Histone deacetylase inhibitors impair innate immune responses

SUMMARYThe innate immune system plays a central role in host defenses against invading pathogens. Innate immune cells sense the presence of pathogens through pattern recognition receptors that trigger intracellular signaling, leading to the production of pro-inflammatory mediators like cytokines, which shape innate and adaptive immune responses. Both by excess and by default inflammation may be detrimental to the host. Indeed, severe sepsis and septic shock are lethal complications of infections characterized by a dysregulated inflammatory response.In recent years, members of the superfamily of histone deacetylases have been the focus of great interest. In mammals, histone deacetylases are broadly classified into two main subfamilies comprising histone deacetylases 1-11 (HDAC1-11) and sirtuins 1-7 (SIRT1-7). These enzymes influence gene expression by deacetylating histones and numerous non-histone proteins. Histone deacetylases have been involved in the development of oncologic, metabolic, cardiovascular, neurodegenerative and autoimmune diseases. Pharmacological modulators of histone deacetylase activity, principally inhibitors, have been developed for the treatment of cancer and metabolic diseases. When we initiated this project, several studies suggested that inhibitors of HDAC 1-11 have anti-inflammatory activity. Yet, their influence on innate immune responses was largely uncharacterized. The present study was initiated to fill in this gap.In the first part of this work, we report the first comprehensive study of the effects of HDAC 1- 11 inhibitors on innate immune responses in vitro and in vivo. Strikingly, expression studies revealed that HDAC1-11 inhibitors act essentially as negative regulators of basal and microbial product- induced expression of critical immune receptors and antimicrobial products by mouse and human innate immune cells like macrophages and dendritic cells. Furthermore, we describe a new molecular mechanism whereby HDAC1-11 inhibitors repress pro-inflammatory cytokine expression through the induction of the expression and the activity of the transcriptional repressor Μί-2β. HDAC1-11 inhibitors also impair the potential of macrophages to engulf and kill bacteria. Finally, mice treated with an HDAC inhibitor are more susceptible to non-severe bacterial and fungal infection, but are protected against toxic and septic shock. Altogether these data support the concept that HDAC 1-11 inhibitors have potent anti-inflammatory and immunomodulatory activities in vitro and in vivo.Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that plays a central role in innate immune responses, cell proliferation and oncogenesis. In the second part of this manuscript, we demonstrate that HDAC1-11 inhibitors inhibit MIF expression in vitro and in vivo and describe a novel molecular mechanism accounting for these effects. We propose that inhibition of MIF expression by HDAC 1-11 inhibitors may contribute to the antitumorigenic and anti-inflammatory effects of these drugs.NAD+ is an essential cofactor of sirtuins activity and one of the major sources of energy within the cells. Therefore, sirtuins link deacetylation to NAD+ metabolism and energy status. In the last part of this thesis, we report preliminary results indicating that a pharmacological inhibitor of SIRT1-2 drastically decreases pro-inflammatory cytokine production (RNA and protein) and interferes with MAP kinase intracellular signal transduction pathway in macrophages. Moreover, administration of the SIRT1-2 inhibitor protects mice from lethal endotoxic shock and septic shock.Overall, our studies demonstrate that inhibitors of HDAC1-11 and sirtuins are powerful anti-inflammatory molecules. Given their profound negative impact on the host antimicrobial defence response, these inhibitors might increase the susceptibility to opportunistic infections, especially in immunocompromised cancer patients. Yet, these inhibitors might be useful to control the inflammatory response in severely ill septic patients or in patients suffering from chronic inflammatory diseases.

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Nitric oxide: a major determinant of mast cell phenotype and function

Mast cells (MC) are important in the numerous physiological processes of homeostasis and disease. Most notably, MC are critical effectors in the development and exacerbation of allergic disorders. Nitric oxide (NO) is a diatomic radical produced by nitric oxide synthase (NOS), and has pluripotent cell signaling and cytotoxic properties. NO can influence many MC functions. Recent evidence shows the source of this NO can be from the mast cell itself. Governing the production of this endogenous NO, through alterations in the expression of tetrahydrobiopterin (BH4), a NOS cofactor, has stabilizing effects on MC degranulation. Furthermore, NO regulates the synthesis and secretion of de novo generated mediators, including leukotrienes and chemokines. These novel observations add to the growing body of knowledge surrounding the role of NO in the MC.

Seroepidemiological study of herpes simplex virus type 2 in patients with the acquired immunodeficiency syndrome in the city of Niterói, Rio de Janeiro, Brazil

This study was designed to determine the seroprevalence of herpes simplex virus type 2 (HSV-2) and to evaluate its association with age, sex as well as other demographic and behavioural factors in 150 human immunodeficiency virus (HIV) positive adults patients attending the general medical outpatient ward for routine care of Niterói, state of Rio de Janeiro, Brazil. Serum samples were screened for HSV-2 antibodies using an indirect ELISA. Eighty-three patients were men (mean age: 38.8) and 67 were women (mean age: 35.4). The estimated prevalence of HSV-2 was 52% (95% CI: 44-60%) and it was higher among men (53%) than among women (50.7%). Overall, the age of first sexual intercourse and past history of genital herpes were associated with HSV-2 seropositivity. Analysis by gender disclosed significant association of number of lifetime sex partners only among men. Although HSV-2 antibodies were frequent in the study group, genital herpes was reported by 21.8% of the HSV-2 positive subjects, indicating low awareness of the HSV-2 infection. These results may have public health importance for Brazil as the high rate of HSV-2 infection may act as a cofactor of HIV transmission.

