Genetic variation in the horsetail Equisetum variegatum Schleich., an endangered species in the Parisian region

Equisetum variegatum Schleicher is a circumboreale species of horsetail. In France, it typically grows at high elevations but is very rare in lowlands. The genetic variation of these populations is described using isozyme electrophoresis and PCR-RFLP of chloroplast DNA. Sampled sites were chosen to represent central vs. marginal and/or endangered parts of the distribution area. Extensive clonal multiplication of plants together with the absence of local recruitment by sexual reproduction seem to be responsible for the low genetic diversity observed within populations. Since adaptive response to environmental changes ultimately relies on the presence of genetic variability, clonal populations of E. variegatum may be particularly vulnerable to disturbance. Moreover, in lowland populations, isolation gives no chance to recover new genotypes through migration events. The preservation of the two endangered populations is proposed by propagation by cuttings of all extant genetic individuals. In the case of a disappearance of one genotype in the field, a replacement will be possible. This plan may be sufficient to preserve E. variegatum in the French lowland for several years.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host.     

Human defensins as cancer biomarkers and antitumour molecules

Human defensins, which are small cationic peptides produced by neutrophils and epithelial cells, form two genetically distinct alpha and beta subfamilies. They are involved in innate immunity through killing microbial pathogens or neutralizing bacterial toxins and in adaptive immunity by serving as chemoattractants and activators of immune cells. α-defensins are mainly packaged in neutrophil granules (HNP1, HNP2, HNP3) or secreted by intestinal Paneth cells (HD5, HD6), while β-defensins are expressed in mucosa and epithelial cells. Using surface enhanced laser desorption/ionisation time-of-flight (SELDI-TOF) mass spectrometry (MS), α-defensins were found to be expressed in a variety of human tumours, either in tumour cells or at their surface. HNP1–3 peptides are also secreted and their accumulation in biological fluids was proposed as a tumour biomarker. Conversely, β-defensin-1 (HBD-1) is down-regulated in some tumour types in which it could behave as a tumour suppressor protein. Alpha-defensins promote tumour cell growth or, at higher concentration, provoke cell death. These peptides also inhibit angiogenesis, which, in addition to immunomodulation, indicates a complex role in tumour development. This review summarizes current knowledge of defensins to discuss their role in tumour growth, tumour monitoring and cancer treatment.     

Investigation of the antifouling constituents from the brown alga Sargassum muticum (Yendo) Fensholt

One of the most promising alternatives to toxic heavy metal-based paints is offered by the development of antifouling coatings in which the active ingredients are compounds naturally occurring in marine organisms and operating as natural antisettlement agents. Sessile marine macroalgae are remarkably free from settlement by fouling organisms. They produce a wide variety of chemically active metabolites in their surroundings, potentially as an aid to protect themselves against other settling organisms. In this study, a dichloromethane extract from the brown seaweed Sargassum muticum was tested in situ and, after 2 months of immersion, showed less fouling organisms on paints in which the extract was included, compared to paints containing only copper after 2 months of immersion. No barnacles or mussels have been observed on the test rack. Identification by NMR and GC/MS of the effective compound revealed the abundance of palmitic acid, a commonly found fatty acid. Pure palmitic acid showed antibacterial activity at 44 μg mL⁻¹, and also inhibited the growth of the diatom Cylindrotheca closterium at low concentration (EC₅₀ = 45.5 μg mL⁻¹), and the germination of Ulva lactuca spores at 3 μg mL⁻¹. No cytotoxicity was highlighted, which is promising in the aim of the development of an environmentally friendly antifouling paint.     

Structure-Function Perturbation and Dissociation of Tetrameric Urate Oxidase by High Hydrostatic Pressure

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.