Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.     

Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of alpha-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B(2)-bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein- and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.     

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.     

Arsenite oxidase,an ancient bioenergetic enzyme

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.     

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.     

Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.     

13C and 1H Nuclear Magnetic Resonance Study of Glycogen Futile Cycling in Strains of the Genus Fibrobacter

We investigated the carbon metabolism of three strains of Fibrobacter succinogenes and one strain of Fibrobacter intestinalis. The four strains produced the same amounts of the metabolites succinate, acetate, and formate in approximately the same ratio (3.7/1/0.3). The four strains similarly stored glycogen during all growth phases, and the glycogen-to-protein ratio was close to 0.6 during the exponential growth phase. ¹³C nuclear magnetic resonance (NMR) analysis of [1-¹³C]glucose utilization by resting cells of the four strains revealed a reversal of glycolysis at the triose phosphate level and the same metabolic pathways. Glycogen futile cycling was demonstrated by ¹³C NMR by following the simultaneous metabolism of labeled [¹³C]glycogen and exogenous unlabeled glucose. The isotopic dilutions of the CH₂ of succinate and the CH₃ of acetate when the resting cells were metabolizing [1-¹³C]glucose and unlabeled glycogen were precisely quantified by using ¹³C-filtered spin-echo difference ¹H NMR spectroscopy. The measured isotopic dilutions were not the same for succinate and acetate; in the case of succinate, the dilutions reflected only the contribution of glycogen futile cycling, while in the case of acetate, another mechanism was also involved. Results obtained in complementary experiments are consistent with reversal of the succinate synthesis pathway. Our results indicated that for all of the strains, from 12 to 16% of the glucose entering the metabolic pathway originated from prestored glycogen. Although genetically diverse, the four Fibrobacter strains studied had very similar carbon metabolism characteristics.     

The Capsule and S-Layer: Two Independent and Yet Compatible Macromolecular Structures in Bacillus anthracis

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-γ-d-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.

Bacillus anthracis, a gram-positive spore-forming bacterium, is the causative agent of anthrax. This disease, to which many animals, including humans, are susceptible, involves toxemia and septicemia. In the mammalian host, B. anthracis bacilli synthesize two toxins (lethal and edema toxins) (31) and a capsule (18) encoded by two large plasmids, pXO1 and pXO2, respectively (12, 21). The capsule is composed of poly-γ-d-glutamic acid and has antiphagocytic properties (13, 31, 37). Although unusual, a similar capsule is also found on Bacillus licheniformis bacilli (9). In the absence of pXO2 or the inducer bicarbonate, the cell does not produce a capsule and the cell wall appears layered. These layers are composed of fragments displaying a highly patterned ultrastructure (10, 16). This type of cell surface is now referred to as the surface layer (S-layer).S-layers are present on the surfaces of many archaea and bacteria (for reviews, see references 29 and 30). Most are formed by noncovalent, entropy-driven assembly of a single (glyco)protein protomer on the bacterial surface, giving rise to proteinaceous paracrystalline layers. Generally, a single S-layer is present, constituting 5 to 10% of total cell protein. Its synthesis is thus presumably energy consuming for the bacterium. Numerous bacteria have S-layers, suggesting that they play important roles in the interaction between the cell and its environment. Various functions have been proposed for S-layers, including shape maintenance and molecular sieving, and they can serve in phage fixation. The S-layer may be a virulence factor, protecting pathogenic bacteria against complement killing, facilitating binding of bacteria to host molecules, or enhancing their ability to associate with macrophages (for reviews, see references 27 and 29).Some bacteria, such as cyanobacteria or Azotobacter spp., possess both a capsule and an S-layer; however, to our knowledge, their structural relationships have not been analyzed through simultaneous genetic and cytologic studies. Both of these features have been independently described for the surface of the pathogenic bacterium B. anthracis. The components of the B. anthracis S-layer are two abundant surface proteins, EA1 and Sap (6, 20). Previous analyses of the B. anthracis S-layer used plasmid-cured strains; consequently, the interaction, if any, between the capsule and the S-layer could not be studied. Temporal or environmental regulation could be such that only one or the other structure is ever present at the cell surface. However, we show that S-layer proteins are synthesized under conditions where the bacilli are capsulated. We determined the localizations of capsule and S-layer components and analyzed whether the S-layer is necessary for proper capsulation. Finally, the assembly of the S-layer proteins in a two-dimensional crystal was examined in the presence of the capsule.     

Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles

The effect of physiological concentrations of KCl and MgCl₂ on the chemical stability of double-stranded and single-stranded DNA has been studied at temperatures typical for hyperthermophiles. These two salts protect both double and single-stranded DNA against heat-induced cleavage by inhibiting depurination. High KCl concentrations also protect DNA cleavage at apurinic sites, while high MgCl₂ concentrations stimulate this cleavage. It has been previously proposed that salt protects double-stranded DNA against depurination by stabilizing the double helix. However, the inhibition of the depurination of single-stranded DNA by KCl and MgCl₂ indicates that this effect is more probably due to a direct interaction of salts with purine nucleotides. These results suggest that the number and nature of heat-induced DNA lesions which have to be repaired might be quite different from one hyperthermophile to another, depending on their intracellular salt concentration. High salt concentrations might be also useful to protect DNA in long polymerase chain reaction (PCR) experiments and for long-term preservation. Received: October 12, 1997 / Accepted: January 29, 1998