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

Deficient selenium status of a healthy adult Spanish population.

INTRODUCTION Selenium is an essential micronutrient for human health, being a cofactor for enzymes with antioxidant activity that protect the organism from oxidative damage. An inadequate intake of this mineral has been associated with the onset and progression of chronic diseases such as hypertension, diabetes, coronary diseases, asthma, and cancer. For this reason, knowledge of the plasma and erythrocyte selenium levels of a population makes a relevant contribution to assessment of its nutritional status. OBJECTIVE The objective of the present study was to determine the nutritional status of selenium and risk of selenium deficiency in a healthy adult population in Spain by examining food and nutrient intake and analyzing biochemical parameters related to selenium metabolism, including plasma and erythrocyte levels and selenium-dependent glutathione peroxidase (GPx) enzymatic activity. MATERIAL AND METHODS We studied 84 healthy adults (31 males and 53 females) from the province of Granada, determining their plasma and erythrocyte selenium concentrations and the association of these levels with the enzymatic activity of glutathione peroxidase (GPx) and with life style factors. We also gathered data on their food and nutrient intake and the results of biochemical analyses. Correlations were studied among all of these variables. RESULTS The mean plasma selenium concentration was 76.6 ± 17.3 μg/L (87.3 ± 17.4 μg/L in males, 67.3 ± 10.7 μg/L in females), whereas the mean erythrocyte selenium concentration was 104.6 μg/L (107.9 ± 26.1 μg/L in males and 101.7 ± 21.7 μg/L in females). The nutritional status of selenium was defined by the plasma concentration required to reach maximum GPx activity, establishing 90 μg/L as reference value. According to this criterion, 50% of the men and 53% of the women were selenium deficient. CONCLUSIONS Selenium is subjected to multiple regulation mechanisms. Erythrocyte selenium is a good marker of longer term selenium status, while plasma selenium appears to be a marker of short-term nutritional status. The present findings indicate a positive correlation between plasma selenium concentration and the practice of physical activity. Bioavailability studies are required to establish appropriate reference levels of this mineral for the Spanish population.

When zero doesn't mean it and other geomathematical mischief

There is almost not a case in exploration geology, where the studied data doesn’tincludes below detection limits and/or zero values, and since most of the geological dataresponds to lognormal distributions, these “zero data” represent a mathematicalchallenge for the interpretation.We need to start by recognizing that there are zero values in geology. For example theamount of quartz in a foyaite (nepheline syenite) is zero, since quartz cannot co-existswith nepheline. Another common essential zero is a North azimuth, however we canalways change that zero for the value of 360°. These are known as “Essential zeros”, butwhat can we do with “Rounded zeros” that are the result of below the detection limit ofthe equipment?Amalgamation, e.g. adding Na2O and K2O, as total alkalis is a solution, but sometimeswe need to differentiate between a sodic and a potassic alteration. Pre-classification intogroups requires a good knowledge of the distribution of the data and the geochemicalcharacteristics of the groups which is not always available. Considering the zero valuesequal to the limit of detection of the used equipment will generate spuriousdistributions, especially in ternary diagrams. Same situation will occur if we replace thezero values by a small amount using non-parametric or parametric techniques(imputation).The method that we are proposing takes into consideration the well known relationshipsbetween some elements. For example, in copper porphyry deposits, there is always agood direct correlation between the copper values and the molybdenum ones, but whilecopper will always be above the limit of detection, many of the molybdenum values willbe “rounded zeros”. So, we will take the lower quartile of the real molybdenum valuesand establish a regression equation with copper, and then we will estimate the“rounded” zero values of molybdenum by their corresponding copper values.The method could be applied to any type of data, provided we establish first theircorrelation dependency.One of the main advantages of this method is that we do not obtain a fixed value for the“rounded zeros”, but one that depends on the value of the other variable.Key words: compositional data analysis, treatment of zeros, essential zeros, roundedzeros, correlation dependency

Integrative analysis of RUNX1 downstream pathways and target genes.

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications

Altered glycogen metabolism in cultured astrocytes from mice with chronic glutathione deficit; relevance for neuroenergetics in schizophrenia.

