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.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-gamma- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response. (C) 2010 Elsevier GmbH. All rights reserved.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric IAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory. (C) 2010 Elsevier Inc. All rights reserved.

Neurophysiological findings of the late-onset, dominant, proximal spinal muscular atrophies with dysautonomia because of the VAPB PR056SER mutation

The vesicle-associated membrane protein/synaptobrevin-associated membrane protein B (VAPB) Pro56Ser Mutation has been identified in Brazilian families showing various motor neuron syndromes. However, the neurophysiological characteristics of these patients have not been detailed, and some questions Still need to be solved, such as the possible presence of myotonia and the origin of the abdominal protrusion seen in most patients. The eventual finding of suggestive electrophysiological characteristics would be helpful not only for clinical diagnosis but also to selection of the appropriate DNA test. To clarify these questions we carried out sensory and motor conduction Studies, including symphatetic skin response, and needle examination in six genetically proven affected members. The electromyographic findings were those of a slowly progressive motor neuron disorder. Topographically, the abdominal muscles were severely affected, but the facial and laryngeal muscles were preserved or very mildly involved. Sensory conduction studies and sympathetic Skin responses were normal. No myotonic discharge was recorded. These findings are indistinguishable from those of other motor neuron disorders, although the predominant involvement of the proximal limbs and of the abdominal muscles may be of some help in the appropriate clinical setting.

Protease activity and the ultrastructure of broiler chicken PSE (pale, soft, exudative) meat

The biological cause of broiler PSE meat seems to be an excessive release of Ca(2+), promoted by a genetic mutation of ryanodine receptors located in the sarcoplasmic reticulum of skeletal muscle cells. Excessive Ca(2+), associated with protein denaturation in meat, enhances protease activity and influences the functional properties of PSE meat. Twenty-four-hour post-mortem Pectoralis major m. samples exhibited lower values for pH, water-holding capacity, and shear force than did control samples, in contrast to colour (L*) and cooking loss values. Protease activity, measured as myofibril fragmentation index, presented higher values in PSE meat than in control samples. Ultrastructural examination revealed shrinking and depolymerisation of myofilaments and Z-lines disorganisation within the sarcomere in PSE meat. Intense calpain activity was also observed, indicating that the process may initiate at the filaments, because of protein denaturation, and spread through Z-lines, resulting in the collapse of the sarcomere structure. (C) 2009 Elsevier Ltd. All rights reserved.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae

Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis.

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Changes in Calcium-Binding Protein Expression in Human Cortical Contusion Tissue

Traumatic brain injury (TBI) produces several cellular changes, such as gliosis, axonal and dendritic plasticity, and inhibition-excitation imbalance, as well as cell death, which can initiate epileptogenesis. It has been demonstrated that dysfunction of the inhibitory components of the cerebral cortex after injury may cause status epilepticus in experimental models; we proposed to analyze the response of cortical interneurons and astrocytes after TBI in humans. Twelve contusion samples were evaluated, identifying the expression of glial fibrillary acidic protein (GFAP) and calcium-binding proteins (CaBPs). The study was made in sectors with and without preserved cytoarchitecture evaluated with NeuN immunoreactivity (IR). In sectors with total loss of NeuN-IR the results showed a remarkable loss of CaBP-IR both in neuropil and somata. In sectors with conserved cytoarchitecture less drastic changes in CaBP-IR were detected. These changes include a decrease in the amount of parvalbumin (PV-IR) neurons in layer II, an increase of calbindin (CB-IR) neurons in layers III and V, and an increase in calretinin (CR-IR) neurons in layer II. We also observed glial fibrillary acidic protein immunoreactivity (GFAP-IR) in the white matter, in the gray-white matter transition, and around the sectors with NeuN-IR total loss. These findings may reflect dynamic activity as a consequence of the lesion that is associated with changes in the excitatory circuits of neighboring hyperactivated glutamatergic neurons, possibly due to the primary impact, or secondary events such as hypoxia-ischemia. Temporal evolution of these changes may be the substrate linking severe cortical contusion and the resulting epileptogenic activity observed in some patients.

