TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and-7

Caspases are central players in proteolytic pathways that regulate cellular processes Such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAH15 as a novel caspase Substrate in a trial Study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence (106)DQPD/Y(110) as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells. (C) 2009 Elsevier Inc. All rights reserved.

Low CD4(+) T-Cell Levels and B-Cell Apoptosis in Vertically HIV-exposed Noninfected Children and Adolescents

Lymphocyte subsets, activation markers and apoptosis were assessed in 20 HIV-exposed noninfected (ENI) children born to HIV-infected women who were or not exposed to antiretroviral (ARV) drugs during pregnancy and early infancy. ENI children and adolescents were aged 6-18 years and they were compared to 25 age-matched healthy non-HIV-exposed children and adolescents (Control). ENI individuals presented lower CD4(+) T cells/mm(3) than Control group (control: 1120.3 vs. ENI: 876.3; t-test, p=0.030). ENI individuals had higher B-cell apoptosis than Control group (Control: 36.6%, ARV exposed: 82.3%, ARV nonexposed: 68.5%; Kruskal-Wallis, p < 0.05), but no statistical difference was noticed between those exposed and not exposed to ARV. Immune activation in CD4(+) T, CD8(+) T and in B cells was comparable in ENI and in Control children and adolescents. Subtle long-term immune alterations might persist among ENI individuals, but the clinical consequences if any are unknown, and these children require continued monitoring.

Holoprosencephaly and Holoprosencephaly-Like Phenotypes: Review of Facial and Molecular Findings in Patients From a Craniofacial Hospital in Brazil

Here we report on the clinical and genetic data for a large sample of Brazilian patients studied at the Hospital de Reabilitacao de Anomalas Craniofaciais-Universidade de Sao Paulo (HRAC-USP) who presented with either the classic holoprosencephaly or the holoprosencephaly-like (HPE-L) phenotype. The sample included patients without detected mutations in some HPE determinant genes such as SHH, GLI2, SIX3, TGIF, and PTCH, as well as the photographic documentation of the previously reported patients in our Center. The HPE-L phenotype has been also called of HPE ``minor forms"" or ""microforms,"" The variable phenotype, the challenge of genetic counseling, and the similarities to patients with isolated cleft lip/palate are discussed. (c) 2010 Wiley-Liss, Inc.

Group IV Graphene- and Graphane-Like Nanosheets

We performed a first-principles investigation on the structural and electronic properties of group IV (C, SiC, Si, Ge, and Sn) graphene-like sheets in flat and buckled configurations and the respective hydrogenated or fluorinated graphane-like ones. The analysis on the energetics, associated with the formation of those structures, showed that fluorinated graphane-like sheets are very stable and should be easily synthesized in the laboratory. We also studied the changes of the properties of the graphene-like sheets as a result of hydrogenation or fluorination. The interatomic distances in those graphane-like sheets are consistent with the respective crystalline ones, a property that may facilitate integration of those sheets within three-dimensional nanodevices.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Synthesis, Structure, and Phosphatase-Like Activity of a New Trinuclear Gd Complex with the Unsymmetrical Ligand H(3)L As a Model for Nucleases

The new trinuclear gadolinium complex [Gd(3)L(2)(NO(3))(2)(H(2)O)(4)]NO(3)center dot 8H(2)O (1) with the unsymmetrical ligand 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl] phenol (H(3)L) was synthesized and characterized. The new ligand H(3)L was obtained in good yield. Complex I crystallizes in an orthorhombic cell, space group Pcab. Kinetic studies show that complex 1 is highly active in the hydrolysis of the substrate 2,4-bis(dinitrophenyl)phosphate (K(m) = 4.09 mM, V(max) = 2.68 x 10(-2) mM s(-1), and k(cat) = V(max)/[1] = 0.67 s(-1)). Through a potentiometric study and determination of the kinetic behavior of 1 in acetonitrile/water solution, the species present in solution could be identified, and a trinuclear monohydroxo species appears to be the most prominent catalyst under mild conditions. Complex 1 displays high efficiency in DNA hydrolytic cleavage, and complete kinetic studies were carried out (K(m) = 4.57 x 10(-4) M, K(cat)` = 3.42 h(-1), and k(cat)`/K(m) = 7.48 x 10(3) M(-1) h(-1)). Studies with a radical scavenger (dimethyl sulfoxide, DMSO) showed that it did not inhibit the activity, indicating the hydrolytic action of 1 in the cleavage of DNA, and studies on the incubation of distamycin with plasmid DNA suggest that 1 is regio-specific, interacting with the minor groove of DNA.

