Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene.

Stable ternary transcription complexes assembled in vitro, using a HeLa whole-cell extract, have been isolated and visualized by electron microscopy. The formation of these stable complexes on the DNA fragment used as template, the 5' end region of the Xenopus laevis vitellogenin gene B2, depends on factors present in the whole-cell extract, RNA polymerase II and at least two nucleotides. Interestingly, bending in the DNA fragment was frequently observed at the binding site of RNA polymerase II. Dinucleotides that can prime initiation within a short sequence of approximately 10 contiguous nucleotides centered around the initiation site used in vivo, also favour the formation of stable complexes. In addition, pre-initiation complexes were isolated and it was shown that factors in the extract involved in their formation are more abundant than the RNA polymerase II molecules available for binding. The possible implication of this observation relative to the in vivo situation is discussed.

The TRAF3-binding site of human molluscipox virus FLIP molecule MC159 is critical for its capacity to inhibit Fas-induced apoptosis.

Members of the viral Flice/caspase-8 inhibitory protein (v-FLIP) family prevent induction of apoptosis by death receptors through inhibition of the processing and activation of procaspase-8 and -10 at the level of the receptor-associated death-inducing signaling complex (DISC). Here, we have addressed the molecular function of the v-FLIP member MC159 of the human molluscum contagiosum virus. MC159 FLIP powerfully inhibited both caspase-dependent and caspase-independent cell death induced by Fas. The C-terminal region of MC159 bound TNF receptor-associated factor (TRAF)3, was necessary for optimal TRAF2 binding, and mediated the recruitment of both TRAFs into the Fas DISC. TRAF-binding-deficient mutants of MC159 showed impaired inhibition of FasL-induced caspase-8 processing and Fas internalization, and had reduced antiapoptotic activity. Our findings provide evidence that a MC159/TRAF2/TRAF3 complex regulates a new aspect of Fas signaling, and identify MC159 FLIP as a molecule that targets multiple features of Fas-induced cell death.

Association between mannose-binding lectin (MBL) deficiency and cytomegalovirus (CMV) infection after kidney transplantation

MBLdeficiency is thought to be a risk factor for the development of viral infection, such as genital herpes and HSV-2 meningitis. However, there is limited data on the possible interaction between MBL and CMV, especially after organ transplantation. Between 2003 and 2005, we measured MBL levels in 16 kidney transplant recipients with high-risk CMV serostatus (donor positive/recipient negative, D+/R−). All patients receivedCMV prophylaxis of valganciclovir 450 mg/day for 3 months after transplantation. After stopping valganciclovir, CMV-DNA was measured in whole blood by real time PCR every 2 weeks for 3 months. CMV infections were diagnosed according to the recommendations of the AST. MBL levels were measured in stored pre-transplantation sera by an investigator blinded to the CMV complications. MBL levels below 500 ng/ml were considered as being functionally deficient. After a follow-up of at least 10 months, seven patients out of 16 developed CMV disease (three CMV syndrome, and four probable invasive disease, i.e. two colitis and two hepatitis), four patients developed asymptomatic CMV infection, and five patients never developed any sign of CMV replication. Peak CMV-DNA was higher in patients with CMV disease than in those with asymptomatic infection (4.64 versus 2.72 mean log copy CMV-DNA/106 leukocytes, p < 0.05). Overall, 9/16 patients (56%) had MBL deficiency: 5/7 (71%) of patients with CMV disease, 4/4 (100%) of patients with asymptomatic CMVinfection, and 0/5 (0%) of patients withoutCMVinfection (p < 0.005, between CMV infection/disease versus no infection or control blood donors). There were no significant differences in age, gender or immunosuppressive regimens between the groups. MBL deficiency may be a significant risk factor for the development of post-prophylaxisCMVinfection in D+/R−kidney recipients, suggesting a new role of innate immunity in the control of CMV infection after organ transplantation.

Fibrinogen and fibronectin binding cooperate for valve infection and invasion in Staphylococcus aureus experimental endocarditis.

