Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

The genus Brycon, the largest subunit of the Bryconinae, has 42 valid species distributed from southern Mexico to the La Plata River in Argentina. Henochilus is a monotypic genus, comprising a single species (H. wheatlandii) found in the upper Rio Doce basin. In the present study, partial sequences of the mitochondrial gene 16S were obtained for fifteen species of Brycon and for Henochilus wheatlandii. The results showed that the genus Brycon is paraphyletic, since Henochilus is the sister-group of B. ferox and B. insignis. The most basal species analyzed were the trans-Andean species B. henni, B. petrosus, and B. chagrensis.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Soy isoflavones reduce heat shock proteins in postmenopausal women

Background: Atherosclerosis and its complications remain the most common cause of death in postmenopausal women. But there are few studies evaluating in hormonal theraphy can affect the autoimmune response involved in atherosclerosis. Objective to evaluate the effects to soy germ isoflavones and hormone replacement theraphy on antibodies against heat shock proteins (HPSP60, HPSP70 and HSC70) in moderately hypertensive hypercholesterolemic postmenopausal women. Methods: Women were treated with soy germ (2g/day) 17'beta'-estradiol(2 mg/day) or 17'beta'-estradiol (2mg/day)+noretisterone acetate (1mg/day), for 3 months after taking placebo for 1 month. The plasma autoantibodies to HSP60, HSP70 and HSC70 were determined by ELISA. Results: Data showed a reduction of autoantibodies against HSC70 after treatment in the 3 studies groups in relation to the placebo. The antibodies reactive to HSP70 were reduced only in women receiving soy germ. No significant differences were found for antibodies against HSP60. Conclusion: The soy germ isoflavones and 17'beta'-estradiol, alone or associated with noretisterone acetate, had similar effects on reduction of antibodies reactive to HSP70 in moderately hypertensive hypercholesterolemic postmenopausal women after 3 months of treatment. Thus, there results indicate that soy isoflavnes and hormone theraphy may modulate some pathways of the immune-inflammatory process in postmenopausal women at high risk for atherosclerosis.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and - 3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P. J. Hudson. 2005. Nat. Biotechnol. 23: 1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Proteome analysis of schizophrenia patients Wernicke's area reveals an energy metabolism dysregulation

Background: Schizophrenia is likely to be a consequence of DNA alterations that, together with environmental factors, will lead to protein expression differences and the ultimate establishment of the illness. The superior temporal gyrus is implicated in schizophrenia and executes functions such as the processing of speech, language skills and sound processing. Methods: We performed an individual comparative proteome analysis using two-dimensional gel electrophoresis of 9 schizophrenia and 6 healthy control patients' left posterior superior temporal gyrus (Wernicke's area - BA22p) identifying by mass spectrometry several protein expression alterations that could be related to the disease. Results: Our analysis revealed 11 downregulated and 14 upregulated proteins, most of them related to energy metabolism. Whereas many of the identified proteins have been previously implicated in schizophrenia, such as fructose-bisphosphate aldolase C, creatine kinase and neuron-specific enolase, new putative disease markers were also identified such as dihydrolipoyl dehydrogenase, tropomyosin 3, breast cancer metastasis-suppressor 1, heterogeneous nuclear ribonucleoproteins C1/C2 and phosphate carrier protein, mitochondrial precursor. Besides, the differential expression of peroxiredoxin 6 (PRDX6) and glial fibrillary acidic protein (GFAP) were confirmed by western blot in schizophrenia prefrontal cortex. Conclusion: Our data supports a dysregulation of energy metabolism in schizophrenia as well as suggests new markers that may contribute to a better understanding of this complex disease.

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.

Characterization of mitochondrial genotypes in the foundation herd of the Canchim beef cattle breed

The Canchim (5/8 Charolais + 3/8 Zebu) beef cattle breed was developed at Southeast-Embrapa Cattle to take advantage of hybrid vigor and to combine the higher growth rate and beef quality of Charolais with tropical adaptations of Zebu. The development of three lineages (old, new, and crossbred) has increased its genetic basis. The genotypic origin (Bos taurus or Bos indicus) of the mitochondrial DNA (mtDNA) of the Canchim breed was unknown. We characterized the mtDNA genotype of this founder herd by allele-specific polymerase chain reaction. The 173 founder Zebu females (62 Indubrasil, 3 Guzerat, and 108 Nellore) and their 6749 offspring were identified. The frequency of B. indicus mtDNA ranged from 1.15 to 2.05% among the descendants (N = 6404) of each maternal line with available DNA, and among animals that were alive (N = 689) in December 2007 among the three lineages. Though mtDNA characterization can be used to direct animal selection, the low frequency of B. indicus mtDNA impairs the evaluation of its effects on production traits in these animals. The high prevalence of B. taurus mtDNA in Canchim proves that the founder Zebu females from the Indubrasil, Guzerat and Nellore breeds were obtained from crosses of Zebu sires with local B. taurus dams.

