Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.     

Utilization of acidic amino acids and their amides by pseudomonads: role of periplasmic glutaminase-asparaginase

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) support rapid growth of a variety of Pseudomonas strains when provided as the sole source of carbon and nitrogen. All key enzymes of glutamate metabolism were detected in P. fluorescence, with glutaminase and asparaginase showing the highest specific activities. A periplasmic glutaminase/asparaginase activity (PGA) was found in all pseudomonads examined, including a number of root-colonizing biocontrol strains. The enzyme was purified and shown to be identical with the ansB gene product described previously. In addition to PGA, P. fluorescens contains a cytoplasmic asparaginase with marked specificity for Asn. PGA is strongly and specifically induced by its substrates (Asn, Gln) but also by the reaction products (Asp, Glu). In addition, PGA is subject to efficient carbon catabolite repression by glucose and by citrate cycle metabolites. A mutant of P. putida KT2440 with a disrupted ansB gene was unable to utilize Gln, whereas growth of the mutant on other amino acids was normal.     

Experimental evidence for a beta beta alpha-Me-finger nuclease motif to represent the active site of the caspase-activated DNase

The caspase-activated DNase (CAD) is an important nuclease involved in apoptotic DNA degradation. Results of a sequence comparison of CAD proteins with beta beta alpha-Me-finger nucleases in conjunction with a mutational and chemical modification analysis suggest that CAD proteins constitute a new family of beta beta alpha-Me-finger nucleases. Nucleases of this family have widely different functions but are characterized by a common active-site fold and similar catalytic mechanisms. According to our results and comparisons with related nucleases, the active site of CAD displays features that partly resemble those of the colicin E9 and partly those of the T4 endonuclease VII active sites. We suggest that the catalytic mechanism of CAD involves a conserved histidine residue, acting as a general base, and another histidine as well as an aspartic acid residue required for cofactor binding. Our findings provide a first insight into the likely active-site structure and catalytic mechanism of a nuclease involved in the degradation of chromosomal DNA during programmed cell death.     

The Microbiome of the Red Sea Coral Stylophora pistillata Is Dominated by Tissue-Associated Endozoicomonas Bacteria

Endozoicomonas bacteria were found highly associated with the coral Stylophora pistillata, and these bacteria are also ubiquitously associated with diverse corals worldwide. Novel Endozoicomonas-specific probes revealed that Endozoicomonas bacteria were abundant in the endodermal tissues of S. pistillata and appear to have an intimate relationship with the coral.     

Canine RBC osmotic tolerance and membrane permeability

The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity (L(p)), the permeability coefficients (P(s)) of common cryoprotectant agents (CPAs), the associated reflection coefficient (sigma), the activation energies (E(a)) of L(p) and P(s) and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L(p), P(s), and sigma for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 degrees C). Using the Arrhenius equation, the activation energies of L(p) and P(s) in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150mOsm (1.67V(iso)) and 1200mOsm (0.45V(iso)), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal.     

Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.     

Determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine by capillary electrophoresis using ultraviolet absorbance and laser-induced fluorescence detection

Two capillary electrophoresis methods have been developed for the direct determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine. Analytes were detected using conventional UV detection as well as laser-induced fluorescence (LIF) detection with an HeCd-laser operating at a wavelength of 325 nm. The results of both detection techniques were compared. Indeed, the limit of quantification was eightfold lower using LIF detection (50 ng/ml) in comparison to UV detection (400 ng/ml). As no interference due to endogenous urine compounds was observed, direct urine analysis was feasible. Analysis was very simple and fast-one run could be performed within less than 10 min (CE-UV method) and 2.5 min (CE-LIF method), respectively. Both assays were fully validated and applied to urine samples from a human volunteer. The results of the application of the CE-LIF method to human urine samples are presented in this publication.     

Molecular phylogeny of French Guiana Hylinae: implications for the systematic and biodiversity of the Neotropical frogs

In this study we used nucleotide sequences from a segment of mitochondrial 16S ribosomal DNA gene to investigate the evolutionary relationships of some French Guiana Hylinae. New sequences, representing the members of different French Guiana frogs-five specimens of the Scinax genus, two Hyla, one Osteocephalus, one Hyalinobatrachium and two Rana as out-group-were examined. In addition, 26 sequences available from GenBank database representing the other subfamilies of the Hylidae were added to our study. This work allowed us to clarify relationships within the four hylids subfamilies (Pelodryadinae, Phyllomedusinae, Hemiphractinae and Hylinae) and the phylogenetic placement of the enigmatic Scinax genus within the Hylidae. We found that: (1) the Scinax genus displays a high level of differentiation in comparison to two other genera (Litoria and Hyla) belonging to 'Hylidae' family; (2) the Hylinae are paraphyletic given the position of the Litoria, which was the sister-group of the Hyla and the Osteocephalus genera; (3) the anterior works and our results (based on two different data sets) showed the paraphyly of the Hylidae questioning the validity of this family; (4) the reassessment of these different taxonomic groups will induce a huge implication on the estimation (past, present and future) of the biodiversity (in Neotropical frogs).