Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Proteomic analysis of total cellular proteins of human neutrophils

Background: Neutrophils are the most abundant leukocytes in peripheral blood and represent one of the most important elements of innate immunity. Recent subcellular proteomic studies have focused on the identification of human neutrophil proteins in various subcellular membrane and granular fractions. Although there are relatively few studies dealing with the analysis of the total extract of human neutrophils, many biological problems such as the role of chemokines, adhesion molecules, and other activating inputs involved in neutrophil responses and signaling can be approached on the basis of the identification of the total cellular proteins. Results: Using gel-LC-MS/MS, 251 total cellular proteins were identified from resting human neutrophils. This is more than ten times the number of proteins identified by an initial proteome analysis of human neutrophils and almost five times the number of proteins identified by the first 2-DE map of extracts of rat polymorphonuclear leukocytes. Most of the proteins identified in the present study are well-known, but some of them, such as neutrophil-secreted proteins and centaurin beta-1, a cytoplasmic protein involved in the regulation of NF-kappa B activity, are described here for the first-time. Conclusion: The present report provides new information about the protein content of human neutrophils. Importantly, our study resulted in the discovery of a series of proteins not previously reported to be associated with human neutrophils. These data are relevant to the investigation of comparative pathological states and models for novel classes of pharmaceutical drugs that could be useful in the treatment of inflammatory disorders in which neutrophils participate.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

In utero protein restriction causes growth delay and alters sperm parameters in adult male rats

Background: Recent studies have supported the concept of ""fetal programming"" which suggests that during the intrauterine development the fetus may be programmed to develop diseases in adulthood. The possible effects of in utero protein restriction on sexual development of rat male offspring were evaluated in the present study. Methods: Pregnant Wistar rats were divided into two experimental groups: one group treated with standard chow (SC, n = 8, 17% protein) and the other group treated with hypoproteic chow (HC, n = 10, 6% protein) throughout gestation. After gestation the two experimental groups received standard chow. To evaluate the possible late reproductive effects of in utero protein restriction, the male offspring of both groups were assessed at different phases of sexual development: prepubertal (30 days old); peripubertal (60 days old); adult (90 days old). Student's t test and Mann-Whitney test were utilized. Differences were considered significant when p < 0.05. Results: We found that in utero protein restriction reduced the body weight of male pups on the first postnatal day and during the different sexual development phases (prepubertal, peripubertal and adult). During adulthood, Sertoli cell number, sperm motility and sperm counts in the testis and epididymal cauda were also reduced in HC. Furthermore, the numbers of sperm presenting morphological abnormalities and cytoplasmic drop retention were higher in HC. Conclusions: In conclusion, in utero protein restriction, under these experimental conditions, causes growth delay and alters male reproductive-system programming in rats, suggesting impairment of sperm quality in adulthood.

SPITZER OBSERVATIONS OF A1763. II. CONSTRAINING THE NATURE OF ACTIVITY IN THE CLUSTER-FEEDING FILAMENT WITH VLA AND XMM-NEWTON DATA

The A1763 superstructure at z = 0.23 contains the first galaxy filament to be directly detected using mid-infrared observations. Our previous work has shown that the frequency of starbursting galaxies, as characterized by 24 mu m emission is much higher within the filament than at either the center of the rich galaxy cluster, or the field surrounding the system. New Very Large Array and XMM-Newton data are presented here. We use the radio and X-ray data to examine the fraction and location of active galaxies, both active galactic nuclei (AGNs) and starbursts (SBs). The radio far-infrared correlation, X-ray point source location, IRAC colors, and quasar positions are all used to gain an understanding of the presence of dominant AGNs. We find very few MIPS-selected galaxies that are clearly dominated by AGN activity. Most radio-selected members within the filament are SBs. Within the supercluster, three of eight spectroscopic members detected both in the radio and in the mid-infrared are radio-bright AGNs. They are found at or near the core of A1763. The five SBs are located further along the filament. We calculate the physical properties of the known wide angle tail (WAT) source which is the brightest cluster galaxy of A1763. A second double lobe source is found along the filament well outside of the virial radius of either cluster. The velocity offset of the WAT from the X-ray centroid and the bend of the WAT in the intracluster medium are both consistent with ram pressure stripping, indicative of streaming motions along the direction of the filament. We consider this as further evidence of the cluster-feeding nature of the galaxy filament.

A New Approach for Video Adaptation through Overlay Services Networks

Video adaptation is an extensively explored content providing technique aimed at appropriately suiting several usage scenarios featured by different network requirements and constraints, user`s terminal and preferences. However, its usage in high-demand video distribution systems, such as CNDs, has been badly approached, ignoring several aspects of optimization of network use. To address such deficiencies, this paper presents an approach for implementing the adaptation service by exploring the concept of overlay services networks. As a result of demonstrate the benefits of this proposal, it is made a comparison of this proposed adaptation service with other strategies of video adaptation.

