Hydrocarbon-metabolizing activity of various mammalian cells in culture

Summary Two methods for determining the hydrocarbon-metabolizing enzyme activity of cultured mammalian cells were compared. The method designed to measure benzo[a]an-thracene-induced aryl hydrocarbon hydroxylase activity could detect and quantify enzyme activities in low passage rodent cells, but could not reproducibly detect levels in intermediate or high passage mouse, rat, or human cells. The method designed to measure the ability of a cell to convert benzo[a]pyrene from an organic-soluble to an aqueous acetone-soluble form proved to be more reproducible. This technique, when modified, was demonstrated to be an effective screening test for the detection of those lines with higher levels of hydrocarbon-metabolizing enzymes. Supported by the Council for Tobacco Research and Contract NIH 70-2068 within the Virus Cancer Program, National Cancer Institute, National Institutes of Health.     

Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

The Growth Response of the Stems of Genetically Modified Tobacco Plants (Nicotiana tabacum'Samsun') to Flexural Stimulation

Genetically modified tobacco plants (Nicotiana tabacum‘Samsun’)with antisense cinnamyl alcohol dehydrogenase DNA, produce secondaryxylem of a reduced tensile stiffness. These plants were grownalongside control plants. The stems of the plants were flexedor protected from flexing over a period of several weeks. Thetensile moduli and second moments of areas of the differenttissues inside the stems were measured and used to calculatethe bending stiffness of the plants. In tobacco, the cylinderof xylem was found to be the most important tissue in determiningthe bending stiffness of the plants. The thickness of the xylemtissue cylinder increased when plants were subjected to flexuralstimulation. This increased the bending stiffness of the stems.The response to mechanical stimulation was found to be correlatedwith tissue strain and the genetically modified plants wereable to exactly compensate for the reduced modulus of theirxylem tissue by increasing the thickness of the xylem tissuecylinder more than in control plants.Copyright 1999 Annals ofBotany Company. Tobacco plants, stem bending, xylem tissue, second moment of area, thigmomorphogenesis, mechanical strain.     

Sexual receptivity and post-emergence ovarian development in females of Coproica vagans (Diptera: Sphaeroceridae)

Abstract .Males and females of the dung fly species Coproica vagans Haliday 1833 (Diptera: Sphaeroceridae) mate soon after emergence from the puparium. At this time females still have immature ovaries. Therefore, mating precedes vitellogenesis in this species. Data presented here show that mating enhances oogenesis in C. vagans females. Mated females mature their first egg batch sooner and oviposit four days earlier than virgin conspecifics. Mating-related enhancement of oogenesis could be explained either through nutritional benefits to females or male chemical or stimulatory manipulation of the females. Oogenesis was divided into six arbitrary stages, with vitellogenesis beginning in stage 4. Ovarian development beyond stage 4 is rapid compared with pre-vitellogenetic development. Virgin females pause oogenesis in stage 4. The genital opening of mated females is blocked by a mating plug that persists until oviposition begins. The plug seems to ensure the paternity of the last male to mate by preventing females from remating. The operational sex ratio in C. vagans populations is presumed to be strongly male-biased.