The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.

Dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (Holmberg, 1887)

Lysine is the reference essential amino acid in fish feeds and usually the most limiting amino acid in feedstuffs. The dietary lysine requirement of juvenile pacu Piaractus mesopotamicus (4.3 g) was determined using five isonitrogenous (32% CP) test diets containing graded levels of lysine (0.9, 1.17, 1.44, 1.69 and 1.96% of dry diet) fed three times a day to four groups of 18 fish for 74 days. Growth, body composition, nutrient retention and hematological parameters of pacu were analyzed. Analysis of variance showed that all growth performance parameters were significantly affected by dietary treatments. The lysine requirements estimated using regression analysis for maximum weight gain and feed efficiency were 1.45 and 1.51% of dry diet, respectively. Nitrogen retention efficiency increased with increasing levels of dietary lysine up to 1.43% (p<0.05). Whole-body protein increased (p<0.05) and whole-body lipid decreased (p<0.05) with increasing dietary lysine level. Thus, the lysine requirement of juvenile pacu was estimated as being 1.4-1.5% of dry diet or 4.4-4.7% of dietary protein. (c) 2009 Elsevier B.V. All rights reserved.

Estimating Amino Acid Requirement of Brazilian Freshwater Fish from Muscle Amino Acid Profile

Information on nutritional requirement of some Brazilian farmed fish species, especially essential amino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based on amino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, and curimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. The objective of the present study was to estimate amino acid requirements of these species and analyze similarities among amino acid composition of different fish species by cluster analysis. To estimate amino acid requirement, the following formula was used: amino acid requirement = [(amount of an individual amino acid in fish muscle tissue) x (average totalEAA requirement among channel catfish, Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(average fish muscle totalEAA). Most values found lie within the range of requirements determined for other omnivorous fish species, in exception of leucine requirement estimated for both species, and arginine requirement estimated for matrinxa alone. Rather than writing off the need for regular dose-response assays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa, results set solid base for the study of tropical species dietary amino acids requirements.

Requirement for PLC gamma 2 in IL-3 and GM-CSF-Stimulated MEK/ERK Phosphorylation in Murine and Human Hematopoietic Stem/Progenitor Cells

Even though the involvement of intracellular Ca(2+) (Ca(i)(2+)) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3)) levels and Ca(i)(2+) release in murine and human hematopoietic stem/ progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+)-dependent kinases in MEK activation. In addition, we identify phospholipase C gamma 2 (PLC gamma 2) as a PLC gamma responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCg inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLC gamma 2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/ progenitor cells. J. Cell. Physiol. 226: 1780-1792, 2011. (C) 2010 Wiley-Liss, Inc.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

In Vivo Effects of Bothrops jararaca Venom on Metabolic Profile and on Muscle Protein Metabolism in Rats

This study investigated the in vivo effects of the Bothrops Jararaca venom (BjV) on general metabolic profile and, specifically. oil muscle protein metabolism in rats. The crude venom (0.4 mg/kg body weight, IV) was infused in awake rats, and plasma activity of enzymes and metabolites levels were determined after 1, 2, 3, and 4 hours. BjV increased urea, lactate, and activities of creatine kinase. lactate dehydrogenase. and aspartate aminotransferase after 4 hours. The content of liver glycogen was reduced by BjV. Protein metabolism was evaluated by means of microdialysis technique and in isolated muscles. BjV induced increase in the muscle interstitial-arterial tyrosine concentration difference. indicating a high protein catabolism. The myotoxicity induced by this venom is associated with reduction of protein synthesis and increase in rates of overall proteolysis, which was accompanied by activation of lysosomal and ubiquitin-proteasome systems without changes in protein levels of cathepsins and ubiquitin-protein conjugates.

Effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) on the viability, proliferation and differentiation of osteoblast-like cells cultured on a chemically modified titanium surface

Aim: The aim of the present study was to assess the influence of the chemical characteristics and roughness of titanium surfaces on the viability, proliferation and differentiation of osteoblast-like cells cultured in a medium supplemented with recombinant human bone morphogenetic protein-7 (rhBMP-7). Material and methods: Osteo-1 cells were grown on titanium disks presenting with the following surfaces: (1) machined, (2) coarse grit-blasted and acid-attacked (SLA) and (3) chemically modified SLA (SLAmod) in the absence or presence of 20 ng/ml rhBMP-7 in culture medium. The viability and number of osteo-1 cells were evaluated after 24 h. Analyses of total protein content (TP) and alkaline phosphatase (AP) activity at 7, 14 and 21 days, collagen content at 7 and 21 days and mineralized matrix formation at 21 days were performed. Results: Cell viability (P=0.5516), cell number (P=0.3485), collagen content (P=0.1165) and mineralized matrix formation (P=0.5319) were not affected by the different surface configurations or by the addition of rhBMP-7 to the medium. Osteo-1 cells cultured on SLA surfaces showed a significant increase in TP at 21 days. The ALPase/TP ratio (P=0.00001) was affected by treatment and time. Conclusion: The results suggest that the addition of rhBMP-7 to the culture medium did not exert any effect on the viability, proliferation or differentiation of osteoblast-like cells grown on the different surfaces tested. All titanium surfaces analyzed allowed the complete expression of the osteoblast phenotype such as matrix mineralization by osteo-1 cells.