Prion Protein and Its Ligand Stress Inducible Protein 1 Regulate Astrocyte Development

Prion protein (PrP(C)) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrP(C) in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild-type astrocytes in a protein kinase A-dependent manner, whereas PrP(C)-null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrP(C)-null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild-type, suggesting a slower rate of astrocyte maturation in PrP(C)-null animals. Furthermore, PrP(C)-null astrocytes treated with STI1 did not differentiate from a flat to a process-bearing morphology, as did wild-type astrocytes. Remarkably, STI1 inhibited proliferation of both wild-type and PrP(C)-null astrocytes in a protein kinase C-dependent manner. Taken together, our data show that PrP(C) and STI1 are essential to astrocyte development and act through distinct signaling pathways.(C) 2009 Wiley-Liss, Inc.

Effect of arginine on the development of the pectoralis muscle and the diameter and the protein:deoxyribonucleic acid rate of its skeletal myofibers in broilers

This work aimed at evaluating the effects of the supplementation of starter diet with Arg on breast muscle development in broilers and the activation of satellite cells and the aggregation of myofibrillar protein. Male Cobb chicks (n = 990) were randomly assigned to 1 of 5 treatments in a complete random design. Measurements of 33 chicks per treatment were made in 6 repetitions. The treatments consisted of a basal diet with 1.390% digestible Arg (without supplementation) and 4 dietary levels of Arg (1.490, 1.590, 1.690, and 1.790%) with Arg:Lys ratios of 1.103, 1.183, 1.262, 1.341, and 1.421, respectively. Arginine supplementation was used only in the starter phase (1 to 21 d). Dietary supplementation with Arg had a positive effect (P < 0.05) on breast and breast fillet weight on d 7 and 21 and on myofiber diameter on d 14 and 21. However, no effect was observed (P > 0.05) on the protein: DNA ratio, which demonstrates that Arg does not interfere with the mitotic activity of the satellite cells. Independently from mechanism, Arg affected muscle growth in the starter phase positively. Dietary supplementation with Arg in the starter phase had no effect (P > 0.05) on the carcass yield of broilers on d 42. Diet supplementation with Arg at levels above the ones recommended for the starter phase may be necessary for improved muscle development in broilers.

Free Cyclitol, Soluble Carbohydrate and Protein Contents in Vigna unguiculata and Phaseolus vulgaris Bean Sprouts

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H(2)O(2)) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3 beta/GSK-3 beta ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H(2)O(2) (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3 beta phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H(2)O(2) levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

P>Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette-Guerin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) and IL-12 and of the anti-apoptotic protein Bcl-2 were measured at day 3 and day 7 post-infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high-dose group, while on the 7th day post-infection macrophage apoptosis was reduced in the low-dose group. Inhibition of apoptosis was correlated with increased production of IL-10, reduced production of TNF-alpha and increased production of Bcl-2. In addition, the production of IL-12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations. HPS presented a smaller effect at concentrations above 1.5 mM. At 10 mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant-BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue. (C) 2011 Elsevier B.V. All rights reserved.

Cholesterol-Lowering Properties of Whole Cowpea Seed and Its Protein Isolate in Hamsters

Hypercholesterolemic hamsters were fed for 4 wk on diets rich in saturated fatty acids and cholesterol, differing only in protein source (20%): casein (control group, HC), whole cowpea seed (HWS), and cowpea protein isolate (HPI). Hamsters fed on HWS and HPI presented significant reductions in plasma total cholesterol and non-HDL cholesterol. HPI and HC presented similar protein digestibility, which were significantly higher than that of HWS. Animals fed on HWS presented significantly higher levels of bile acids and cholesterol in feces than did the animals fed on casein or HPI diets. Histological analyses of the liver showed that HC diet resulted in steatosis widely distributed throughout the hepatic lobule, while HWS and HPI diets promoted reductions in liver steatosis. The effectiveness of HWS for modulating lipid metabolism was greater than that of HPI, as measured by plasma cholesterol reduction and liver steatosis.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)