Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Physical, epidemiological, and molecular evaluation of infection by Cryptosporidium galli in Passeriformes

Due to the scarcity of information related to the epidemiology of Cryptosporidium infection in passerine birds, this study aimed to determine the periodicity of fecal shedding of Cryptosporidium spp. oocysts, after natural infection, and its clinical signs, mortality, and molecular characterization. Four hundred eighty fecal samples were collected from 40 birds, including 372 samples from 31 adult birds and 108 samples from nine young birds (up to 12 months old), housed in five aviaries, monthly from September 2007 to September 2008, with the exception of April. The birds originated from aviaries in which the following species were raised: great-billed seed-finch (Oryzoborus maximiliani), lesser seed-finch (Oryzoborus angolensis), ultramarine grosbeak (Cyanocompsa brissonii), and rusty-collared seedeater (Sporophila collaris). The samples were preserved in 2.5% potassium dichromate at 4A degrees C until processing. The oocysts were purified by centrifugal flotation in Sheather`s solution, followed by genomic DNA extraction and molecular characterization of oocysts using the nested polymerase chain reaction for amplification of fragments of the 18S subunit of rRNA gene. Intermittent shedding of oocysts was observed by positive amplification for Cryptosporidium spp. in 91 (24.5%) samples of adult birds and 14 (13%) of young birds. The sequencing of the amplified fragments enabled the identification of Cryptosporidium galli. Although all the aviaries had birds positive for C. galli, morbidity or mortality was observed in only one aviary and was associated with concomitant infection with Escherichia coli and Isospora sp.

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

The aim of this study was to determine the prevalence of Cryptosporidium species and genotypes in birds kept in captivity in Brazil. A total of 966 samples from 18 families of birds was collected and stored in 5% potassium dichromate solution at 4 degrees C until processing. Oocysts were purified in Sheather sugar solution following extraction of genomic DNA. Molecular analyses were performed using nested-PCR for amplification of fragments of the 18S subunit of rRNA gene and of the actin gene. Amplification of Cryptosporidium DNA fragments was obtained in 47 (4.86%) samples. Sequencing of amplified fragments and phylogenetic analyses allowed the identification of Cryptosporidium baileyi in a black vulture (Coragyps atratus), a domestic chicken (Gallus gallus domesticus) and a saffron finch (Sicalis flaveola); Cryptosporidium galli in canaries (Serinus canaria), a cockatiel (Nymphicus hollandicus) and lesser seed-finches (Oryzoborus angolensis); Cryptosporidium meleagridis in a domestic chicken (G. g. domesticus); Cryptosporidium parvum in a cockatiel (N. hollandicus); Cryptosporidium avian genotype I in a canary (S. canaria) and an Indian peafowl (Pavo cristatus); Cryptosporidium avian genotype II in ostriches (Struthio camelus) and Cryptosporidium avian genotype III in a cockatiel (N. hollandicurs) and a peach-faced lovebird (Agapornis roseicolis). (C) 2009 Elsevier B.V. All rights reserved.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies. Published by Elsevier Ltd.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

Histological and molecular temporomandibular joint analyses after mandibular advancement surgery: study in minipigs

Objectives. The objective of this study was to elucidate the changes occurring in the temporomandibular joint (TMJ) after surgical mandibular advancement with different fixation techniques: bicortical screws (rigid fixation) and miniplates (semi-rigid fixation). Study design. Eighteen minipigs were equally and randomly divided into 3 groups: Group I (control), nonoperated animals; Group II, animals submitted to surgical advancement surgery and osteosynthesis by bicortical screws; and Group III, animals submitted to surgical advancement surgery and osteosynthesis by miniplates. Four months after the surgeries, the presence of interleukin (IL)-6 and IL-10 in synovial fluid samples was assessed in ELISA experiments. TMJs were histologically prepared. Results. Higher levels of IL-10 (P = .0436) were found for Group II. Descriptive histological analysis was compatible with the ELISA findings. Conclusions. Rigid fixation evokes more pronounced signs of bone remodeling in the TMJ, whereas malleable fixation promotes a more intense inflammatory activity. Therefore, rigid fixation seems to transmit a higher impact of postoperative masticatory forces to the TMJ.

Molecular and cellular biology of pre-malignancy in the gastrointestinal tract

Important pathogenic alterations within established cancers are acquired during the premalignant stage. These genetic alterations can be grouped into specific neoplastic pathways that differ within and between anatomical sites. By understanding the mechanisms that determine the initiation and progression of each pathway, it will be possible to develop novel approaches to the diagnosis, prevention and treatment of cancer. This chapter outlines the principles underlying the molecular characterization of pre-malignant lesions, taking colorectal neoplasia as the main model.

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Towards a molecular epidemiology of alcohol dependence analysing the interplay of genetic and environmental risk factors

Background Progress in identifying genetic factors protective against alcohol dependence (AlcD) requires a paradigm shift in psychiatric epidemiology. Aims To integrate analysis of research into the genetics of alcoholism. Method Data from prospective questionnaire and interview surveys of the Australian twin panel, and from a subsample who underwent alcohol challenge, were analysed. Results In men, effects of alcohol dehydrogenase ADH2*1/*2 genotype or high alcohol sensitivity (risk-decreasing), and of history of childhood conduct disorder, or having monozygotic co-twin or twin sister with AlcD (risk-increasing) were significant and comparable in magnitude. Religious affiliation (Anglican versus other) was associated with the ADH2 genotype, but did not explain the associations with AlcD symptoms. No protective effect of the ADH2*1/*2 genotype was observed in women. Conclusions The early onset and strong familial aggregation of AlcD, and opportunity for within-family tests of genetic association to avoid confounding effects, make epidemiological family studies of adolescents and young adults and their families a priority.

Interpretation of the reversible inhibition of adenosine deaminase by small cosolutes in terms of molecular crowding

Published results on the inhibitory effects of small cosolutes on adenosine deamination by adenosine deaminase [Kurz. L. C.. Weitkamp, E., and Frieden, C. (1987) Biochemistry 26, 3027-3032; Dzingeleski, G., and Wolfenden, R. (1993) Biochemistry 32, 9143 -9147] have been reexamined. Results for sucrose, dioxane, methanol, and ethanol are shown to be qualitatively consistent with thermodynamic interpretation in terms of molecular crowding effects arising from the occurrence of a minor increase in enzyme volume and/or asymmetry during the kinetic reaction-a conformational transition that could be either preexisting or ligand induced. Direct evidence for the existence of the putative isomeric transition is provided by active enzyme gel chromatography on Sephadex G-100, which demonstrates a negative dependence of enzyme elution volume upon substrate concentration and is therefore consistent with substrate-mediated conformational changes that favor a larger (or more asymmetric) isomeric state of the enzyme. There are thus experimental grounds for adopting the present description of the inhibitory effects of unrelated cosolutes on the kinetics of adenosine deamination by adenosine deaminase in terms of thermodynamic nonideality.

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b

The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as we:ll as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding try trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T reversible arrow R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.