Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma

Background Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. Methods This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. Results The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. Conclusion The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. (c) 2011 Wiley Periodicals, Inc. Head Neck, 2012

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

The contribution of HGAL/GCET2 in immunohistological algorithms: a comparative study in 424 cases of nodal diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma can be subclassified into at least two molecular subgroups by gene expression profiling: germinal center B-cell like and activated B-cell like diffuse large B-cell lymphoma. Several immunohistological algorithms have been proposed as surrogates to gene expression profiling at the level of protein expression, but their reliability has been an issue of controversy. Furthermore, the proportion of misclassified cases of germinal center B-cell subgroup by immunohistochemistry, in all reported algorithms, is higher compared with germinal center B-cell cases defined by gene expression profiling. We analyzed 424 cases of nodal diffuse large B-cell lymphoma with the panel of markers included in the three previously described algorithms: Hans, Choi, and Tally. To test whether the sensitivity of detecting germinal center B-cell cases could be improved, the germinal center B-cell marker HGAL/GCET2 was also added to all three algorithms. Our results show that the inclusion of HGAL/GCET2 significantly increased the detection of germinal center B-cell cases in all three algorithms (P<0.001). The proportions of germinal center B-cell cases in the original algorithms were 27%, 34%, and 19% for Hans, Choi, and Tally, respectively. In the modified algorithms, with the inclusion of HGAL/GCET2, the frequencies of germinal center B-cell cases were increased to 38%, 48%, and 35%, respectively. Therefore, HGAL/GCET2 protein expression may function as a marker for germinal center B-cell type diffuse large B-cell lymphoma. Consideration should be given to the inclusion of HGAL/GCET2 analysis in algorithms to better predict the cell of origin. These findings bear further validation, from comparison to gene expression profiles and from clinical/therapeutic data. Modern Pathology (2012) 25, 1439-1445; doi: 10.1038/modpathol.2012.119; published online 29 June 2012

Interactions between the NO-Citrulline Cycle and Brain-derived Neurotrophic Factor in Differentiation of Neural Stem Cells

The diffusible messenger NO plays multiple roles in neuroprotection, neurodegeneration, and brain plasticity. Argininosuccinate synthase (AS) is a ubiquitous enzyme in mammals and the key enzyme of the NO-citrulline cycle, because it provides the substrate L-arginine for subsequent NO synthesis by inducible, endothelial, and neuronal NO synthase (NOS). Here, we provide evidence for the participation of AS and of the NO-citrulline cycle in the progress of differentiation of neural stem cells (NSC) into neurons, astrocytes, and oligodendrocytes. AS expression and activity and neuronal NOS expression, as well as L-arginine and NOx production, increased along neural differentiation, whereas endothelial NOS expression was augmented in conditions of chronic NOS inhibition during differentiation, indicating that this NOS isoform is amenable to modulation by extracellular cues. AS and NOS inhibition caused a delay in the progress of neural differentiation, as suggested by the decreased percentage of terminally differentiated cells. On the other hand, BDNF reversed the delay of neural differentiation of NSC caused by inhibition of NOx production. Alikely cause is the lack of NO, which up-regulated p75 neurotrophin receptor expression, a receptor required for BDNF-induced differentiation of NSC. We conclude that the NO-citrulline cycle acts together with BDNF for maintaining the progress of neural differentiation.

The methylation patterns of the IGF2 and IGF2R genes in bovine spermatozoa are not affected by flow-cytometric sex sorting