The expression of Staphylococcus aureus adhesins in Lactococcus lactis identified clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) as critical for valve colonization in rats with experimental endocarditis. This study further analyzed their role in disease evolution. Infected animals were followed for 3 d. ClfA-positive lactococci successfully colonized damaged valves, but were spontaneously eradicated over 48 h. In contrast, FnBPA-positive lactococci progressively increased bacterial titers in vegetations and spleens. At imaging, ClfA-positive lactococci were restricted to the vegetations, whereas FnBPA-positive lactococci also invaded the adjacent endothelium. This reflected the capacity of FnBPA to trigger cell internalization in vitro. Because FnBPA carries both fibrinogen- and fibronectin-binding domains, we tested the role of these functionalities by deleting the fibrinogen-binding domain of FnBPA and supplementing it with the fibrinogen-binding domain of ClfA in cis or in trans. Deletion of the fibrinogen-binding domain of FnBPA did not alter fibronectin binding and cell internalization in vitro. However, it totally abrogated valve infectivity in vivo. This ability was restored in cis by inserting the fibrinogen-binding domain of ClfA into truncated FnBPA, and in trans by coexpressing full-length ClfA and truncated FnBPA on two separate plasmids. Thus, fibrinogen and fibronectin binding could cooperate for S. aureus valve colonization and endothelial invasion in vivo.

Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.

AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 x 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites.

The ability to determine the location and relative strength of all transcription-factor binding sites in a genome is important both for a comprehensive understanding of gene regulation and for effective promoter engineering in biotechnological applications. Here we present a bioinformatically driven experimental method to accurately define the DNA-binding sequence specificity of transcription factors. A generalized profile was used as a predictive quantitative model for binding sites, and its parameters were estimated from in vitro-selected ligands using standard hidden Markov model training algorithms. Computer simulations showed that several thousand low- to medium-affinity sequences are required to generate a profile of desired accuracy. To produce data on this scale, we applied high-throughput genomics methods to the biochemical problem addressed here. A method combining systematic evolution of ligands by exponential enrichment (SELEX) and serial analysis of gene expression (SAGE) protocols was coupled to an automated quality-controlled sequence extraction procedure based on Phred quality scores. This allowed the sequencing of a database of more than 10,000 potential DNA ligands for the CTF/NFI transcription factor. The resulting binding-site model defines the sequence specificity of this protein with a high degree of accuracy not achieved earlier and thereby makes it possible to identify previously unknown regulatory sequences in genomic DNA. A covariance analysis of the selected sites revealed non-independent base preferences at different nucleotide positions, providing insight into the binding mechanism.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes.

We present a new lab-on-a-chip system for electrophysiological measurements on Xenopus oocytes. Xenopus oocytes are widely used host cells in the field of pharmacological studies and drug development. We developed a novel non-invasive technique using immobilized non-devitellinized cells that replaces the traditional "two-electrode voltage-clamp" (TEVC) method. In particular, rapid fluidic exchange was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimetre-sized cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of human epithelial sodium channels (ENaC). Our results show that the response time of this ion channel to a specific reactant is about an order of magnitude faster than could be estimated with the traditional TEVC technique.

Mutations in penicillin-binding protein (PBP) genes and in non-PBP genes during selection of penicillin-resistant Streptococcus gordonii.

Penicillin resistance in Streptococcus spp. involves multiple mutations in both penicillin-binding proteins (PBPs) and non-PBP genes. Here, we studied the development of penicillin resistance in the oral commensal Streptococcus gordonii. Cyclic exposure of bacteria to twofold-increasing penicillin concentrations selected for a progressive 250- to 500-fold MIC increase (from 0.008 to between 2 and 4 microg/ml). The major MIC increase (> or = 35-fold) was related to non-PBP mutations, whereas PBP mutations accounted only for a 4- to 8-fold additional increase. PBP mutations occurred in class B PBPs 2X and 2B, which carry a transpeptidase domain, but not in class A PBP 1A, 1B, or 2A, which carry an additional transglycosylase domain. Therefore, we tested whether inactivation of class A PBPs affected resistance development in spite of the absence of mutations. Deletion of PBP 1A or 2A profoundly slowed down resistance development but only moderately affected resistance in already highly resistant mutants (MIC = 2 to 4 microg/ml). Thus, class A PBPs might facilitate early development of resistance by stabilizing penicillin-altered peptidoglycan via transglycosylation, whereas they might be less indispensable in highly resistant mutants which have reestablished a penicillin-insensitive cell wall-building machinery. The contribution of PBP and non-PBP mutations alone could be individualized in DNA transformation. Both PBP and non-PBP mutations conferred some level of intrinsic resistance, but combining the mutations synergized them to ensure high-level resistance (> or = 2 microg/ml). The results underline the complexity of penicillin resistance development and suggest that inhibition of transglycosylase might be an as yet underestimated way to interfere with early resistance development.

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.

