A low molecular weight substance purified from human placenta inhibits cAMP-dependent protein kinase and activates protein kinase C

We have purified from human placenta a low molecular mass substance that inhibits cAMP-dependent protein kinase and activates protein kinase C. This protein kinase regulator was purified in three steps: (1) homogenizing placentas in chloroform/methanol and extracting the regulator into water; (2) eluting a strong anion exchange high performance liquid chromatography (HPLC) column with a quaternary gradient; and (3) eluting a reversed-phase HPLC column with a binary gradient. The regulator was found to be highly purified by HPLC, thin-layer chromatography (TLC) and laser desorption ionization mass spectrometry with a molecular mass of 703 Daltons by the latter procedure. The physical and biochemical properties of this protein kinase regulator suggest that it is a phospholipid but it did not co-elute by HPLC or by TLC with any of the known phospholipid activators of protein kinase C.     

Decreased phosphorylation of a low molecular weight protein by cGMP-dependent protein kinase in variant HL-60 cells resistant to nitric oxide- and cGMP-induced differentiation

We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs [7]. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by ³⁵S-methionine/³⁵S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

Strategies for Mixed-Grass Prairie Restoration: Herbicide, Tilling, and Nitrogen Manipulation

Large areas of North American prairie have been planted with grasses introduced from Eurasia. We examined three strategies (herbicide, tilling, and nitrogen manipulation) for enhancing the establishment of seedlings of native species and suppressing the introduced grasses Agropyron cristatum (crested wheat grass) and Bromus inermis (smooth brome). Plots (5 × 15 m) were subjected to one of three levels of tilling (none, intermediate, complete) and four levels of nitrogen (none, intermediate, high, and sawdust added to immobilize nitrogen). Treatments were applied in a factorial design with twelve treatments and ten replicates. Seeds of 41 native species were drilled into the plots in May 1992. Following the failure of seeds to establish in 1992, a subplot (5 × 13 m) within each main plot was sprayed with the herbicide glyphosate in April 1993. The nitrogen treatments were repeated in Spring 1993. In August 1993, the density of native seedlings in sprayed subplots was 20 times that in unsprayed subplots. Within sprayed subplots, native seedling density and the cover of bare ground decreased significantly with increasing nitrogen availability. Plots receiving sawdust had significantly higher mean cover of bare ground and significantly lower concentrations of soil available nitrogen. Native seedling density was significantly higher in plots receiving the highest intensity of tilling. The responses of native seedlings to all these factors point to the importance of neighbor-free establishment sites as a prerequisite for prairie restoration.     

A generic schema and data collection forms applicable to diverse entomological studies of mosquitoes

Background

Standardized schemas, databases, and public data repositories are needed for the studies of malaria vectors that encompass a remarkably diverse array of designs and rapidly generate large data volumes, often in resource-limited tropical settings lacking specialized software or informatics support.

Results

Data from the majority of mosquito studies conformed to a generic schema, with data collection forms recording the experimental design, sorting of collections, details of sample pooling or subdivision, and additional observations. Generically applicable forms with standardized attribute definitions enabled rigorous, consistent data and sample management with generic software and minimal expertise. Forms use now includes 20 experiments, 8 projects, and 15 users at 3 research and control institutes in 3 African countries, resulting in 11 peer-reviewed publications.

Conclusion

We have designed generic data schema that can be used to develop paper or electronic based data collection forms depending on the availability of resources. We have developed paper-based data collection forms that can be used to collect data from majority of entomological studies across multiple study areas using standardized data formats. Data recorded on these forms with standardized formats can be entered and linked with any relational database software. These informatics tools are recommended because they ensure that medical entomologists save time, improve data quality, and data collected and shared across multiple studies is in standardized formats hence increasing research outputs.