The objectives of this study were to investigate the effect of sexing by flow cytometry on the methylation patterns of the IGF2 and IGF2R genes. Frozen-thawed, unsorted, and sex-sorted sperm samples from four Nellore bulls were used. Each ejaculate was separated into three fractions: non-sexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Sperm were isolated from the extender, cryoprotectant, and other cell types by centrifugation on a 40:70% Percoll gradient, and sperm pellets were used for genomic DNA isolation. DNA was used for analyses of the methylation patterns by bisulfite sequencing. Methylation status of the IGF2 and IGF2R genes were evaluated by sequencing 195 and 147 individual clones, respectively. No global differences in DNA methylation were found between NS, SX, and SY groups for the IGF2 (P=0.09) or IGF2R genes (P=0.38). Very specific methylation patterns were observed in the 25th and 26th CpG sites in the IGF2R gene. representing higher methylation in NS than in the SX and SY groups compared with the other CpG sites. Further, individual variation in methylation patterns was found among bulls. In conclusion, the sex-sorting procedure by flow cytometry did not affect the overall DNA methylation patterns of the IGF2 and IGF2R genes, although individual variation in their methylation patterns among bulls was observed. Mol. Reprod. Dev. 79:7784, 2012. (C) 2011 Wiley Periodicals, Inc.

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs. (c) 2012 Elsevier B.V. All rights reserved.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

Regulatory variation in a TBX5 enhancer leads to isolated congenital heart disease

Recent studies have identified the genetic underpinnings of a growing number of diseases through targeted exome sequencing. However, this strategy ignores the large component of the genome that does not code for proteins, but is nonetheless biologically functional. To address the possible involvement of regulatory variation in congenital heart diseases (CHDs), we searched for regulatory mutations impacting the activity of TBX5, a dosage-dependent transcription factor with well-defined roles in the heart and limb development that has been associated with the HoltOram syndrome (hearthand syndrome), a condition that affects 1/100 000 newborns. Using a combination of genomics, bioinformatics and mouse genetic engineering, we scanned approximate to 700 kb of the TBX5 locus in search of cis-regulatory elements. We uncovered three enhancers that collectively recapitulate the endogenous expression pattern of TBX5 in the developing heart. We re-sequenced these enhancer elements in a cohort of non-syndromic patients with isolated atrial and/or ventricular septal defects, the predominant cardiac defects of the HoltOram syndrome, and identified a patient with a homozygous mutation in an enhancer approximate to 90 kb downstream of TBX5. Notably, we demonstrate that this single-base-pair mutation abrogates the ability of the enhancer to drive expression within the heart in vivo using both mouse and zebrafish transgenic models. Given the population-wide frequency of this variant, we estimate that 1/100 000 individuals would be homozygous for this variant, highlighting that a significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the HoltOram syndrome.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50 mu M of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circulardichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized. (C) 2012 Elsevier Inc. All rights reserved.

Time course involvement of matrix metalloproteinases in the vascular alterations of renovascular hypertension

Increased vascular matrix metalloproteinases (MMPs) levels play a role in late phases of hypertensive vascular remodeling. However, no previous study has examined the time course of MMPs in the various phases of two-kidney, one-clip hypertension (2K1C). We examined structural vascular changes, collagen and elastin content, vascular oxidative stress, and MMPs levels/activities during the development of 2K1C hypertension. Plasma angiotensin converting enzyme (ACE) activity was measured to assess renin-angiotensin system activation. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10 weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NADPH activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. 2K1C hypertension was associated with increased ACE activity, which decreased to normal after 10 weeks. We found increased aortic collagen and elastin content in the early phase of hypertension, which were associated with vascular hypertrophy, increased vascular MMP-2 and MMP-14 (but not TIMP-2) levels, and increased gelatinolytic activity, possibly as a result of increased vascular NADPH oxidase activity and oxidative stress. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with early increases in MMPs activities, enhanced matrix deposition and oxidative stress. Using antioxidants or MMPs inhibitors in the early phase of hypertension may prevent the vascular alterations of hypertension. (C) 2012 Elsevier B.V. All rights reserved.

Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone

We aimed to investigate the possible role of creatine (CR) supplementation in counteracting dexamethasone-induced muscle wasting and insulin resistance in rats. Also, we examined whether CR intake would modulate molecular pathways involved in muscle remodeling and insulin signaling. Animals were randomly divided into four groups: (1) dexamethasone (DEX); (2) control pair-fed (CON-PF); (3) dexamethasone plus CR (DEX-CR); and (4) CR pair-fed (CR-PF). Dexamethasone (5 mg/kg/day) and CR (5 g/kg/day) were given via drinking water for 7 days. Plantaris and extensor digitorum longus (EDL) muscles were removed for analysis. Plantaris and EDL muscle mass were significantly reduced in the DEX-CR and DEX groups when compared with the CON-PF and CR-PF groups (P < 0.05). Dexamethasone significantly decreased phospho-Ser(473)-Akt protein levels compared to the CON-PF group (P < 0.05) and CR supplementation aggravated this response (P < 0.001). Serum glucose was significantly increased in the DEX group when compared with the CON-PF group (DEX 7.8 +/- A 0.6 vs. CON-PF 5.2 +/- A 0.5 mmol/l; P < 0.05). CR supplementation significantly exacerbated hyperglycemia in the dexamethasone-treated animals (DEX-CR 15.1 +/- A 2.4 mmol/l; P < 0.05 vs. others). Dexamethasone reduced GLUT-4 translocation when compared with the CON-PF and CR-PF (P < 0.05) groups and this response was aggravated by CR supplementation (P < 0.05 vs. others). In conclusion, supplementation with CR resulted in increased insulin resistance and did not attenuate muscle wasting in rats treated with dexamethasone. Given the contrast with the results of human studies that have shown benefits of CR supplementation on muscle atrophy and insulin sensitivity, we suggest caution when extrapolating this animal data to human subjects.

Microcystin-producing genotypes from cyanobacteria in Brazilian reservoirs

The aim of this study was to evaluate the use of new oligonucleotide primers (mcyB-F/R, mcyB-F/R-A, and mcyB-F/R-B) designed from Brazilian cyanobacteria for the detection of microcystin-producing genotypes in 27 environmental samples from water reservoirs and 11 strains of Microcystis. Microcystins were found using HPLC in all 11 strains and 19 of the environmental samples. The new oligonucleotide primers amplified fragments of microcystin-producing genes, including the eight environmental samples in which no microcystins were detected by HPLC, but which presented amplified fragments, thereby demonstrating the existence of microcystin-producing genes. The new oligonucleotide primers exhibited better specificity when used with environmental samples and were more reliable in comparison with those described in the literature (mcyB-FAA/RAA and mcyA-Cd/FR), which generate false-negative results. The better performance of these new oligonucleotide primers underline the need for designing molecular markers that are well fitted to the regional biological diversity. As this is a fast predictive technique for determining the presence or absence of microcystins, it could be used either alone or in conjunction with other techniques, such as the screening of samples to be sent for quantitative toxicological analysis using HPLC, thereby reducing monitoring cost and time. (c) 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Mutations in the Mitochondrial Methionyl-tRNA Synthetase Cause a Neurodegenerative Phenotype in Flies and a Recessive Ataxia (ARSAL) in Humans

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.

The COP9 signalosome counteracts the accumulation of cullin SCF ubiquitin E3 RING ligases during fungal development

Defects in the COP9 signalosome (CSN) impair multicellular development, including embryonic plant or animal death or a block in sexual development of the fungus Aspergillus nidulans. CSN deneddylates cullin-RING ligases (CRLs), which are activated by covalent linkage to ubiquitin-like NEDD8. Deneddylation allows CRL disassembly for subsequent reassembly. An attractive hypothesis is a consecutive order of CRLs for development, which demands repeated cycles of neddylation and deneddylation for reassembling CRLs. Interruption of these cycles could explain developmental blocks caused by csn mutations. This predicts an accumulation of neddylated CRLs exhibiting developmental functions when CSN is dysfunctional. We tested this hypothesis in A. nidulans, which tolerates reduced levels of neddylation for growth. We show that only genes for CRL subunits or neddylation are essential, whereas CSN is primarily required for development. We used functional tagged NEDD8, recruiting all three fungal cullins. Cullins are associated with the CSN1/CsnA subunit when deneddylation is defective. Two CRLs were identified which are specifically involved in differentiation and accumulate during the developmental block. This suggests that an active CSN complex is required to counteract the accumulation of specific CRLs during development.