CONTRASTING PHYLOGEOGRAPHIC PATTERNS IN MITOCHONDRIAL DNA AND MICROSATELLITES: EVIDENCE OF FEMALE PHILOPATRY AND MALE-BIASED GENE FLOW AMONG REGIONAL POPULATIONS OF THE BLUE-AND-YELLOW MACAW (PSITTACIFORMES: ARA ARARAUNA) IN BRAZIL

Comparing the patterns of population differentiation among genetic markers with different modes of inheritance call provide insights into patterns of sex-biased dispersal and gene flow. The blue-and-yellow Macaw (Ara ararauna) is a Neotropical parrot with a broad geographic distribution ill South America. However, little is known about the natural history and current status Of remaining wild populations, including levels of genetic variability. The progressive decline and possible fragmentation of populations may endanger this species in the near future. We analyzed mitochondrial DNA (mtDNA) control-region sequences and six microsatellite 106 Of Blue-and-yellow Macaws sampled throughout their geographic range ill Brazil to describe population genetic Structure, to make inferences about historical demography and dispersal behavior, and to provide insight for conservation efforts. Analyses of population genetic structure based on mtDNA showed evidence of two major populations ill western and eastern Brazil that share a few low-frequency haplotypes. This phylogeographic pattern seems to have originated by the historical isolation of Blue-and-yellow Macaw populations similar to 374,000 years ago and has been maintained by restricted gene flow and female philopatry. By contrast, variation ill biparentally inherited microsatellites was not structured geographically, Male-biased dispersal and female philopatry best explain the different patterns observed in these two markers. Because females disperse less than males, the two regional populations with well-differentiated mtDNA haplogroups should be considered two different management units for conservation purposes. Received 4 November 2007 accepted 10 December 2008.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.

Phylogeography and historical demography of the neotropical stingless bee Melipona quadrifasciata (Hymenoptera, Apidae): incongruence between morphology and mitochondrial DNA

The stingless bees are among the most abundant and ecologically important social invertebrates in tropical communities. The Neotropical stingless bee Melipona quadrifasciata has two subspecies: M. quadrifasciata quadrifasciata and M. quadrifasciata anthidioides. The main difference between subspecies are the yellow metassomal stripes, which are continuous in M. q. quadrifasciata and discontinuous in M. q. anthidioides. Recently, two populations were described with continuous stripes and inhabiting clearly disjunct areas in relation to M. q. quadrifasciata. We sequenced 852 bp of the mtDNA COI gene from 145 colonies from 56 localities, and for the first time performed a detailed phylogeographic study of a neotropical stingless bee. Phylogenetic analyses revealed the existence of two clades exhibiting a south to north distribution: southern populations comprise the subspecies M. q. quadrifasciata, and northern populations are composed of M. q. anthidioides and two disjunct populations with continuous stripes. The divergence time of these two phylogroups was estimated between 0.233 and 0.840 million years ago in the Pleistocene, a period of climatic changes and geomorphological alterations in the Neotropical region. No evidence of genetic structure in relation to the tergal stripes was found, indicating that the morphological trait regarding the pattern of stripes on tergites is not an accurate diagnostic for the subspecies of M. quadrifasciata.

Determination of the molecular weight of proteins in solution from a single small-angle X-ray scattering measurement on a relative scale

This paper describes a new and simple method to determine the molecular weight of proteins in dilute solution, with an error smaller than similar to 10%, by using the experimental data of a single small-angle X-ray scattering (SAXS) curve measured on a relative scale. This procedure does not require the measurement of SAXS intensity on an absolute scale and does not involve a comparison with another SAXS curve determined from a known standard protein. The proposed procedure can be applied to monodisperse systems of proteins in dilute solution, either in monomeric or multimeric state, and it has been successfully tested on SAXS data experimentally determined for proteins with known molecular weights. It is shown here that the molecular weights determined by this procedure deviate from the known values by less than 10% in each case and the average error for the test set of 21 proteins was 5.3%. Importantly, this method allows for an unambiguous determination of the multimeric state of proteins with known molecular weights.

Identification of archaeal proteins that affect the exosome function in vitro

Background: The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results: Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions: Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.