Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Mechanisms of Sperm Oxidation: Breaking Down the Origins of Oxidative Activity

Oxidative stress has been long considered an important cause of male infertility, and sperm‐derived reactive oxygen species (ROS) have been considered one of the major sources. In article number 1900205, Issue 2, Netherton et al. have improved upon current technologies for detection of true ROS‐derived oxidative events from those originated by mitochondrial compounds. Moreover, the authors have isolated proteins that bear lipid aldehyde adducts, in order to verify their origin. In their paper, Netherton et al. demonstrate that sperm‐derived ROS do not contribute to sperm oxidative stress, which is an important finding, given that most studies consider altered sperm a major source of seminal ROS. Moreover, adducted proteins are mostly of prostatic origin, which leads to questions regarding how and where the oxidative events occur. Under the light of this study and the methodological improvements it has brought, it may be important to revisit some foundation studies in semen and sperm oxidative stress.     

Laboratory-scale evidence for lightning-mediated gene transfer in soil

Electrical fields and current can permeabilize bacterial membranes, allowing for the penetration of naked DNA. Given that the environment is subjected to regular thunderstorms and lightning discharges that induce enormous electrical perturbations, the possibility of natural electrotransformation of bacteria was investigated. We demonstrated with soil microcosm experiments that the transformation of added bacteria could be increased locally via lightning-mediated current injection. The incorporation of three genes coding for antibiotic resistance (plasmid pBR328) into the Escherichia coli strain DH10B recipient previously added to soil was observed only after the soil had been subjected to laboratory-scale lightning. Laboratory-scale lightning had an electrical field gradient (700 versus 600 kV m(-1)) and current density (2.5 versus 12.6 kA m(-2)) similar to those of full-scale lightning. Controls handled identically except for not being subjected to lightning produced no detectable antibiotic-resistant clones. In addition, simulated storm cloud electrical fields (in the absence of current) did not produce detectable clones (transformation detection limit, 10(-9)). Natural electrotransformation might be a mechanism involved in bacterial evolution.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Plant genome complexity may be a factor limiting in situ the transfer of transgenic plant genes to the phytopathogen Ralstonia solanacearum

The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 μg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 μg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 μg/mL OPN (bull 2 = 85 ± 4% and bull 5 = 78 ± 4%) than without OPN (bull 2 = 75 ± 4% and bull 5 = 69 ± 4%). Those bulls also had increase in cleavage and embryo development (bull 2 = 85 ± 3%, 41 ± 1.9%; bull 5 = 76 ± 2%, 37 ± 1.8%) compared with control (bull 2 = 75 ± 3%, 30 ± 2%; bull 5 = 68 ± 2%, 29 ± 2%). Incubating matured oocytes in 10 μg/mL OPN (87 ± 3%) and 100 μg/mL OPN (88 ± 3%) significantly increased fertilization than control (73 ± 3%). OPN also improve cleavage, and embryo development in treatments with 10 μg/mL OPN (82.7 ± 1.3%; 31.7 ± 1.4%) and 100 μg/mL OPN (85.8 ± 1.3%; 33.8 ± 1.5%) when compared with control (74.1 ± 1.3%; 24.2 ± 1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.     

Conditions for natural transformation of Ralstonia solanacearum.

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.     

Impact of the Microscale Distribution of a Pseudomonas Strain Introduced into Soil on Potential Contacts with Indigenous Bacteria

Soil bioaugmentation is a promising approach in soil bioremediation and agriculture. Nevertheless, our knowledge of the fate and activity of introduced bacteria in soil and thus of their impact on the soil environment is still limited. The microscale spatial distribution of introduced bacteria has rarely been studied, although it determines the encounter probability between introduced cells and any components of the soil ecosystem and thus plays a role in the ecology of introduced bacteria. For example, conjugal gene transfer from introduced bacteria to indigenous bacteria requires cell-to-cell contact, the probability of which depends on their spatial distribution. To quantitatively characterize the microscale distribution of an introduced bacterial population and its dynamics, a gfp-tagged derivative of Pseudomonas putida KT2440 was introduced by percolation in repacked soil columns. Initially, the introduced population was less widely spread at the microscale level than two model indigenous functional communities: the 2,4-dichlorophenoxyacetic acid degraders and the nitrifiers (each at 10⁶ CFU g⁻¹ soil). When the soil was percolated with a substrate metabolizable by P. putida or incubated for 1 month, the microscale distribution of introduced bacteria was modified towards a more widely dispersed distribution. The quantitative data indicate that the microscale spatial distribution of an introduced strain may strongly limit its contacts with the members of an indigenous bacterial community. This could constitute an explanation to the low number of indigenous transconjugants found most of time when a plasmid-donor strain is introduced into soil.