Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta

Phospholipid scramblase induces nonspecific bidirectional movement of phospholipids across the membrane during cell activation and has been proposed to mediate the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet during apoptosis, a cell surface change that is critical for apoptotic cell removal. We report here that protein kinase C (PKC) delta plays an important role in activated transbilayer movement of phospholipids and surface PS exposure by directly enhancing the activity of phospholipid scramblase. Specific inhibition of PKCdelta by rottlerin prevented both apoptosis- and activation-induced scramblase activity. PKCdelta was either selectively cleaved and activated in a caspase 3-dependent manner (during apoptosis) or translocated to the plasma membrane (in stimulated cells) and could directly phosphorylate scramblase immunoprecipitated from Jurkat cells. Furthermore, reconstitution of PKCdelta and scramblase, but not scramblase or PKCdelta alone in Chinese hamster ovary cells demonstrated enhanced scramblase activity.     

Cpr6 and Cpr7, two closely related Hsp90-associated immunophilins from Saccharomyces cerevisiae, differ in their functional properties

Hsp90 is an abundant cytosolic molecular chaperone. It controls the folding of target proteins including steroid hormone receptors and kinases in complex with several partner proteins. Prominent members of this protein family are large peptidyl prolyl cis/trans isomerases (PPIases), which catalyze the cis/trans isomerization of prolyl peptide bonds in proteins and possess chaperone activity. In Saccharomyces cerevisiae, two closely related large Hsp90-associated PPIases, Cpr6 and Cpr7, exist. We show here that these homologous proteins bind with comparable affinity to Hsp90 but exhibit significant structural and functional differences. Cpr6 is more stable than Cpr7 against thermal denaturation and displays an up to 100-fold higher PPIase activity. In contrast, the chaperone activity of Cpr6 is much lower than that of Cpr7. Based on these results we suggest that the two immunophilins perform overlapping but not identical tasks in the Hsp90 chaperone cycle.     

Refolding, purification, and characterization of human recombinant PDE4A constructs expressed in Escherichia coli

A 5'-truncated PDE4A-cDNA corresponding to the amino acid positions 200-886 of the "full-length" sequence (Accession No. L20965) was generated from human leukocyte mRNA by RT-PCR. Several PDE4A constructs containing the catalytic region and differing in their degree of N- and/or C-terminal truncation (amino acid positions 200-886, 200-704, 342-886, and 342-704) were expressed in Escherichia coli to investigate the effect of truncations on purification characteristics, long-term stability, and aggregation. All peptides accumulated as inclusion bodies, necessitating refolding prior to purification by dye and metal chelate affinity chromatography. The constructs differed in long-term stability due to variable levels of protease contamination. The position of the His-tag also influenced the purification results. The best results were obtained with the N- and C-truncated form C-terminally His-tagged, appropriate quantities of which were obtained in pure form and was found to be stable against proteolysis at 4 degrees C for at least 6 weeks. The comparison of the molecular mass of the investigated PDE4A constructs obtained by SDS electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation indicated that C-terminal truncated PDE4A forms dimers whereas PDE4A constructs with a complete C-terminus tend to form larger aggregates.     

Tracing the D-pathway in reconstituted site-directed mutants of cytochrome c oxidase from Paracoccus denitrificans

Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.     

A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis

We developed a modified allele-specific PCR procedure for assaying single nucleotide polymorphisms (SNPs) and used the procedure (called SNAP for single-nucleotide amplified polymorphisms) to generate 62 Arabidopsis mapping markers. SNAP primers contain a single base pair mismatch within three nucleotides from the 3' end of one allele (the specific allele) and in addition have a 3' mismatch with the nonspecific allele. A computer program called SNAPER was used to facilitate the design of primers that generate at least a 1,000-fold difference in the quantity of the amplification products from the specific and nonspecific SNP alleles. Because SNAP markers can be readily assayed by electrophoresis on standard agarose gels and because a public database of over 25,000 SNPs is available between the Arabidopsis Columbia and Landsberg erecta ecotypes, the SNAP method greatly facilitates the map-based cloning of Arabidopsis genes defined by a mutant phenotype.     

Membrane association of presynaptic cytomatrix protein bassoon

Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.     

Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species.     

Effect of Prior Exposure to Noninfected Ticks on Susceptibility of Mice to Lyme Disease Spirochetes

To determine whether prior exposure to Nearctic Ixodes vector ticks protects native reservoir mice from tick-borne infection by Lyme disease spirochetes, we compared their infectivities for white-footed mice and laboratory mice that had been repeatedly infested by noninfected deer ticks. Nymphal ticks readily engorged on tick-exposed laboratory mice, but their feeding success on white-footed mice progressively declined. Tick-borne spirochetes readily infected previously tick-infested mice. Thus, prior infestation by Nearctic ticks does not protect sympatric reservoir mice or Palearctic laboratory mice from infection by sympatric tick-borne spirochetes.     

Characterization of the properties of differentially modified titanium surfaces for dental implantology. 2: In vitro biocompatibility studies]

The aim of the present study was to determine whether specific surface modifications are capable of improving the biocompatibility of a titanium surface, and whether there is a correlation between the physico-chemical properties of the implant material and its biocompatibility. To this end, the properties of titanium surfaces were modified using various methods or the latter were coated with various materials. Plasma treatments under different atmospheric conditions (N2-plasma, SO2-plasma, acetylene plasma) as well as plasma polymerization were used to affect the biological response. Characterization of the physico-chemical surface properties by means of X-ray photoelectron spectroscopy (XPS), contact angle measurements and the calculation of surface tensions or surface energy provided important information on the interactions at the interface between the implant material and the aqueous environment. The influence of the respective surface modification on cell proliferation, cell viability and the activity of mitochondrial dehydrogenases was evaluated in specific in vitro tests with human gingiva fibroblasts. It was show that different modifications of the titanium samples induce different biological responses of the gingiva fibroblasts. The results confirm the existence of correlations between thermodynamic surface properties and cellular reactions under in vitro conditions.     

Bassoon,a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 M _r, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.