Comparison of oxidative stress markers in HIV-infected patients on efavirenz or atazanavir/ritonavir-based therapy.

INTRODUCTION Chronic low-grade inflammation and immune activation may persist in HIV patients despite effective antiretroviral therapy (ART). These abnormalities are associated with increased oxidative stress (OS). Bilirubin (BR) may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UGT1A1, thus increasing unconjugated BR levels, a distinctive feature of this drug. We compared changes in OS markers in HIV patients on ATV/r versus efavirenz (EFV)-based first-line therapies. MATERIALS AND METHODS Cohort of the Spanish Research Network (CoRIS) is a multicentre, open, prospective cohort of HIV-infected patients naïve to ART at entry and linked to a biobank. We identified hepatitis C virus/hepatitis B virus (HCV/HBV) negative patients who started first-line ART with either ATV/r or EFV, had a baseline biobank sample and a follow-up sample after at least nine months of ART while maintaining initial regimen and being virologically suppressed. Lipoprotein-associated Phospholipase A2 (Lp-PLA2), Myeloperoxidase (MPO) and Oxidized LDL (OxLDL) were measured in paired samples. Marker values at one year were interpolated from available data. Multiple imputations using chained equations were used to deal with missing values. Change in the OS markers was modelled using multiple linear regressions adjusting for baseline marker values and baseline confounders. Correlations between continuous variables were explored using Pearson's correlation tests. RESULTS 145 patients (97 EFV; 48 ATV/r) were studied. Mean (SD) baseline values for OS markers in EFV and ATV/r groups were: Lp-PLA2 [142.2 (72.8) and 150.1 (92.8) ng/mL], MPO [74.3 (48.2) and 93.9 (64.3) µg/L] and OxLDL [76.3 (52.3) and 82.2 (54.4) µg/L]. After adjustment for baseline variables patients on ATV/r had a significant decrease in Lp-PLA2 (estimated difference -16.3 [CI 95%: -31.4, -1.25; p=0.03]) and a significantly lower increase in OxLDL (estimated difference -21.8 [-38.0, -5.6; p<0.01] relative to those on EFV, whereas no differences in MPO were found. Adjusted changes in BR were significantly higher for the ATV/r group (estimated difference 1.33 [1.03, 1.52; p<0.01]). Changes in BR and changes in OS markers were significantly correlated. CONCLUSIONS In virologically suppressed patients on stable ART, OS was lower in ATV/r-based regimens compared to EFV. We hypothesize these changes could be in part attributable to increased BR plasma levels.

Association of irisin with fat mass, resting energy expenditure, and daily activity in conditions of extreme body mass index.

FNDC5/irisin has been recently postulated as beneficial in the treatment of obesity and diabetes because it is induced in muscle by exercise, increasing energy expenditure. However, recent reports have shown that WAT also secretes irisin and that circulating irisin is elevated in obese subjects. The aim of this study was to evaluate irisin levels in conditions of extreme BMI and its correlation with basal metabolism and daily activity. The study involved 145 female patients, including 96 with extreme BMIs (30 anorexic (AN) and 66 obese (OB)) and 49 healthy normal weight (NW). The plasma irisin levels were significantly elevated in the OB patients compared with the AN and NW patients. Irisin also correlated positively with body weight, BMI, and fat mass. The OB patients exhibited the highest REE and higher daily physical activity compared with the AN patients but lower activity compared with the NW patients. The irisin levels were inversely correlated with daily physical activity and directly correlated with REE. Fat mass contributed to most of the variability of the irisin plasma levels independently of the other studied parameters. Conclusion. Irisin levels are influenced by energy expenditure independently of daily physical activity but fat mass is the main contributing factor.

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

CB1 blockade potentiates down-regulation of lipogenic gene expression in perirenal adipose tissue in high carbohydrate diet-induced obesity.

