Analysis of MLL2 gene in the first Brazilian family with Kabuki syndrome

Most patients with Kabuki syndrome (KS) are the only person in their family with the condition. However, familial cases of KS have been described showing evidence that this syndrome can be inherited as a dominant trait with variable expressivity. We report on two related individuals with facial findings characteristic of KS. The proposita had arched eyebrows, long and upward slanting palpebral fissures, cleft lip and palate, retromicrognathia, brachydactyly of hands and feet, stubby fingers, nail hypoplasia, and prominent finger pads. Her mother had eyebrows with dispersed lateral half, long and upward slanting palpebral fissures, retrognathia, abnormal and posteriorly rotated ears, prominent finger pads, brachydactyly of feet, learning difficulties, and psychomotor development delay. DNA sequencing revealed a novel missense mutation in the MLL2 gene in both the proposita and her mother. The mutation (p.R5432Q) was found in the exon 51, within the SET domain of the gene, which confers methyltransferase activity on the protein. Therefore, the epigenetic and transcriptional regulatory properties of this protein may be altered and this suggests that the mutation is the cause of phenotype observed in both the patient and her mother. The clinical signs and the molecular evidence in this family further support the notion that KS is an autosomal dominant condition with variable expressivity. To our knowledge this is the first report of a Brazilian family with recurrence of this syndrome. (C) 2012 Wiley Periodicals, Inc.

The I405V and Taq1B polymorphisms of the CETP gene differentially affect sub-clinical carotid atherosclerosis

Background: Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample. Methods: The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-a concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography. Results: No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed. Conclusions: None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of gamma-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas. Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4 mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation. (C) 2012 Elsevier B.V. All rights reserved.

Administration of a murine diet supplemented with conjugated linoleic acid increases the expression and activity of hepatic uncoupling proteins

Daily intake of conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and to increase body metabolism; this latter effect has been often associated with the up-regulation of uncoupling proteins (UCPs). Here we addressed the effects of a CLA-supplemented murine diet (similar to 2 % CLA mixture, cis-9, trans-10 and trans-10, cis-12 isomers; 45 % of each isomer on alternating days) on mitochondrial energetics, UCP2 expression/activity in the liver and other associated morphological and functional parameters, in C57BL/6 mice. Diet supplementation with CLA reduced both lipid accumulation in adipose tissues and triacylglycerol plasma levels, but did not augment hepatic lipid storage. Livers of mice fed a diet supplemented with CLA showed high UCP2 mRNA levels and the isolated hepatic mitochondria showed indications of UCP activity: in the presence of guanosine diphosphate, the higher stimulation of respiration promoted by linoleic acid in mitochondria from the CLA mice was almost completely reduced to the level of the stimulation from the control mice. Despite the increased generation of reactive oxygen species through oxi-reduction reactions involving NAD(+)/NADH in the Krebs cycle, no oxidative stress was observed in the liver. In addition, in the absence of free fatty acids, basal respiration rates and the phosphorylating efficiency of mitochondria were preserved. These results indicate a beneficial and secure dose of CLA for diet supplementation in mice, which induces UCP2 overexpression and UCP activity in mitochondria while preserving the lipid composition and redox state of the liver.

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Diversity of the KIR gene cluster in an urban Brazilian population

The activity of natural killer cells depends on the balance between activating and inhibitory signals coming from their receptors. Among these are the killer cell immunoglobulin-like receptors (KIR) that recognize specific HLA class I allotypes. Here we characterized KIR genetic diversity and their HLA ligands in the population of Curitiba, Parana State (n = 164), and compared it with other worldwide populations. The distribution of 2DL4 alleles was also analyzed. The Curitiba population did not differ significantly from European and Euro-descendant populations, but as an admixed population showed higher genetic diversity. We found 27 KIR profiles, many of them uncommon in European populations, in agreement with the elevated historically recent gene flow in the study population. The frequencies of KIR genes and their respective HLA ligands were distributed independently and none of the analyzed individuals lacked functional KIR-HLA ligand combinations. KIR gene frequencies of 33 worldwide populations were consistent with geographic and ethnic distribution, in agreement with demography being the major factor shaping the observed gene content diversity of the KIR locus.

