Chloroplast protein import : quantitative analysis of precursor binding

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0°C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.     

The murine p53 protein blocks replication of SV40 DNA in vitro by inhibiting the initiation functions of SV40 large T antigen

We have characterized the effect of murine p53 on SV40 DNA replication in vitro. Purified wild-type murine p53 dramatically inhibited the ability of SV40 T antigen to mediate the replication of a plasmid bearing the viral origin (ori-DNA) in vitro. In contrast, polyoma ori-DNA replication in vitro was unaffected by p53. Surprisingly, both unbound p53 and SV40 T antigen-bound p53 were equally detrimental to SV40 ori-DNA replication. Thus, p53 interferes with interactions between T antigen molecules that are required for DNA synthesis. p53 inhibited the binding to and subsequent unwinding of the SV40 origin by T antigen and thus selectively blocked the initial stages of ori-DNA replication. In contrast to the nononcogenic wild-type murine p53, high concentrations of a mutant transforming p53 failed to block SV40 ori-DNA replication in vitro. These observations may provide insight into a possible role for p53 in the cell.     

Actinomycetes inducing phytotoxic or fungistatic activity in a Douglas-fir Forest and in an adjacent area of repeated regeneration failure in Southwestern Oregon

Actinomycetes were isolated from the upper 1 - 3 cm of the soil layer in a well-developed forest and in an adjacent clearcut area where Douglas-fir [Pseudotsuga menziesii (MIRB.) Franco] regeneration had been impaired for two decades. The population density in the clearcut area was two times as high as that in the forested area. The percentage of actinomycetes that inhibited seed germination of the test plants was significantly higher in isolates obtained from the clearcut area than in those obtained from the forested area, and isolates from the clearcut showed five times the phytotoxic effect of those from the forest. Some actinomycete isolates, 4 % from the clearcut and 2.6 % from the forest, significantly reduced in vitro growth of two common ectomycorrhizal fungi of Douglas-fir,Laccaria laccata andHebeloma ovstuliniforme. Two actinomycete isolates from the clearcut reduced fungal growth by 40 % and 73 %. Reduction of the nutrient in the growth medium did not affect the antifungal activity of the actinomycetes. The data support the idea that, along with other factors, phytotoxic and antifungal actinomycetes may suppress natural regeneration or establishment of planted seedlings - either directly or. indirectly - through inhibition of seed germination or of mycorrhizal fungi.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Benthic algal biomass and productivity in high subarctic streams,Alaska

Year-round measurements of the standing crop of epilithic algae (as chlorophyll a concentration) in two streams — one second and one fourth order (map scale 1:63 360) — in interior Alaska (64°–65° N) were only about one tenth that reported from streams of temperate North America. Cell densities in these streams, however, were similar to those in comparable temperate streams. Year-round domination of the benthic flora by very tiny diatoms (Achnanthes spp.) may explain the apparent disparity between low chlorophyll a content and nearly average cell densities. Chlorophyll a standing crop in a more alkaline groundwater-fed stream, however, was higher and within the range of similarly sized temperate streams. Maximum chlorophyll a standing crop varied positively with alkalinity in 5 clear-water streams where standing crop was measured on natural or artificial substrates. Seasonal mean concentrations of sestonic chlorophyll a (used as estimates of benthic algal chlorophyll a standing crop) varied directly and significantly with alkalinity among ten clear-water streams; and, with total phosphorus among 8 of 10 clear-water and 5 brown-water streams studied. During the summer, when there is little darkness, gross primary productivity (as estimated by the diurnal dissolved-oxygen method) was similar to that of northern temperate streams. Gross primary productivity was also seen to vary directly with alkalinity in 5 clear-water streams of this region.U.S. Fish and Wildlife Service     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

The frequency of the ΔF508 mutation on cystic fibrosis chromosomes in Israeli families: correlation to CF haplotypes in Jewish communities and Arabs

Summary We have analysed the distribution of the ΔF508 mutation and the haplotypes of cystic fibrosis (CF) bearing chromosomes among the Israeli CF population. The population was classified according to its ethnic origin and included 3 groups, Ashkenazi Jews, Sephardic/Oriental Jews and Arabs. Haplotype B (KM19 allele 2, XV2c allele 1) was found to be the predominant haplotype in all groups but in each of them the haplotype distribution was different. The ΔF508 mutation was present in all groups and accounts for 32% of the CF mutations. It was mainly associated with the B haplotype but only one third of the CF chromosomes with this haplotype carry the ΔF508 mutation. This work is dedicated to Dr. Ruth Voss who initiated the CF study in Israel and was tragically killed in a car accident on 7 August 1988     

A catalogue of Clostridium thermocellum endoglucanase, β-glucosidase and xylanase genes cloned in Escherichia coli

Abstract Two independent collections of clones containing Clostridium thermocellum genes involved in cellulose have been previously obtained at IAPGR, Cambridge, and at the Pasteur Institute, Paris. The two collections were compared for cross-hybridization, restriction maps and enzyme phenotypes. Truly distinct genes were one β-glucosidase gene, two xylanase genes, and fifteen endogluconase genes. Two of the cloned fragments contained extraneous DNA which was absent from their respective counterparts isolated in the other collection. The dicrepancies resulted from in vivo rearrangements which had occurred in either of the C. thermocellum NCIB 10682 stocks used to generate the two gene banks.