De novo lipogenesis and hypercaloric diets are thought to contribute to increased fat mass, particularly in abdominal fat depots. CB1 is highly expressed in adipose tissue, and CB1-mediated signalling is associated with stimulation of lipogenesis and diet-induced obesity, though its contribution to increasing fat deposition in adipose tissue is controversial. Lipogenesis is regulated by transcription factors such as liver X receptor (LXR), sterol-response element binding protein (SREBP) and carbohydrate-responsive-element-binding protein (ChREBP). We evaluated the role of CB1 in the gene expression of these factors and their target genes in relation to lipogenesis in the perirenal adipose tissue (PrAT) of rats fed a high-carbohydrate diet (HCHD) or a high-fat diet (HFD). Both obesity models showed an up-regulated gene expression of CB1 and Lxrα in this adipose pad. The Srebf-1 and ChREBP gene expressions were down-regulated in HFD but not in HCHD. The expression of their target genes encoding for lipogenic enzymes showed a decrease in diet-induced obesity and was particularly dramatic in HFD. In HCHD, CB1 blockade by AM251 reduced the Srebf-1 and ChREBP expression and totally abrogated the remnant gene expression of their target lipogenic enzymes. The phosphorylated form of the extracellular signal-regulated kinase (ERK-p), which participates in the CB1-mediated signalling pathway, was markedly present in the PrAT of obese rats. ERK-p was drastically repressed by AM251 indicating that CB1 is actually functional in PrAT of obese animals, though its activation loses the ability to stimulate lipogenesis in PrAT of obese rats. Even so, the remnant expression levels of lipogenic transcription factors found in HCHD-fed rats are still dependent on CB1 activity. Hence, in HCHD-induced obesity, CB1 blockade may help to further potentiate the reduction of lipogenesis in PrAT by means of inducing down-regulation of the ChREBP and Srebf-1 gene expression, and consequently in the expression of lipogenic enzymes.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

Disruption of GIP/GIPR axis in human adipose tissue is linked to obesity and insulin resistance.

CONTEXT Glucose-dependent insulinotropic peptide (GIP) has a central role in glucose homeostasis through its amplification of insulin secretion; however, its physiological role in adipose tissue is unclear. OBJECTIVE Our objective was to define the function of GIP in human adipose tissue in relation to obesity and insulin resistance. DESIGN GIP receptor (GIPR) expression was analyzed in human sc adipose tissue (SAT) and visceral adipose (VAT) from lean and obese subjects in 3 independent cohorts. GIPR expression was associated with anthropometric and biochemical variables. GIP responsiveness on insulin sensitivity was analyzed in human adipocyte cell lines in normoxic and hypoxic environments as well as in adipose-derived stem cells obtained from lean and obese patients. RESULTS GIPR expression was downregulated in SAT from obese patients and correlated negatively with body mass index, waist circumference, systolic blood pressure, and glucose and triglyceride levels. Furthermore, homeostasis model assessment of insulin resistance, glucose, and G protein-coupled receptor kinase 2 (GRK2) emerged as variables strongly associated with GIPR expression in SAT. Glucose uptake studies and insulin signaling in human adipocytes revealed GIP as an insulin-sensitizer incretin. Immunoprecipitation experiments suggested that GIP promotes the interaction of GRK2 with GIPR and decreases the association of GRK2 to insulin receptor substrate 1. These effects of GIP observed under normoxia were lost in human fat cells cultured in hypoxia. In support of this, GIP increased insulin sensitivity in human adipose-derived stem cells from lean patients. GIP also induced GIPR expression, which was concomitant with a downregulation of the incretin-degrading enzyme dipeptidyl peptidase 4. None of the physiological effects of GIP were detected in human fat cells obtained from an obese environment with reduced levels of GIPR. CONCLUSIONS GIP/GIPR signaling is disrupted in insulin-resistant states, such as obesity, and normalizing this function might represent a potential therapy in the treatment of obesity-associated metabolic disorders.

Estudio de la albúmina sérica y del índice de masa corporal como marcadores nutricionales en pacientes en hemodiálisis.