Morphological variation and reproductive incompatibility of three coconut-mite-associated populations of predatory mites identified as Neoseiulus paspalivorus (Acari: Phytoseiidae)

Predatory mites identified as Neoseiulus paspalivorus DeLeon (Phytoseiidae) have been considered as agents for classical biological control of the coconut mite, Aceria guerreronis Keifer (Eriophyidae), in Africa and elsewhere. Preliminary identification of geographically distinct populations as belonging to the same species (N. paspalivorus) was based on their morphological similarity. However, laboratory studies recently conducted have shown large differences in feeding behaviors and biological characteristics among individuals collected from three geographic origins: Brazil (South America), Benin and Ghana (West Africa). As morphologically similar specimens do not necessarily belong to the same species, we evaluated under laboratory conditions, reproductive compatibility between the specimens from three geographic locations to ascertain their conspecificity. Morphological measurements were also made to determine whether there is a means of discriminating between them. Inter-population crosses showed complete reproductive isolation between the three geographic populations, but interpopulation discontinuities in morphometric characters were absent. These results indicate that the tested specimens are distinct biological entities despite morphological similarity. Further molecular genetic studies are therefore proposed, including screening for endosymbionts and assessment of genetic differentiation, to determine the cause of reproductive incompatibility and to clarify the taxonomic relationship between those populations.

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection Rust caused by the fungus Puccinia psidii is currently the most important disease of eucalyptus. It is widely disseminated in Brazil, and causes serious damage in nurseries and plantation areas. The identification of resistant germplasm along with knowledge of the genetic basis of resistance heredity are the first requirements for the success of breeding programs aiming to develop resistant varieties. Earlier studies carried out under controlled conditions suggested a monogenic control as well as the participation of at least two genes promoting resistance to rust. The goal of this study was to evaluate the resistance to P. psidii under field conditions in fourteen progenies from controlled crosses and self-crosses among four hybrid clones of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla ST Blake that contrast for resistance to the fungus. Results indicated that resistance could be explained by one locus with main effects and at least three different alleles. However, loci with minor effects may influence the resistance, since variation on severity classes was observed. Differences in segregation of resistance between reciprocal crosses were not observed, indicating absence of cytoplasmic effects.

In silico analysis of candidate genes involved in light sensing and signal transduction pathways in soybean

Several aspects of photoperception and light signal transduction have been elucidated by studies with model plants. However, the information available for economically important crops, such as Fabaceae species, is scarce. In order to incorporate the existing genomic tools into a strategy to advance soybean research, we have investigated publicly available expressed sequence tag ( EST) sequence databases in order to identify Glycine max sequences related to genes involved in light-regulated developmental control in model plants. Approximately 38,000 sequences from open-access databases were investigated, and all bona fide and putative photoreceptor gene families were found in soybean sequence databases. We have identified G. max orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses, although some important Arabidopsis phytochrome-signaling components are absent. Moreover, soybean and Arabidopsis gene-family homologs appear to have undergone a distinct expansion process in some cases. We propose a working model of light perception, signal transduction and response-eliciting in G. max, based on the identified key components from Arabidopsis. These results demonstrate the power of comparative genomics between model systems and crop species to elucidate several aspects of plant physiology and metabolism.

Orchid fleck symptoms may be caused naturally by two different viruses transmitted by Brevipalpus

Brevipalpus-transmitted viruses (BTV) cause chlorotic, necrotic and/or ringspot lesions in leaves and stems of orchids, citrus, coffee and several other plant species. There are two different types of BTVs, the nuclear and the cytoplasmic, based on maturation locale in the cell and particle morphology. The orchid fleck virus (OFV) is a BTV that infects orchids. Its short rodlike particles are 32-40 nm in diameter, 100-150 nm in length. OFV is found in the nucleus and is associated with intranuclear electronlucent viroplasms. In 1999, transmission electron microscopy analysis revealed a distinct type of virus causing orchid fleck symptoms. The bacilliform particles, 70-80 nm in diameter and 110-120 nm in length, induced electron-dense viroplasm inclusions in infected cells and resembled the cytoplasmic type associated with BTV, such as the citrus leprosis virus C. Our objective in the present study was to verify whether the cytoplasmic type virus found in orchids could be amplified using primers for other cytoplasmic BTVs, such as CiLV-C and Solanum violaefolium ringspot virus (SvRSV). Additionally, we aimed to differentiate the two BTVs found in orchids: the nuclear and the cytoplasmic types of OFV using microscopy and molecular and serological tools. This virus was not amplified by the CiLV-C and SvRSV primers, and neither the molecular nor the serological tools available to the OFV diagnosis reacted with it, demonstrating that they are definitely different viruses.

