorinating bacteria. Phylotype SF1 dechlorinated all of the 2345-substituted congeners, mostly in the double-flanked meta position and 2356-, 236-, and 235-substituted congeners in the ortho-flanked meta position, with a few exceptions. A phylotype with 100% sequence identity to PCB-dechlorinating bacterium o-17 was responsible for an ortho and a double-flanked meta dechlorination reaction. Most of the dechlorination pathways supported the growth of all three phylotypes based on competitive PCR enumeration assays, which indicates that PCB-impacted environments have the potential to sustain populations of these PCB-dechlorinating microorganisms. The results demonstrate that the variation in dechlorination patterns of congener mixtures typically observed at different PCB impacted sites can potentially be mediated by the synergistic activities of relatively few dechlorinating species.     

Egg color as an adaptation for thermoregulation

ABSTRACT.   Avian embryos are incubated at temperatures only 2–6 °C below that at which hyperthermia begins to influence survival. In habitats where sunlight directly strikes the eggs, even for short periods, heat gain may be a substantial threat to survival, and reflective pigmentation may reduce the rate of heat gain. The results of previous studies suggest that light-colored eggs acquire heat slower than dark eggs, but artificial pigments were used to create differences in egg coloration. This approach is problematic because natural eggshell pigments have low absorbance in the near-infrared waveband that encompasses about half of incident solar radiation. We used naturally-pigmented eggs to measure the influence of egg coloration on heat gain. Triads ( N = 18) of eggs from Brewer's ( Euphagous cyanocephalus ), Red-winged ( Agelaius phoeniceus ), and Yellow-headed ( Xanthocephalus xanthocephalus ) blackbirds were crossed with six nests of each species and either exposed to full sunlight or placed under a diffusing umbrella. Thermisters recorded internal egg temperature every minute until an asymptotic temperature was reached. Eggs in full sunlight acquired heat more rapidly than eggs in the shaded environment, but heat gain did not vary with egg color in either environment. Eggs placed in Yellow-headed Blackbird nests took longer to reach asymptotic temperature, but there was no significant egg-by-nest interaction. Thus, it appears that differences in reflectivity of eggshell pigments in the visible range (400–700 nm) do not result in different rates of heat acquisition. The thermoregulation hypothesis was not supported.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.     

Amino acid–base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level

To assess whether there are universal rules that govern amino acid–base recognition, we investigate hydrogen bonds, van der Waals contacts and water-mediated bonds in 129 protein–DNA complex structures. DNA–backbone interactions are the most numerous, providing stability rather than specificity. For base interactions, there are significant base–amino acid type correlations, which can be rationalised by considering the stereochemistry of protein side chains and the base edges exposed in the DNA structure. Nearly two-thirds of the direct read-out of DNA sequences involves complex networks of hydrogen bonds, which enhance specificity. Two-thirds of all protein–DNA interactions comprise van der Waals contacts, compared to about one-sixth each of hydrogen and water-mediated bonds. This highlights the central importance of these contacts for complex formation, which have previously been relegated to a secondary role. Although common, water-mediated bonds are usually non-specific, acting as space-fillers at the protein–DNA interface. In conclusion, the majority of amino acid–base interactions observed follow general principles that apply across all protein–DNA complexes, although there are individual exceptions. Therefore, we distinguish between interactions whose specificities are ‘universal’ and ‘context-dependent’. An interactive Web-based atlas of side chain–base contacts provides access to the collected data, including analyses and visualisation of the three-dimensional geometry of the interactions.     

Applicability of phosphorus budget models to southern African man-made lakes

An investigation of the phosphorus loading characteristics of 31 southern African man-made was lakes made. The lakes were characterized by low water retention times, with most of the lakes having retention times of less than one year. Catchment phosphorus export rates showed wide variation (1–162 mg P m^-2 y^-1) with those lakes experiencing excessive municipal wastewater inputs having export rates in excess of 53 mg m^-2 y^-1. The phosphorus data were tested against the Vollenweider (1976) and Dillon & Rigler (1974) phosphorus budget models which predict in-lake steady state concentrations of phosphorus. It was found that both models displayed good potential for the prediction of steady state concentrations of phosphorus, with better results being obtained from the Dillon & Rigler (1974) model. However, because phosphorus concentrations within these lakes may not necessarily be related to trophic status the use of these models as a predictive tool for eutrophication control still requires further development.     

Saprophytic competence of acid tolerant strains ofRhizobium trifolii in acid soil

Summary Laboratory prescreening ofRhizobium trifolii for acid tolerance, based upon the ability of rhizobia to grow in acid media (pH 4.2) containing Al (15 M), was successful for the selection of strains capable of survival in acid soil.Both sterile and non-sterile soils of varying acidity were inoculated with several strains ofR. trifolii.Acid tolerant strains generally had significantly higher populations at every sample period than an acid sensitive strain. Amelioration of soil acidity by liming improved persistence of all strains. Soil sterilization by autoclaving adversely affected survival of all strains at each soil acidity level.Paper Number 8766 of the Journal Series, North Carolina Agricultural Research Service, Raleigh, NC 27650, USA.