Identification and purification of two distinct complexes containing the five RAD51 paralogs.

Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.

Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

New and emerging treatment of Staphylococcus aureus infections in the hospital setting.

Methicillin-resistant Staphylococcus aureus (MRSA), both hospital-acquired and community-acquired, is a dangerous pathogen that is involved in an increasing number of serious infections with high risk for morbidity and mortality. Community-acquired MRSA strains have epidemic potential and can be particularly virulent. Vancomycin has been the standard hospital treatment for the past 40 years, but vancomycin-resistant isolates of S. aureus have emerged in the USA, and vancomycin-intermediate isolates are increasingly being reported worldwide. New antimicrobial agents with activity against multidrug-resistant S. aureus and other resistant pathogens are urgently needed. Despite great strides, further advances in our understanding of the molecular and biochemical mechanisms responsible for antimicrobial resistance are still required. Several agents have been recently approved for the treatment of serious Gram-positive infections, including linezolid, daptomycin, and tigecycline. The novel investigational cephalosporin, ceftobiprole, is one of the first penicillinase-resistant agents to target penicillin-binding protein 2a (or PBP2a), an acquired PBP with low beta-lactam-affinity that confers intrinsic beta-lactam resistance to S. aureus and other staphylococci. This mechanism of PBP binding, including inhibition of PBP2a, confers broad-spectrum activity against clinically important Gram-negative and Gram-positive pathogens, including MRSA. Phase III clinical trials comparing ceftobiprole with vancomycin alone and in combination with ceftazidime for the treatment of complicated skin and skin structure infections showed ceftobiprole to have efficacy similar to the efficacy of these comparators as evidenced by non-inferior clinical cure and microbiological eradication rates.

In silico prediction of human carboxylesterase-1 (hCES1) metabolism combining docking analyses and MD simulations.

Metabolic problems lead to numerous failures during clinical trials, and much effort is now devoted in developing in silico models predicting metabolic stability and metabolites. Such models are well known for cytochromes P450 and some transferases, whereas little has been done to predict the hydrolytic activity of human hydrolases. The present study was undertaken to develop a computational approach able to predict the hydrolysis of novel esters by human carboxylesterase hCES1. The study involves both docking analyses of known substrates to develop predictive models, and molecular dynamics (MD) simulations to reveal the in situ behavior of substrates and products, with particular attention being paid to the influence of their ionization state. The results emphasize some crucial properties of the hCES1 catalytic cavity, confirming that as a trend with several exceptions, hCES1 prefers substrates with relatively smaller and somewhat polar alkyl/aryl groups and larger hydrophobic acyl moieties. The docking results underline the usefulness of the hydrophobic interaction score proposed here, which allows a robust prediction of hCES1 catalysis, while the MD simulations show the different behavior of substrates and products in the enzyme cavity, suggesting in particular that basic substrates interact with the enzyme in their unprotonated form.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Vaccination against schistosomiasis and fascioliasis with the new recombinant antigen Sm14: potential basis of a multi-valent anti-helminth vaccine?

Molecular cloning of components of protective antigenic preparations have suggested that related parasite fatty acid binding proteins could form the basis of the well documented protective, immune cross reactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. We have now confirmed the cross protective potential of parasite fatty acid binding proteins and suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni of veterinary and human importance respectively.

Novel mechanisms of immune evasion by Schistosoma mansoni

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28-A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56-A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70-A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1-A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.