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, BcI-2 and NF-kappa B pathways

During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappa B p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappa B activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappa B activation in rats submitted to the RH model was observed. in agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappa B pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer.

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappa B is kept from binding to its consensus sequence by the inhibitor I kappa B alpha, which retains NF-kappa B in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappa B alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappa B. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-a by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappa B alpha and NF-kappa B, NF-kappa B activation and TNF-alpha mRNA and protein synthesis inmacrophages. Two-month-old male BALB/Cmice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-a mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappa B activation after LPS stimulation. These results led us to conclude that PEM changes NF-kappa B signalling pathway in macrophages to LPS stimulus.

Chemoprevention of rat hepatocarcinogenesis with histone deacetylase inhibitors: Efficacy of tributyrin, a butyric acid prodrug

Hepatocellular carcinoma (HCC) ranks in prevalence and mortality among top 10 cancers worldwide. Butyric acid (BA), a member of histone deacetylase inhibitors (HDACi) has been proposed as an anticareinogenic agent. However, its short half-life is a therapeutical limitation. This problem could be circumvented with tributyrin (TB), a proposed BA prodrug. To investigate TB effectiveness for chemoprevention, rats were treated with the compound during initial phases of ""resistant hepatocyte"" model of hepatocarcinogenesis, and cellular and molecular parameters were evaluated. TB inhibited (p < 0.05) development of hepatic preneoplastic lesions (PNL) including persistent ones considered HCC progression sites. TB increased (p < 0.05) PNL remodeling, a process whereby they tend to disappear. TB did not inhibit cell proliferation in PNL, but induced (p < 0.05) apoptosis in remodeling ones. Compared to controls, rats treated with TB presented increased (P < 0.05) hepatic levels of BA indicating its effectiveness as a prodrug. Molecular mechanisms of TB-induced hepatocarcinogenesis chemoprevention were investigated. TB increased (p < 0.05) hepatic nuclear histone H3K9 hyperacetylation specifically in PNL and p21 protein expression, which could be associated with inhibitory HDAC effects. Moreover, it reduced (p < 0.05) the frequency of persistent PNL with aberrant cytoplasmic p53 accumulation, an alteration associated with increased malignancy. Original data observed in our study support the effectiveness of TB as a prodrug of BA and as an HDACi in hepatocarcinogenesis chemoprevention. Besides histone acetylation and p21 restored expression, molecular mechanisms involved with TB anticarcinogenic actions could also be related to modulation of p53 pathways. (C) 2008 Wiley-Liss, Inc.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

EULAR/PRINTO/PRES criteria for Henoch-Schonlein purpura, childhood polyarteritis nodosa, childhood Wegener granulomatosis and childhood Takayasu arteritis: Ankara 2008. Part II: Final classification criteria

Objectives To validate the previously proposed classification criteria for Henoch-Schonlein purpura (HSP), childhood polyarteritis nodosa (c-PAN), c-Wegener granulomatosis (c-WG) and c-Takayasu arteritis (c-TA). Methods Step 1: retrospective/prospective webdata collection for children with HSP, c-PAN, c-WG and c-TA with age at diagnosis <= 18 years. Step 2: blinded classification by consensus panel of a representative sample of 280 cases. Step 3: statistical (sensitivity, specificity, area under the curve and.-agreement) and nominal group technique consensus evaluations. Results 827 patients with HSP, 150 with c-PAN, 60 with c-WG, 87 with c-TA and 52 with c-other were compared with each other. A patient was classified as HSP in the presence of purpura or petechiae (mandatory) with lower limb predominance plus one of four criteria: (1) abdominal pain; (2) histopathology (IgA); (3) arthritis or arthralgia; (4) renal involvement. Classification of c-PAN required a systemic inflammatory disease with evidence of necrotising vasculitis OR angiographic abnormalities of medium-/small-sized arteries (mandatory criterion) plus one of five criteria: (1) skin involvement; (2) myalgia/muscle tenderness; (3) hypertension; (4) peripheral neuropathy; (5) renal involvement. Classification of c-WG required three of six criteria: (1) histopathological evidence of granulomatous inflammation; (2) upper airway involvement; (3) laryngo-tracheo-bronchial involvement; (4) pulmonary involvement (x-ray/CT); (5) antineutrophilic cytoplasmic antibody positivity; (6) renal involvement. Classification of c-TA required typical angiographic abnormalities of the aorta or its main branches and pulmonary arteries (mandatory criterion) plus one of five criteria: (1) pulse deficit or claudication; (2) blood pressure discrepancy in any limb; (3) bruits; (4) hypertension; (5) elevated acute phase reactant. Conclusion European League Against Rheumatism/Paediatric Rheumatology International Trials Organisation/Paediatric Rheumatology European Society propose validated classification criteria for HSP, c-PAN, c-WG and c-TA with high sensitivity/specificity.