Neurodegenerative and psychiatric disorders including Alzheimer's, Parkinson's or Huntington's diseases and schizophrenia have been associated with a deficit in glutathione (GSH). In particular, a polymorphism in the gene of glutamate cysteine ligase modulatory subunit (GCLM) is associated with schizophrenia. GSH is the most important intracellular antioxidant and is necessary for the removal of reactive by-products generated by the utilization of glucose for energy supply. Furthermore, glucose metabolism through the pentose phosphate pathway is a major source of NADPH, the cofactor necessary for the regeneration of reduced glutathione. This study aims at investigating glucose metabolism in cultured astrocytes from GCLM knockout mice, which show decreased GSH levels. No difference in the basal metabolism of glucose was observed between wild-type and knockout cells. In contrast, glycogen levels were lower and its turnover was higher in knockout astrocytes. These changes were accompanied by a decrease in the expression of the genes involved in its synthesis and degradation, including the protein targeting to glycogen. During an oxidative challenge induced by tert-Butylhydroperoxide, wild-type cells increased their glycogen mobilization and glucose uptake. However, knockout astrocytes were unable to mobilize glycogen following the same stress and they could increase their glucose utilization only following a major oxidative insult. Altogether, these results show that glucose metabolism and glycogen utilization are dysregulated in astrocytes showing a chronic deficit in GSH, suggesting that alterations of a fundamental aspect of brain energy metabolism is caused by GSH deficit and may therefore be relevant to metabolic dysfunctions observed in schizophrenia.

The challenge of modeling nuclear receptor regulatory networks in mammalian cells.

Nuclear receptors are a major component of signal transduction in animals. They mediate the regulatory activities of many hormones, nutrients and metabolites on the homeostasis and physiology of cells and tissues. It is of high interest to model the corresponding regulatory networks. While molecular and cell biology studies of individual promoters have provided important mechanistic insight, a more complex picture is emerging from genome-wide studies. The regulatory circuitry of nuclear receptor regulated gene expression networks, and their response to cellular signaling, appear highly dynamic, and involve long as well as short range chromatin interactions. We review how progress in understanding the kinetics and regulation of cofactor recruitment, and the development of new genomic methods, provide opportunities but also a major challenge for modeling nuclear receptor mediated regulatory networks.

ParA2, a Vibrio cholerae chromosome partitioning protein, forms left-handed helical filaments on DNA.

Most bacterial chromosomes contain homologs of plasmid partitioning (par) loci. These loci encode ATPases called ParA that are thought to contribute to the mechanical force required for chromosome and plasmid segregation. In Vibrio cholerae, the chromosome II (chrII) par locus is essential for chrII segregation. Here, we found that purified ParA2 had ATPase activities comparable to other ParA homologs, but, unlike many other ParA homologs, did not form high molecular weight complexes in the presence of ATP alone. Instead, formation of high molecular weight ParA2 polymers required DNA. Electron microscopy and three-dimensional reconstruction revealed that ParA2 formed bipolar helical filaments on double-stranded DNA in a sequence-independent manner. These filaments had a distinct change in pitch when ParA2 was polymerized in the presence of ATP versus in the absence of a nucleotide cofactor. Fitting a crystal structure of a ParA protein into our filament reconstruction showed how a dimer of ParA2 binds the DNA. The filaments formed with ATP are left-handed, but surprisingly these filaments exert no topological changes on the right-handed B-DNA to which they are bound. The stoichiometry of binding is one dimer for every eight base pairs, and this determines the geometry of the ParA2 filaments with 4.4 dimers per 120 A pitch left-handed turn. Our findings will be critical for understanding how ParA proteins function in plasmid and chromosome segregation.

Vitamin C requirements in parenteral nutrition.

Some biochemical functions of vitamin C make it an essential component of parenteral nutrition (PN) and an important therapeutic supplement in other acute conditions. Ascorbic acid is a strong aqueous antioxidant and is a cofactor for several enzymes. The average body pool of vitamin C is 1.5 g, of which 3%-4% (40-60 mg) is used daily. Steady state is maintained with 60 mg/d in nonsmokers and 140 mg/d in smokers. Shocked surgical, trauma, and septic patients have a drastic reduction of circulating plasma ascorbate concentrations. These low concentrations require 3-g doses/d to restore normal plasma ascorbate concentrations, questioning the recommended PN dose of 100 mg/d. Determination of intravenous requirements is usually based on plasma concentrations, which are altered during the inflammatory response. There is no clear indicator of deficiency: serum or plasma ascorbate concentrations <0.3 mg/dL (20 micromol/L) indicates inadequate vitamin C status. On the basis of available pharmacokinetic data the 100 mg/d dose for patients receiving home PN and 200 mg/d for stable adult patients receiving PN are adequate, but requirements have been shown to be higher in perioperative, trauma, burn, and critically ill patients, paralleling oxidative stress. One recommendation cannot fit all categories of patients. Large vitamin C supplements may be considered in severe critical illness, major trauma, and burns because of increased requirements resulting from oxidative stress and wound healing. Future research should distinguish therapeutic use of high-dose ascorbic acid antioxidant therapy from nutritional PN requirements.

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Summary.  Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.