Monocyte Chemoattractant-1 as a Urinary Biomarker for the Diagnosis of Activity of Lupus Nephritis in Brazilian Patients

Objective. Monocyte chemotactic protein (MCP-1), involved in the pathogenesis of lupus nephritis (LN), has recently been indicated as a new biomarker of kidney activity in systemic lupus erythematosus (SLE). Our aim was to assess urinary MCP-1 (uMCP-1) as a biomarker of renal activity in patients with SLE and to compare it to other disease activity markers, using the ELISA. Methods. Seventy-five female Brazilian patients with SLE and a control group participated in our study. Patients with SLE were distributed among 3 groups according to kidney involvement and classified according to disease activity based on clinical and laboratory measures such as urinary sediment, proteinuria, kidney function, C3, C4, anti-dsDNA, disease activity index, and renal SLE disease activity index. The serum and uMCP-1 concentrations were measured by sandwich ELISA. Results. In the A-LN group (active lupus nephritis: SLE with kidney involvement), the concentration of uMCP-1 was significantly higher than in other groups. A cutoff point was established using the results of the control group to apply this test in the detection of LN. A-LN had a higher frequency of positive results for uMCP-1 in comparison to the other groups (p < 0.001). To detect disease activity in patients with LN, a new cutoff was determined based on the results of patients with SLE with kidney involvement. Setting specificity at 90%, the sensitivity of the test was 50%. Conclusion. The high specificity makes uMCP-1 a useful test as a predictor of kidney activity in SLE, especially when associated to other measures used in clinical practice. (First Release Sept 1 2012; J Rheumatol 2012;39:1948-54; doi :10.3899/jrheum.110201)

Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population

de Oliveira Alvim R, Lima Santos PCJ, Goncalves Dias R, Rodrigues MV, de Sa Cunha R, Mill JG, Junior WN, Krieger JE, Pereira AC. Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population. Physiol Genomics 44: 587-592, 2012. First published April 10, 2012; doi:10.1152/physiolgenomics.00122.2011.-NADPH oxidase p22phox subunit is responsible for the production of reactive oxygen species in the vascular tissue. The C242T polymorphism in the p22phox gene has been associated with diverse coronary artery disease phenotypes, but the findings about the protective or harmful effects of the T allele are still controversial. Our main aim was to assess the effect of p22phox C242T genotypes on arterial stiffness, a predictor of late morbidity and mortality, in individuals from the general population. We randomly selected 1,178 individuals from the general population of Vitoria City, Brazil. Genotypes for the C242T polymorphism were detected by PCR-RFLP, and pulse wave velocity (PWV) values were measured with a noninvasive automatic device Complior. p22phox and TNF-alpha gene expression were quantified by real-time PCR in human arterial mammary smooth muscle cells. In both the entire and nonhypertensive groups: individuals carrying the TT genotype had higher PWV values and higher risk for increased arterial stiffness [odds ratio (OR) 1.93, 95% confidence interval (CI) 1.27-2.92 and OR 1.78, 95% CI 1.07-2.95, respectively] compared with individuals carrying CC + CT genotypes, even after adjustment for covariates. No difference in the p22phox gene expression according C242T genotypes was observed. However, TNF-alpha gene expression was higher in cells from individual carrying the T allele, suggesting that this genetic marker is associated with functional phenotypes at the gene expression level. In conclusion, we suggest that p22phox C242T polymorphism is associated with arterial stiffness evaluated by PWV in the general population. This genetic association shed light on the understanding of the genetic modulation on vascular dysfunction mediated by NADPH oxidase.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Correlations of CTLA-4 gene polymorphisms and hepatitis C chronic infection