Effect of free or protein-associated soy isoflavones on the antioxidant status in rats

BACKGROUND: The objective of this study was to investigate the effect of chronic ingestion of free and protein-associated soy isoflavones on the antioxidant status in male Wistar rats. Free isoflavone (iso), protein-associated soy isoflavone (iso + prot) and soy protein (prot) extracts were administered for 30 days by gavage to the rats at a dosage of 1 mg aglycone isoflavones per 200 g body weight, adjusted daily, and the prot group was given the same concentration of soy protein received by the iso + prot group. Antioxidant capacity of plasma, thiobarbituric acid-reactive substance (TBARS) and glutathione (GSH) levels and catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in plasma, erythrocytes and tissues and gene expression levels in liver and kidney were evaluated. RESULTS: Chronic ingestion of free but not of protein-associated soy isoflavones nor of solely soy protein increased plasma antioxidant capacity and GPx activity in erythrocytes. Soy protein increased CAT activity and gene expression in liver. SOD activity in erythrocytes was increased by all treatments. CONCLUSION: The overall results confirm that dietary soy isoflavones have a positive effect on antioxidant status, enhancing antioxidant capacity of plasma and antioxidant enzymes in various tissues, but the effects are dependent on the form of administration and on a complex mechanism of antioxidant status balance on the organism. (C) 2010 Society of Chemical Industry

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Heterozygosity for the S180L Variant of MAL/TIRAP, a Gene Expressing an Adaptor Protein in the Toll-Like Receptor Pathway, Is Associated with Lower Risk of Developing Chronic Chagas Cardiomyopathy

Background. Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. Among T. cruzi-infected individuals, only a subgroup develops severe chronic Chagas cardiomyopathy (CCC); the majority remain asymptomatic. T. cruzi displays numerous ligands for the Toll-like receptors (TLRs), which are an important component of innate immunity that lead to the transcription of proinflammatory cytokines by nuclear factor-kappa B. Because proinflammatory cytokines play an important role in CCC, we hypothesized that single-nucleotide polymorphisms (SNPs) in the genes that encode proteins in the TLR pathway could explain differential susceptibility to CCC among T. cruzi-infected individuals. Methods. For 169 patients with CCC and 76 T. cruzi-infected, asymptomatic individuals, we analyzed SNPs by use of polymerase chain reaction-restriction fragment length polymorphism analysis for the genes TLR1, TLR2, TLR4, TLR5, TLR9, and MAL/TIRAP, which encodes an adaptor protein. Results. Heterozygous carriers of the MAL/TIRAP variant S180L were more prevalent in the asymptomatic group (24 [32%] of 76 subjects) than in the CCC group (21 [12%] of 169) (chi(2) = 12.6; P = .0004 [adjusted P (P(c)) = .0084]; odds ratio [OR], 0.31 [95% confidence interval {CI}, 0.16-0.60]). Subgroup analysis showed a stronger association when asymptomatic patients were compared with patients who had severe CCC (i.e., patients with left-ventricular ejection fraction <= 40%) (chi(2) = 11.3; P = .0008 [P(c) = .017]; OR, 0.22 [95% CI, 0.09-0.56]) than when asymptomatic patients were compared with patients who had mild CCC (i.e., patients with left-ventricular ejection fraction >40%) (chi(2) = 7.7; P = .005 [P(c) = .11]; OR, 0.33 [95% CI, 0.15-0.73]). Conclusion. T. cruzi-infected individuals who are heterozygous for the MAL/TIRAP S180L variant that leads to a decrease in signal transduction upon ligation of TLR2 or TLR4 to their respective ligand may have a lower risk of developing CCC.

Low temperature induced changes in activity and protein levels of the enzymes associated to conversion of starch to sucrose in banana fruit

Storage at low temperature is the most frequently used method to extend the shelf life of banana fruit, and is fundamental for extended storage and transport over long distances. However, storage and transport conditions must be carefully controlled because of the high susceptibility of many commercial cultivars to chilling injury. The physiological behavior of bananas at low temperatures has been studied to identify possible mechanisms of resistance to chilling injury. The aim of this work was to evaluate differences in the starch-to-sucrose metabolism of a less tolerant and susceptible (Musa acuminata, AAA cv. Nanicao) and a more tolerant (M. acuminata x Musa balbusiana, AAB, cv. Prata) banana cultivar to chilling injury. Fruits of these cultivars were stored in chambers at 13 degrees C for 15 d, at which point they were transferred to 19 degrees C, where they were left until complete ripening. The low temperature induced significant changes in the metabolism of starch and sucrose in comparison to fruit ripened only at 19 degrees C. The sucrose accumulation was slightly higher in cv. Prata, and different patterns of starch degradation, sucrose synthesis, activity and protein levels of the alpha-and beta-amylases, starch phosphorylase, sucrose synthase and sucrose phosphate synthase were detected between the cultivars. Our results suggest that starch-to-sucrose metabolism is likely part of the mechanism for cold acclimation in banana fruit, and the cultivar-dependent differences contribute to their ability to tolerate cold temperatures. (C) 2011 Elsevier B.V. All rights reserved.