Enzymatic activity of dystrophic chicken sarcoplasmic reticulum

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.     

Transforming growth factor beta 1 inhibits epidermal growth factor receptor endocytosis and down-regulation in cultured fetal rat hepatocytes.

Incubation of fetal rat hepatocytes (FRH) with transforming growth factor beta 1 (TGF-beta 1) resulted in growth arrest and a biphasic effect on epidermal growth factor (EGF) receptor. After 2 h of exposure, EGF receptor (EGFR) was reduced by 43%. From 6 to 24 h, TGF-beta 1 exposure resulted in progressive increase in EGFR up to 74% over control. The increased binding was due to increase in high affinity EGF binding sites. FRH grown in medium containing EGF exhibited down-regulated EGFR with loss of high affinity EGF binding sites. With TGF-beta 1 exposure, high affinity EGFR was not down-regulated by EGF. Since down-regulation of EGFR involves internalization, the kinetics of EGF receptor-mediated endocytosis were examined. In TGF-beta 1-exposed FRH, EGF endocytosis was inhibited, with a reduction in the first order rate constant for the process from 0.078 to 0.043 min-1. Despite inhibition of growth, receptor down-regulation, and EGF endocytosis after TGF-beta 1 exposure, EGF-induced receptor autophosphorylation was preserved as demonstrated by [32P]phosphate-labeling of immunoprecipitated EGFR. These observations provide direct evidence that TGF-beta 1 regulates growth of fetal cells. Further, they suggest that TGF-beta 1 regulates endocytosis of EGF and possibly of other ligands.     

The inhibition of cytochrome oxidase by diaminomaleonitrile

Diaminomaleonitrile, a tetramer of cyanide, was examined as a possible antagonist to cyanide inhibition of cytochrome oxidase (EC 1.9.3.1). This compound was found to inhibit cytochrome oxidase in vitro; however, despite their structural similarities, diaminomaleonitrile and cyanide inhibit cytochrome oxidase by different mechanisms and bind to the enzyme at different sites. Diaminomaleonitrile inhibition of cytochrome oxidase is described in terms of a partially competitive mechanism. Biological oxidation of diaminomaleonitrile may lead to the formation of cyanide.     

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.     

Sarcomere length determination using laser diffraction. Effect of beam and fiber diameter

An experimental and theoretical analysis is presented involving the effect of variation in fiber and beam diameter upon the determination of average sarcomere length in isolated single muscle fibers using laser light diffraction. The muscle diffraction phenomenon is simplified by first considering diffraction order position and intensity to be the result of grating and Bragg diffraction. It is the product of the intensity profiles, which results from these types of diffraction, that produces the diffracted order. These simplifying assumptions are then extended to the case of the real muscle. Based on these considerations and the theory that we recently presented, conditions are set forth under which grating information (i.e., sarcomere length) can be maximally expressed to yield accurate average sarcomere length values.     

Antigen responsiveness of an established human B-lymphoid cell line I. Evidence for a surface receptor for Candida antigen detected by direct migration inhibition

Responsiveness to a soluble crude Candida albicans antigen (CaAg) by a long-term human lymphoid cell line with B-cell characteristics (PGLC-33H) is demonstrated by the direct migration inhibition (DMI) assay. An inability to detect comparable CaAg responses in other tested B- and T-lymphoid cell lines suggests that this may be a specific response for PGLC-33H cells. CaAg responsiveness is completely abrogated by trypsinization of cells. Regeneration of CaAg responsiveness is temperature dependent, detectable in 8–14 hr in cells maintained at 37°C. Trypsinized cells kept at 4°C do not regain CaAg response. The rate of cell-CaAg interaction is rapid and not temperature dependent. Cells pulsed with CaAg at 37, 25, and 4°C demonstrate comparable DMI responses following a 2-min pulse with CaAg. These findings suggest that PGLC-33H cells have surface receptors for CaAg which result in the inhibition of cell migration upon exposure to antigen.     

Seed dormancy of Cardiospermum halicacabum (Sapindaceae) from three precipitation zones in Sri Lanka

• This study investigated seed germination of Cardiospermum halicacabum, a medicinally important invasive species.
• We compared mass, moisture content (MC), dormancy and dormancy‐breaking treatments and imbibition and germination of scarified and non‐scarified seeds of C. halicacabum from a low‐elevation dry zone (DZ), low‐elevation wet zone (WZ1) and mid‐elevation wet zone (WZ2) in Sri Lanka to test the hypothesis that the percentage of seeds with water‐impermeable seed coats (physical dormancy, PY) decreases with increased precipitation.
• Seed mass was higher in WZ2 than in DZ and WZ1, while seed MC did not vary among the zones. All scarified DZ, WZ1 and WZ2 and non‐scarified DZ and WZ1 seeds imbibed water, but only a few non‐scarified WZ2 seeds did so. When DZ and WZ1 seeds were desiccated, MC and percentage imbibition decreased, showing that these seeds have the ability to develop PY. GA₃ promoted germination of embryos excised from fresh DZ and WZ1 seeds and of scarified WZ2 seeds.
• At maturity, seeds from DZ and WZ1 had only physiological dormancy (PD), while those from WZ2 had combinational dormancy (PY+PD). Thus, our hypothesis was not supported. Since a high percentage of excised embryos developed into normal seedlings; this is a low‐cost method to produce C. halicacabum plants for medicinal and ornamental purposes.