Gender differences in whole body fat oxidation kinetics during exercise

Introduction Discrepancies appear in studies comparing fat oxidation between men and women during exercise (1). Therefore, this study aimed to quantitatively describe and compare whole body fat oxidation kinetics between genders during exercise using a sinusoidal model (SIN) (2). Methods Twelve men and 11 women matched for age, body mass index (23.4±0.6 kg.m-2 and 21.5±0.8 kg.m-2, respectively) and aerobic fitness [maximal oxygen uptake ( ) (58.5±1.6 mL.kg FFM-1.min-1 and 55.3±2.0 mL.kg FFM-1.min-1, respectively) and power output ( ) per kilogram of fat-free mass (FFM)] performed submaximal incremental tests (Incr) with 5-min stages and 7.5% increment on a cycle ergometer. Respiratory and HR values were averaged over the last 2 minutes of each stage. All female study participants were eumenorrheic, reported regular menstrual cycles (28.6 ± 0.8 days) and were not taking oral contraceptives (OC) or other forms of exogenous ovarian hormones. Women were studied in the early follicular phase (FP) of their menstrual cycle (between days 3 and 8, where day 1 is the first day of menses). Fat oxidation rates were determined using indirect calorimetry and plotted as a function of exercise intensity. The SIN model (2), which includes three independent variables (dilatation, symmetry, translation), was used to mathematically describe fat oxidation kinetics and to determine the intensity (Fatmax) eliciting the maximal fat oxidation (MFO). Results During Incr, women exhibited greater fat oxidation rates from 35 to 85% , MFO (6.6 ± 0.9 vs. 4.5 ± 0.3 mgkg FFM-1min-1) and Fatmax (58.1 ± 1.9 vs. 50.0 ± 2.7% ) (P<0.05) than men. While men and women showed similar global shapes of fat oxidation kinetics in terms of dilatation and symmetry (P>0.05), the fat oxidation curve tended to be shifted towards higher exercise intensities in women (rightward translation, P=0.08). Conclusion These results showed that women, eumenorrheic, not taking OC and tested in FP, have a greater reliance on fat oxidation than men during submaximal exercise, but they also indicate that this greater fat oxidation is shifted towards higher exercise intensities in women compared with men. References 1. Blaak E. Gender differences in fat metabolism. Curr Opin Clin Nutr Metab Care 4: 499-502, 2001. 2. Cheneviere X, Malatesta D, Peters EM, and Borrani F. A mathematical model to describe fat oxidation kinetics during graded exercise. Med Sci Sports Exerc 41: 1615-1625, 2009.

Biological activity of ectodysplasin A is conditioned by its collagen and heparan sulfate proteoglycan-binding domains.

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

Comparative localization of glioma-reactive monoclonal antibodies in vivo in an athymic mouse human glioma xenograft model.

Radioiodinated murine monoclonal antibodies (Mabs) 81C6, Me 1-14, C12, D12, and E9, made against or reactive with human gliomas but not normal brain, and Mab UJ13A, a pan-neuroectodermal Mab reactive with normal human glial and neural cells, were evaluated in paired label studies in the D-54 MG subcutaneous human glioma xenograft model system in nude mice. Following intravenous injection in the tail vein of mice bearing 200-400 mm3 tumors, specific localization of Mabs to tumor over time (6 h-9 days) was evaluated by tissue counting; each Mab demonstrated a unique localization profile. The comparison of localization indices (LI), determined as a ratio of tissue level of Mab to control immunoglobulin with simultaneous correction for blood levels of each, showed Mabs 81C6 and Me 1-14 to steadily accumulate in glioma xenografts, maintaining LI from 5-20 at 7-9 days after Mab injection. Mab UJ13A peaked at day 1, maintaining this level through day 2, and declining thereafter. Mabs D12 and C12 peaked at days 3 and 4, respectively, and E9 maintained an LI of greater than 3 from days 3-9. Percent injected dose localized/g of tumor varied from a peak high of 16% (81C6) to a low of 5% (Me 1-14 and UJ13A). Immunoperoxidase histochemistry, performed with each Mab on a battery of primary human brain neoplasms, revealed that Mabs 81C6 and E9, which demonstrated the highest levels of percent injected dose localized/g of tumor over time, reacted with antigens expressed in the extracellular matrix. This finding suggests that extracellular matrix localization of antigen represents a biologically significant factor affecting localization and/or binding in the xenograft model used. The demonstration of significant localization, varied kinetics and patterns of localization of this localizing Mab panel warrants their continued investigation as potential imaging and therapeutic agents for human trials.

9-cis-retinoic acid enhances fatty acid-induced expression of the liver fatty acid-binding protein gene.

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.