Estudi d'adipoquines com a marcadors plasmàtics de síndrome metabòlica

La síndrome metabòlica s’associa amb un risc elevat de desenvolupar diabetis tipus 2 i malaltia cardiovascular. La síndrome metabòlica es defineix com un clúster d’anormalitats metabòliques i, d’entre totes, l’obesitat abdominal constitueix el factor de risc més prevalent i crític en el desenvolupament de la síndrome metabòlica, el risc cardiovascular augmentat i la resistència a la insulina. La prevalença augmentada de l’obesitat en la població a nivell mundial ha portat el teixit adipós al primer pla dels estudis epidemiològics. Anteriorment es considerava el reservori energètic de l’organisme, actualment es parla del teixit adipós com un òrgan endocrí, metabòlicament molt actiu, implicat en diferents vies i processos metabòlics. L’etiologia de l’obesitat és complexa i multifactorial, però es fa evident en la disfuncionalitat del teixit adipós. Un teixit adipós disfuncional veu superada la seva capacitat d’emmagatzemar lípid i respon amb la hipersecreció de diferents molècules (adipoquines, citoquines i mediadors inflamatoris) a favor de la resistència a la insulina, proinflamatòries i proaterogèniques. La fatty acid-binding protein 4 (FABP4) i la retinol-binding protein 4 (RBP4) són dues adipoquines que en circulació, es desconeix la funció exacta que duen a terme. Estudis recents han suggerit la FABP4 com a marcador d’adipositat, síndrome metabòlica i diabetis tipus 2. I, RBP4, malgrat que les dades de diferents estudis en humans desperten certa controvèrsia, s’ha associat amb la resistència a la insulina i el desenvolupament de la diabetis tipus 2. En aquesta memòria es recullen els treballs en què es va estudiar el paper d’aquestes adipoquines en relació a malalties de base metabòlica amb afectació del teixit adipós com són la síndrome metabòlica, la diabetis tipus 2, la hiperlipèmia familiar combinada i la, lipodistrofia associada a tractament combinat antiretroviral de la infecció pel virus de la immunodeficiència humana (VIH).

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Variation in novel exons (RACEfrags) of the MECP2 gene in Rett syndrome patients and controls.

The study of transcription using genomic tiling arrays has lead to the identification of numerous additional exons. One example is the MECP2 gene on the X chromosome; using 5'RACE and RT-PCR in human tissues and cell lines, we have found more than 70 novel exons (RACEfrags) connecting to at least one annotated exon.. We sequenced all MECP2-connected exons and flanking sequences in 3 groups: 46 patients with the Rett syndrome and without mutations in the currently annotated exons of the MECP2 and CDKL5 genes; 32 patients with the Rett syndrome and identified mutations in the MECP2 gene; 100 control individuals from the same geoethnic group. Approximately 13 kb were sequenced per sample, (2.4 Mb of DNA resequencing). A total of 75 individuals had novel rare variants (mostly private variants) but no statistically significant difference was found among the 3 groups. These results suggest that variants in the newly discovered exons may not contribute to Rett syndrome. Interestingly however, there are about twice more variants in the novel exons than in the flanking sequences (44 vs. 21 for approximately 1.3 Mb sequenced for each class of sequences, p=0.0025). Thus the evolutionary forces that shape these novel exons may be different than those of neighboring sequences.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Structural and functional interaction sites between Na,K-ATPase and FXYD proteins.

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K(+) and Na(+) affinity. Little is known about the interaction sites between the Na,K-ATPase alpha subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase alpha subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe(956), Glu(960), Leu(964), and Phe(967) in TM9 of the Na,K-ATPase alpha subunit represent one face interacting with the three FXYD proteins. Leu(964) and Phe(967) contribute to the efficient association of FXYD proteins with the Na,K-ATPase alpha subunit, whereas Phe(956) and Glu(960) are essential for the transmission of the functional effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. The relative contribution of Phe(956) and Glu(960) to the K(+) effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. In contrast to the effect on the apparent K(+) affinity, Phe(956) and Glu(960) are not involved in the effect of FXYD2 and FXYD4 on the apparent Na(+) affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe(956), Glu(960), Leu(964), and Phe(967) in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase alpha subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase.

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

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Non-coding small RNAs (sRNAs) have important regulatory functions in bacteria. In Pseudomonas spp., about 40 sRNAs have been reported until the end of 2008, a number that almost certainly is an underestimate. We provide a summary of the coding regions for these sRNAs is Pseudomonas aeruginosa. The functions of some Pseudomonas sRNAs can be deduced from those of homologous well-characterized sRNAs of Escherichia coli, e.g. 6S RNA (a stationary phase regulator of RNA polymerase) and tmRNA (a component of a machinery serving to eliminate truncated polypeptides). Two sRNAs of P. aeruginosa, PrrF1 and PrrF2, whose expression is repressed by the Fur repressor in the presence of iron, inhibit translation initiation of mRNAs specifying superoxide dismutase (sodB), succinate dehydrogenase (sdhABCD) and anthranilate degradation (antABC), via a base-paring mechanism. As a consequence, these mRNAs are poorly expressed under conditions of iron limitation. Two further sRNAs of P. aeruginosa, RsmY and RsmZ, whose expression is positively controlled by the GacS/GacA two-component system in response to unknown signals, act as scavengers of the RNA-binding protein RsmA. In this way, translational repression exerted by RsmA on target mRNAs can be relieved. The Gac/Rsm signal transduction pathway globally regulates motility and the formation of extracellular products in pseudomonas spp.