The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Whole-Cell versus Total RNA Extraction for Analysis of Microbial Community Structure with 16S rRNA-Targeted Oligonucleotide Probes in Salt Marsh Sediments

rRNA-targeted oligonucleotide probes have become powerful tools for describing microbial communities, but their use in sediments remains difficult. Here we describe a simple technique involving homogenization, detergents, and dispersants that allows the quantitative extraction of cells from formalin-preserved salt marsh sediments. Resulting cell extracts are amenable to membrane blotting and hybridization protocols. Using this procedure, the efficiency of cell extraction was high (95.7% ± 3.7% [mean ± standard deviation]) relative to direct DAPI (4′,6′-diamidino-2-phenylindole) epifluorescence cell counts for a variety of salt marsh sediments. To test the hypothesis that cells were extracted without phylogenetic bias, the relative abundance (depth distribution) of five major divisions of the gram-negative mesophilic sulfate-reducing delta proteobacteria were determined in sediments maintained in a tidal mesocosm system. A suite of six 16S rRNA-targeted oligonucleotide probes were utilized. The apparent structure of sulfate-reducing bacteria communities determined from whole-cell and RNA extracts were consistent with each other (r² = 0.60), indicating that the whole-cell extraction and RNA extraction hybridization approaches for describing sediment microbial communities are equally robust. However, the variability associated with both methods was high and appeared to be a result of the natural heterogeneity of sediment microbial communities and methodological artifacts. The relative distribution of sulfate-reducing bacteria was similar to that observed in natural marsh systems, providing preliminary evidence that the mesocosm systems accurately simulate native marsh systems.     

Distribution and palaeoecology of freshwater algae and stromatolites: III, some new forms from the Carboniferous, Permian and Triassic of France and Spain

New collections allow us to describe some new algae forming: a) centimetric, unorganized, masses (Toutinella bifurcata from the Middle Triassic of Provence); b) large, decimetric masses (Zarramanella menorica from the ‘Buntsandstein’ of Minorca, Balearic Islands) and c) interesting, plurispecific biocenosis, laminated and silicified, from the French Massif Central: Lower Stephanian of Saint-Étienne basin (with Hankerpella minima, Baltzerella stephaniense, Ponsinella undulata) and Lower Autunian of Bourbon-l'Archambault basin (Hanskerpella pustulata, Baltzerella stephaniense, Ponsinella multifrons, Sarfatigirella intricata). Other forms are badly preserved, one is possibly a Broutinella sp. and another may be a coccoid form of cyanobacteria or chlorophyceae. Silicified biocenoses macroscopically occur in the form of regular laminations, exhibiting vertical, planar joints (1–2 cm wide) and horizontal, planar joints, 5 mm to 3 cm thick, with various sedimentary infillings. These facies represent early and late silicifications of biological felts (mats), probably previously partly calcified and affected by desiccation cracks partly infilled before the silicification. There is no evidence for an early silicification of living filaments and trichomes. The silicification also affects other types of sediments (petites, sandstones, conglomerates, breccias) associated with the algal formations. Macroscopically, these laminated formations do not show domes or hemispheres; on the other hand, these features clearly appear under the microscope, and these formations are true stromatolites. The basins of Saint-Étienne and Bourbon-l'Archambault contain volcanic rocks; the siliceous series, very localized in the basins, represent an impregnation of various primary materials by silica of hydrothermal origin. The present study is voluntarily limited to the palaeontological and palaeoecological appearances; algal laminations formed in ephemeral pools and small lakes, under very particular conditions, ‘hydrothermal’ sensu lato, are still to be stated more precisely.

Résumé

Des récoltes nouvelles permettent de décrire quelques algues nouvelles formant des amas centimétriques non organisés (Toutinella bifurcata du Trias moyen de Provence), de grosses masses décimétriques (Zarmmanella menorica du Buntsandstein de Minorque) et d'intéressantes biocrenoses plurispécifiques laminées et silicifiées du Massif central français: Stephanien inférieur de Saint-Étienne (à Hankerpella minima, Baltzerella stephaniense, Ponsinella undulata), Autunien inférieur du bassin de Bourbon-l'Archambault (Hanskerpella pustulata, Baltzerella stephaniense, Ponxinella multifrons, Sarfatigirella intricata). D'autres formes sont mal conservées, l'une serait attribuables à une ? Broutinella sp. et une autre à une coccoide (cyanobactéries ou chlorophycées). Les biocœnoses silicificées se présentent macroscopiquement sous forme de laminations régulières, accidentées de fentes planes verticales (1–2 cm de largeur) et de fentes planes horizontales (5 mm–3 cm de hauteur), à remplissages sédimentaires variés. Ces faciès correspondent à des silicifications précoces et tardives de feutres biologiques probablement en grande partie calcifiés précocement et affectés de fentes de dessiccation partiellement remplies avant la silicification. Aucun indice de silicification de filaments et de trichomes du vivant des organismes n'a pu être observé. La silicification affecte aussi d'autres sédiments (pélites, grès, conglomérats, brèches), associés aux anciennes formations algaires. Macrospiquement, ces formations laminées ne montrent pas de dômes ni d'hémisphères; en revanche ces structures apparaissent parfaitement au microscope, et ce sont de véritables stromatolites. Les bassins de Saint-Étienne el de Bourbon-l'Archambault contiennent des formations volcaniques; les formations siliceuses, qui sont très localisées dans les bassins, correspondent certainement à une imprégnation par de la silice d'origine hydrothermale de sédiments variés. La présente étude se limite volontairement à l'aspect paléontologique et paléoécologique; les laminations algaires se sont formées dans des bassins ou de petits lacs éphémères, dans des conditions très particulières, hydrothermales sensu lato, encore à préciser.     

