Olfactory cues and nest recognition in the solitary bee Osmia lignaria

Abstract.  The use of olfactory cues for nest recognition by the solitary bee Osmia lignaria is studied in a greenhouse environment. Glass tubes are provided as nesting cavities to allow the in-nest behaviour of bees to be observed. In addition, each glass tube is cut into three sections for experimental manipulation and for subsequent chemical analysis. Nesting females drag their abdomen along the tube before exiting, spiral inside the tube, and sometimes deposit fluid droplets from the tip of the abdomen. For the manipulation, the outer section, the middle section, or both sections are removed and replaced with similar clean glass tube sections, and the behaviour exhibited by test females is recorded upon arrival in front of the nesting site and inside the nesting tubes. The resulting hesitation behaviour displayed by females after treatments appears to indicate the loss of some olfactory cues used for nest recognition inside the entire nest. Chemical analysis of the depositions inside the nesting tube, as well as analysis of the cuticular lipids of the nesting bees, reveals the presence of free fatty acids, hydrocarbons and wax esters.     

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor βPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.     

The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases

This study sought to evaluate the levels of mRNA expression and protein synthesis of MMP-13, cathepsin K, aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5) and 5-lipoxygenase (5-LOX) in cartilage in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA), and to examine the effects of treatment with licofelone, a 5-lipoxygenase (LOX)/cyclooxygenase (COX) inhibitor, on the levels of these catabolic factors. Sectioning of the ACL of the right knee was performed in three experimental groups: group 1 received no active treatment (placebo group); and groups 2 and 3 received therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day orally, respectively) for 8 weeks, beginning the day following surgery. A fourth group consisted of untreated dogs that were used as normal controls. Specimens of cartilage were selected from lesional areas of OA femoral condyles and tibial plateaus, and were processed for real-time quantitative PCR and immunohistochemical analyses. The levels of MMP-13, cathepsin K, ADAMTS-4, ADAMTS-5 and 5-LOX were found to be significantly increased in OA cartilage. Licofelone treatment decreased the levels of both mRNA expression and protein synthesis of the factors studied. Of note was the marked reduction in the level of 5-LOX gene expression. The effects of the drug were about the same at both tested dosages. In vivo treatment with therapeutic dosages of licofelone has been found to reduce the degradation of OA cartilage in experimental OA. This, coupled with the results of the present study, indicates that the effects of licofelone are mediated by the inhibition of the major cartilage catabolic pathways involved in the destruction of cartilage matrix macromolecules. Moreover, our findings also indicate the possible auto-regulation of 5-LOX gene expression by licofelone in OA cartilage.     

Internalization and trafficking of the human and rat growth hormone-releasing hormone receptor

Internalization and intracellular trafficking of the growth hormone-releasing hormone receptor (GHRH-R) were studied in rat anterior pituitary and human (h) and rat (r) GHRH-R-transfected BHK cells, with the GHRH agonist, [N(alpha)-5-carboxyfluoresceinyl-D-Ala(2), Ala(8), Ala(15), Lys(22)]hGHRH(1-29)NH(2) (Fluo-GHRH). Time- and temperature-dependent internalization of stimulated GHRH-R was blocked by phenyl arsine oxide (PAO) in both cell types. In anterior pituitary and rGHRH-R-transfected BHK cells, only filipin III and cerulenin blocked receptor-mediated internalization of Fluo-GHRH while in hGHRH-R-transfected BHK cells, only hyperosmolar sucrose inhibited this process. These results suggest that hGHRH-R internalization is clathrin-dependent, while fatty acid acylation of rGHRH-R appears to be a prerequisite to caveolin-dependent internalization. Experiments in anterior pituitary using Bodipy-FL-C(5) ganglioside GM1, a specific marker of lipid rafts such as caveolae, confirmed this latter pathway. Co-localization of Fluo-GHRH with LysoTracker indicated that Fluo-GHRH was directed to acidic organelles in both cell types. Finally, studies using cycloheximide and monensin showed that upon stimulation with GHRH, an optimal concentration of functional GHRH-R was maintained at the plasma membrane due to de novo synthesis and recycling in pituitary cells and to de novo synthesis solely in hGHRH-R-transfected BHK cells. This first study on the dynamics of the GHRH/GHRH-R complexes using fluorescence imaging in a native environment compared to cell system models, revealed that both receptor primary structure and concentration at the plasma membrane play important roles in internalization and trafficking of specific G-protein-coupled receptors (GPCR).     

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

Targeting DNA with novel diphenylcarbazoles

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.     

Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also influences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid profiles, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.     

Cellular and biochemical features of skeletal muscle in obese Yucatan minipigs

Objective: To examine cellular and biochemical features of skeletal muscle in response to dietary‐induced obesity in a novel Yucatan minipig model of childhood obesity. Research Methods and Procedures: From 4 to 16 months of age, minipigs were fed either a recommended human‐type diet (NF; n = 4) or were overfed a western‐type diet with saturated fat and high‐glycemic index carbohydrates (OF, n = 4). Muscle samples (biceps femoris) were histochemically stained for the identification of intramuscular adipocytes, intramyocellular lipid aggregates (oil red O), and myofiber types (myosin ATPase, succinate dehydrogenase). Gene expressions and/or activities of factors involved in lipogenesis, lipolysis, or energetic metabolism were quantified in muscle. Results: Cross‐sectional areas of myofibers paralleled pig body weight (r = 0.86, p < 0.01). The size of intramuscular adipocytes, the relative proportion of oil red O‐stained fibers, and total muscle lipid content tended (p ≤ 0.10) to increase in response to OF diet. Hormone‐sensitive lipase, carnitine palmityl transferase‐I, and uncoupling protein 2 mRNA levels were lower (p < 0.05) in OF pigs than in NF pigs. Activities of β‐hydroxyacyl‐coenzyme A dehydrogenase and citrate synthase assessing post‐carnitine palmityl transferase I events and the proportion of oxidative myofibers were not altered by OF diet. Activity and gene expression of fatty acid synthase were lower (p < 0.02) in OF pigs than in NF pigs. Discussion: Overfeeding in Yucatan minipigs reduced the expression levels of three catabolic steps in skeletal muscle that are involved also in the etiology of human obesity.     

Rab35 - A vesicular traffic-regulating small GTPase with actin modulating roles

The Rab family of GTPases are regulators of eukaryotic vesicular membrane traffic, while modulation of actin dynamics is a function conventionally associated with the Rho family of GTPases. Rab35 is a Rab protein with both plasma membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions even in typically non-adherent Jurkat T-cells. Recent evidence indicates that Rab35’s activity, particularly the ability to mediate protrusive outgrowths, is due to its direct influence on actin dynamics. This can occur via activation of the Rho family of GTPases, or through the engagement of its effector fascin, an actin bundling protein.