Interferon-gamma enhances cytotoxic T lymphocyte recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide

Keratinocytes expressing the human papillomavirus (HPV) type 16 E7 protein, as a transgene driven by the K14 promoter, form a murine model of HPV-mediated epithelial cancers in humans. Our previous studies have shown that K14E7 transgenic skin grafts onto syngeneic mice are not susceptible to immune destruction despite the demonstrated presence of a strong, systemic CTL response directed against the E7 protein. Consistent with this finding, we now show that cultured, E7 transgenic keratinocytes (KC) express comparable endogenous levels of E7 protein to a range of CTL-sensitive E7-expressing cell lines but are not susceptible to CTL-mediated lysis in vitro . E7 transgenic and non-transgenic KC are susceptible to conventional mechanisms of CTL-mediated lysis, including perforin and Fas/FasL interaction when an excess of exogenous peptide is provided. The concentration of exogenous peptide required to render a cell susceptible to lysis was similar between KC and other conventional CTL targets (e.g. EL-4), despite large differences in H-2D(b) expression at the cell surface. Furthermore, exposure of KC to IFN-gamma increased H-2D(b) expression, but did not substantially alter the exogenous peptide concentration required to sensitize cells for half maximal lysis. In contrast, the lytic sensitivity of transgenic KC expressing endogenous E7 is modestly improved by exposure to IFN-gamma. Thus, failure of CTL to eliminate KC expressing endogenous E7, and by inference squamous tumours expressing E7, may reflect the need for a sustained, local inflammatory environment during the immune effector phase.

Crystallization of PNMT, the adrenaline-synthesizing enzyme, is critically dependent on a high protein concentration

Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 Angstrom. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 Angstrom.

Constitutively active signal transducer and activator of transcription 5 can replace the requirement for growth hormone in adipogenesis of 3T3-F442A preadipocytes

Although it is the best characterized in vitro model of GH action, the mechanisms used by GH to induce differentiation of murine 3T3-F442A preadipocytes remain unclear. Here we have examined the role of three transcriptional regulators in adipogenesis. These regulators are either rapidly induced in response to GH [Stra13, signal transducer and activator of transcription (Stat) 3] or of central importance to GH signaling (Stat5). Retroviral transfection of 3T3-F442A preadipocytes was used to increase expression of Stra13, Stat3, and Stat5a. Only Stat5a transfection increased the expression of adipogenic markers peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein (C/EBP)alpha, and adipose protein 2/fatty acid-binding protein in response to GH, as determined by quantitative RT-PCR. Transfection with constitutively active Stat3 and Stat5a revealed that constitutively active Stat5a but not Stat3 was able to replace the GH requirement for adipogenesis. Constitutively active Stat5a but not Stat3 was able to increase the formation of lipid droplets and expression of alpha-glycerol phosphate dehydrogenase toward levels seen in mature adipocytes. Constitutively active Stat5a was also able to increase the expression of transcripts for C/EBPalpha to similar levels as GH, and of C/EBPbeta, peroxisome proliferator-activated receptor gamma, and adipose protein 2/fatty acid-binding protein transcripts to a lesser extent. An in vivo role for GH in murine adipogenesis is supported by significantly decreased epididymal fat depot size in young GH receptor-deleted mice, before manifestation of the lipolytic actions of GH. We conclude that Stat5 is a critical factor in GH-induced, and potentially prolactin-induced, murine adipogenesis.

Mechanism of interleukin-8 induction by human cytomegalovirus UL76 protein

Dissertation presented to obtain the Ph.D degree in Biology

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

The endodermis acts as a "second skin" in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry.

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehyde-fixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Role of the host cell's unfolded protein response in arenavirus infection.

Arenaviruses are enveloped RNA viruses with a nonlytic life cycle that cause acute and persistent infections. Here, we investigated the role of the host cell's unfolded protein response (UPR) in infection of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). In mammalian cells, the endoplasmic reticulum (ER) chaperone protein GRP78/BiP functions as the principal sensor for the induction of the UPR and interacts with three mediators: kinase/endonuclease inositol-requiring protein 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Acute infection with LCMV resulted in a selective induction of the ATF6-regulated branch of the UPR, whereas pathways controlled by PERK and IRE1 were neither activated nor blocked. Expression of individual LCMV proteins revealed that the viral glycoprotein precursor (GPC), but not that of other viral proteins, was responsible for the induction of ATF6. Rapid downregulation of the viral GPC during transition from acute to persistent LCMV infection restored basal levels of UPR signaling. To address a possible role of ATF6 signaling in LCMV infection, we used cells deficient in site 2 protease (S2P), a metalloprotease required for the activation of ATF6. Cells deficient in S2P showed significantly lower levels of production of infectious virus during acute but not persistent infection, indicating a requirement for ATF6-mediated signaling for optimal virus multiplication. In summary, acute LCMV infection seems to selectively induce the ATF6-regulated branch of the UPR that is likely beneficial for virus replication and cell viability, but it avoids induction of PERK and IRE1, whose activation may be detrimental for virus and the host cell.

Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana.

Blue light is known to cause rapid phosphorylation of a membrane protein in etiolated seedlings of several plant species, a protein that, at least in etiolated pea seedlings and maize coleoptiles, has been shown to be associated with the plasma membrane. The light-driven phosphorylation has been proposed on the basis of correlative evidence to be an early step in the signal transduction chain for phototropism. In the Arabidopsis thaliana mutant JK224, the sensitivity to blue light for induction of first positive phototropism is known to be 20- to 30-fold lower than in wild type, whereas second positive curvature appears to be normal. While light-induced phosphorylation can be demonstrated in crude membrane preparations from shoots of the mutant, the level of phosphorylation is dramatically lower than in wild type, as is the sensitivity to blue light. Another A. thaliana mutant, JK218, that completely lacks any phototropic responses to up to 2 h of irradiation, shows a normal level of light-induced phosphorylation at saturation. Since its gravitropic sensitivity is normal, it is presumably blocked in some step between photoreception and the confluence of the signal transduction pathways for phototropism and gravitropism. We conclude from mutant JK224 that light-induced phosphorylation plays an early role in the signal transduction chain for phototropism in higher plants.