Background: Protein calorie malnutrition as well as systemic inflammation and metabolic disorders are common among patients with chronic renal failure undergoing renal replacement therapy (haemodialysis), which contributes to its morbidity and mortality. Aims: The aims of this work was to evaluate the nutritional status of patients in a hemodialysis treatment through the assessment of biochemical parameters nutritional as albumin, and anthropometric parameters of body mass index during ten years of follow up. Methods: In this work has been followed 90 patients of both sexes with chronic kidney disease who were treated with hemodialysis regularly on our unit for ten years. All patients were conducted quarterly measurements of plasma albumin (Alb), and other biochemical determinations, and anthropometric measurements of height, weight and body mass index calculated by the formula weight/height², grouped n BMI < 23 kg/m2 and albumin levels <3.8 g/dl according to the consensus of the panel of experts of the International Society for renal Nutrition and metabolism. Results: During the 10 years all patients showed a significant decline in the biochemical parameters and the albumin, change in BMI does not presented significant changes in relation to malnutrition. Conclusions: Malnutrition in patients on dialysis is a fact patent, BMI does not correspond with the biochemical parameters were observed, for what nutritional impairment in these patients is mainly expressed by serum albumin.

Assessing cardiovascular risk in hepatitis C: An unmet need.

Chronic hepatitis C virus (HCV) is associated with significant morbidity and mortality, as a result of the progression towards cirrhosis and hepatocellular carcinoma. Additionally, HCV seems to be an independent risk factor for cardiovascular diseases (CVD) due to its association with insulin resistance, diabetes and steatosis. HCV infection represents an initial step in the chronic inflammatory cascade, showing a direct role in altering glucose metabolism. After achieving sustained virological response, the incidence of insulin resistance and diabetes dramatically decrease. HCV core protein plays an essential role in promoting insulin resistance and oxidative stress. On the other hand, atherosclerosis is a common disease in which the artery wall thickens due to accumulation of fatty deposits. The main step in the formation of atherosclerotic plaques is the oxidation of low density lipoprotein particles, together with the increased production of proinflammatory markers [tumor necrosis factor-α, interleukin (IL)-6, IL-18 or C-reactive protein]. The advent of new direct acting antiviral therapy has dramatically increased the sustained virological response rates of hepatitis C infection. In this scenario, the cardiovascular risk has emerged and represents a major concern after the eradication of the virus. Consequently, the number of studies evaluating this association is growing. Data derived from these studies have demonstrated the strong link between HCV infection and the atherogenic process, showing a higher risk of coronary heart disease, carotid atherosclerosis, peripheral artery disease and, ultimately, CVD-related mortality.

Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.

Alpha-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and alpha-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His6-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the alpha-ketoglutarate-dependent dioxygenases and share the specific motif HXDX(24)TX(131)HX(10)R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent Km values were 99 microM for (R)-mecoprop, 164 microM for (R)-dichlorprop, and 3 microM for alpha-ketoglutarate for RdpA and 132 microM for (S)-mecoprop, 495 microM for (S)-dichlorprop, and 20 microM for alpha-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 microM for SdpA and >230 microM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace alpha-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAalpha-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Plasmacytoid dendritic cells in the skin: to sense or not to sense nucleic acids.

Plasmacytoid dendritic cells (pDCs) are specialized sensors of viral nucleic acids that initiate protective immunity through the production of type I interferons (IFNs). Normally, pDCs fail to sense host-derived self-nucleic acids but do so when self-nucleic acids form complexes with endogenous antimicrobial peptides produced in damaged skin. Whereas regulated expression of antimicrobial peptides may lead to pDC activation and protective immune responses to skin injury, overexpression of antimicrobial peptides in psoriasis drives excessive sensing of self-nucleic acids by pDCs resulting in IFN-driven autoimmunity. In skin tumors, pDCs are unable to sense self-nucleic acids; however, therapeutic activation of pDCs by synthetic nucleic acids or analogues can be exploited to generate antitumor immunity.

Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors.

Three novel members of the Xenopus nuclear hormone receptor superfamily have been cloned. They are related to each other and similar to the group of receptors that includes those for thyroid hormones, retinoids, and vitamin D3. Their transcriptional activity is regulated by agents causing peroxisome proliferation and carcinogenesis in rodent liver. All three Xenopus receptors activate the promoter of the acyl coenzyme A oxidase gene, which encodes the key enzyme of peroxisomal fatty acid beta-oxidation, via a cognate response element that has been identified. Therefore, peroxisome proliferators may exert their hypolipidemic effects through these receptors, which stimulate the peroxisomal degradation of fatty acids. Finally, the multiplicity of these receptors suggests the existence of hitherto unknown cellular signaling pathways for xenobiotics and putative endogenous ligands.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans.

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ∆foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ∆scdA∆echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ∆scdA∆echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.