Background: Cytotoxic T lymphocyte-associated factor 4 (CTLA-4) functions as a negative regulator of T cell-mediated immune response. Molecular changes associated to CTLA-4 gene polymorphisms could reduce its ability to suppress and control lymphocyte proliferation. Aims: To evaluate the frequency of CTLA-4 gene polymorphisms in chronic hepatitis C virus (HCV) infected patients and correlate to clinical and histological findings. Methods: We evaluated 112 HCV-infected subjects prospectively selected and 183 healthy controls. Clinical and liver histological data were analysed. - 318C > T, A49G and CT60 CTLA-4 single-nucleotide polymorphisms (SNPs) were studied by PCR-RFLP and AT(n) polymorphism by DNA fragment analysis by capillary electrophoresis in automatic sequencer. Results: Eight AT repetitions in 3' UTR region were more frequent in HCV-infected subjects. We found a positive association of -318C and + 49G with HCV genotype 3 (P = 0.008, OR 9.13, P = 0.004, OR 2.49 respectively) and an inverse association of both alleles with HCV genotype 1 (P = 0.020, OR 0.19, P = 0.002, OR 0.38 respectively). Allele + 49G was also associated to aminotransferases quotients > 3 (qALT, P = 0.034, qAST, P = 0.041). Allele G of CT60 SNP was also associated with qAST > 3 (P = 0.012). Increased number of AT repetitions was positively associated to severe necroinflammatory activity scores in liver biopsies (P = 0.045, OR 4.62). Conclusion: CTLA-4 gene polymorphisms were associated to HCVinfection. Eight AT repetitions were more prevalent in HCV-infected subjects. - 318C and + 49G alleles were associated to genotypes 1 and 3 infections and increased number of AT repetitions in 3' UTR region favoured severe necroinflammatory activity scores in liver biopsies.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Suppression of Hypoxia-Inducible Factor-1 alpha Contributes to the Antiangiogenic Activity of Red Propolis Polyphenols in Human Endothelial Cells

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1 alpha (HIF1 alpha) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent mariner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1 alpha protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1 alpha protein half-life from similar to 58 min to 38 min under hypoxic conditions. The reduced HIF1 alpha protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1 alpha. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1 alpha. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1 alpha protein accumulation and signaling. J. Nutr. 142: 441-447, 2012.

Effect of ultraviolet filters on skin superoxide dismutase activity in hairless mice after a single dose of ultraviolet radiation

Organic sunscreens may decrease their protective capability and also behave as photo-oxidants upon ultraviolet radiation (UVR) exposure. The present study investigated the effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm(2)). The retention of these UV filters was first evaluated in vivo using hairless mice to guarantee the presence of the filters in the skin layers at the moment of irradiation. The in vivo effect of the UV filters on skin SOD was then assayed spectrophotometrically via the reduction of cytochrome c. The cream gel formulation promoted the penetration of the three UV filters into the epidermis and the dermis at one hour post-application. A significant decrease in SOD activity was observed in irradiated animals treated with sunscreen formulation. However, no effect on SOD activity in skin was observed by the isolated presence of the sunscreens, the formulation components, or the exposure to UVR. The sunscreens may have formed degradation products under UVR that may have either inhibited the enzyme or generated reactive species in the skin. (C) 2011 Elsevier B.V. All rights reserved.

Liposuction induces a compensatory increase of visceral fat which is effectively counteracted by physical activity: a randomized trial

Context: Liposuction is suggested to result in long-term body fat regain that could lead to increased cardiometabolic risk. We hypothesized that physical activity could prevent this effect. Objective: Our objective was to investigate the effects of liposuction on body fat distribution and cardiometabolic risk factors in women who were either exercise trained or not after surgery. Design, Setting, and Participants: Thirty-six healthy normal-weight women participated in this 6-month randomized controlled trial at the University of Sao Paulo, Sao Paulo, Brazil. Interventions: Patients underwent a small-volume abdominal liposuction. Two months after surgery, the subjects were randomly allocated into two groups: trained (TR, n = 18, 4-month exercise program) and nontrained (NT, n = 18). Main Outcome Measures: Body fat distribution (assessed by computed tomography) was assessed before the intervention (PRE) and 2 months (POST2), and 6 months (POST6) after surgery. Secondary outcome measures included body composition, metabolic parameters and dietary intake, assessed at PRE, POST2, and POST6, and total energy expenditure, physical capacity, and sc adipocyte size and lipid metabolism-related gene expression, assessed at PRE and POST6. Results: Liposuction was effective in reducing sc abdominal fat (PRE vs. POST2, P = 0.0001). Despite the sustained sc abdominal fat decrement at POST6 (P = 0.0001), the NT group showed a significant 10% increase in visceral fat from PRE to POST6 (P = 0.04; effect size = -0.72) and decreased energy expenditure (P = 0.01; effect size = 0.95) when compared with TR. Dietary intake, adipocyte size, and gene expression were unchanged over time. Conclusion: Abdominal liposuction does not induce regrowth of fat, but it does trigger a compensatory increase of visceral fat, which is effectively counteracted by physical activity. (J Clin Endocrinol Metab 97: 2388-2395, 2012)