Analysis of the CC chemokine receptor 5 (CCR5) delta-32 polymorphism in inflammatory bowel disease

The inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) are complex multifactorial traits involving both environmental and genetic factors. Recent studies have shown the important role of pro-inflammatory cytokines and chemokines, including RANTES, in IBD. RANTES is the natural ligand for the CC-chemokine receptor 5 (CCR5). The chromosomal location of the CCR5 gene on 3p21 coincides with an IBD-susceptibility locus identified by genome-wide scanning. A 32-bp deletion (A32) in the CCR5 gene results in a nonfunctional receptor and is found with high frequency in Caucasians. In this study, we investigated the presence of the CCR5delta32 allele in a large cohort of IBD patients and in a healthy control population. Blood samples were obtained from 538 unselected IBD cases (433 unrelated IBD patients: 289 CD, 142 UC, 2 indeterminate colitis; 105 affected first-degree relatives) and 135 unaffected first-degree family members. Of the IBD patients, 36% had familial IBD with at least two members being affected. There were no significant differences in the CCR5delta32 mutation frequency between IBD patients and healthy controls, nor between CD and UC patients. There was no correlation between the CCR5delta32 genotype and the age at IBD-diagnosis, the frequency of surgical intervention, or disease localization. Only the association between CCR5delta32 homozygosity and the presence of anal lesions in CD patients was statistically significant (P=0.007). Analysis by the transmission/disequilibrium test showed no significant transmission distortion to the probands or their clinically silent siblings. Based on these results, it is unlikely that the CCR5delta32 allele is an important marker for predisposition to IBD.     

A Sensitive and Inexpensive Yeast Bioassay for the Mycotoxin Zearalenone and Other Compounds with Estrogenic Activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 μg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

Organotypic heart slices for cell transplantation and physiological studies

Recent studies have significantly improved our ability to investigate cell transplantation and study the physiology of transplanted cells in cardiac tissue. Several previous studies have shown that fully-immersed heart slices can be used for electrophysiological investigations. Additionally, ischemic heart slices induced by glucose and oxygen deprivation offer a useful tool to investigate mechanical integration and to measure forces of contraction of engrafted cells, at least for short term analysis. A recent and novel model of heart slices, prepared from rat and human tissues, can be maintained in culture for up to two months. This new heart slice model can be used for long term in vitro cell transplantation studies and for pharmacological evaluation. This review will focus on describing these models and demonstrating the use of organotypic heart slices as a novel tool for drugs for studying electrophysiology and developing cellular therapeutic approaches to alleviate cardiac tissue damage.Key words: heart, organotypic, culture, stem cells, transplantation, electrophysiology, pharmacology     

Influence of pH on the human prion protein: insights into the early steps of misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. Conversion from the normal cellular form (PrP^C) or recombinant PrP (recPrP) to a misfolded form is pH-sensitive, in that misfolding and aggregation occur more readily at lower pH. To gain more insight into the influence of pH on the dynamics of PrP and its potential to misfold, we performed extensive molecular-dynamics simulations of the recombinant PrP protein (residues 90-230) in water at three different pH regimes: neutral (or cytoplasmic) pH (∼7.4), middle (or endosomal) pH (∼5), and low pH (<4). We present five different simulations of 50 ns each for each pH regime, amounting to a total of 750 ns of simulation time. A detailed analysis and comparison with experiment validate the simulations and lead to new insights into the mechanism of pH-induced misfolding. The mobility of the globular domain increases with decreasing pH, through displacement of the first helix and instability of the hydrophobic core. At middle pH, conversion to a misfolded (PrP^Sc-like) conformation is observed. The observed changes in conformation and stability are consistent with experimental data and thus provide a molecular basis for the initial steps in the misfolding process.     

The import competence of a peroxisomal membrane protein is determined by Pex19p before the docking step

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.     

Evidence for Megalake Chad, north-central Africa, during the late Quaternary from satellite data

The existence of a very large Lake Chad during the late Quaternary, Megalake Chad, has long been questioned. A Megalake Chad would present strong evidence for climatic fluctuations of great magnitude during the Holocene in tropical Africa. In this paper we used satellite data from Landsat and Modis sensors to collect and analyse new information on landforms in a 2 000 000 km² region of the Lake Chad Basin. We detected 2300 km of remains marking the ancient shoreline of Megalake Chad. The satellite data also indicated many Saharan rivers and relict deltas leading to the long paleoshoreline. Large dunefield flattenings were observed and interpreted as the result of wave-cut erosion by the paleolake. Similarities were noticed between the landforms observed along the paleoshoreline of Megalake Chad and that of the former Aral Sea. This finding has significant consequences for reconstructing paleohydrology and paleoenvironments through the Lake Chad basin, and continental climate change.