Effects of acute aerobic exercise on leukocyte inflammatory gene expression in systemic lupus erythematosus.

UNLABELLED: Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). METHODS: All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. RESULTS: In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively). CONCLUSION: The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response.

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation.

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

Characterization of bovine transcripts preferentially expressed in testis and with a putative role in spermatogenesis.

Although the number of genes known to be associated with bovine spermatogenesis has increased in the past few years, regulation of this biological process remains poorly understood. Therefore, discovery of new male fertility genetic markers is of great value for assisted selection in commercially important cattle breeds, e.g., Nelore, that have delayed reproductive maturation and low fertility rates. The objective of the present study was to identify sequences associated with spermatogenesis that could be used as fertility markers. With RT-PCR, the following five transcripts preferentially expressed in adult testis were detected: TET(656) detected only in adult testis; TET(868) and TET(515) expressed preferentially in adult testis but also detected in fetal gonads of both sexes; and TET(456) and TET(262,) expressed primarily in the testis, but also present in very low amounts in somatic tissues. Based on their homologies and expression profiles, we inferred that they had putative roles in spermatogenesis. Detection of sequences differentially expressed in testis, ovary, or both, was a useful approach for identifying new genes related to bovine spermatogenesis. The data reported here contributed to discovery of gene pathways involved in bovine spermatogenesis, with potential for prediction of fertility.

Validação comparativa utilizando PCR quantitativo em tempo real (qPCR) e PCR convencional de sêmen bovino centrifugado em gradiente de densidade contínuo / Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR) of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05). The percentage of female embryos in the Percoll and OptiPrep groups was 62.0 percent and 47.1 percent, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

O objetivo do presente estudo foi determinar o enriquecimento de espermatozoides portadores do cromossomo X após a centrifugação em gradiente de densidade contínuo de Percoll ou OptiPrep, utilizando reação em cadeia da polimerase quantitativa em tempo real (qPCR) do DNA do espermatozoide e dos embriões bovinos produzidos in vitro resultantes pela PCR convencional. Espermatozoides descongelado foram depositados em gradientes de densidade e os tubos foram centrifugados. Os sobrenadantes foram gentilmente aspirados e os espermatozoides recuperados do fundo dos tubos. As taxas de clivagem e de blastocisto foram determinadas pela produção in vitro de embriões e a PCR foi realizada para a identificação genética do sexo dos embriões. Verificou-se diferença na taxa de blastocistos entre os grupos Percoll e OptiPrep (P<0,05). A porcentagem de embriões de fêmeas nos grupos Percoll e OptiPrep foi de 62,0 por cento e 47,1 por cento, respectivamente. Estes resultados foram confirmados pela qPCR do DNA de espermatozoides e uma subestimação foi observada no grupo do gradiente de densidade de Percoll. Foi possível a sexagem de espermatozoides utilizando uma metodologia simples.

Development of Y-chromosome-specific SCAR markers conserved in taurine, zebu and bubaline cattle.

Sex pre-selection of bovine offsprings has commercial relevance for cattle breeders and several methods have been used for embryo sex determination. Polymerase chain reaction (PCR) has proven to be a reliable procedure for accomplishing embryo sexing. To date, most of the PCR-specific primers are derived from the few single-copy Y-chromosome-specific gene sequences already identified in bovines. Their detection demands higher amounts of embryonic genomic material or a nested amplification reaction. In order to circumvent this, limitation we searched for new male-specific sequences potentially useful in embryo sexing using random amplified polymorphic DNA (RAPD) analysis. Random amplified polymorphic DNA (RAPD) assay reproducibility problems can be overcome by its conversion into Sequence Characterized Amplified Region (SCAR) markers. In this work, we describe the identification of two bovine male-specific markers (OPC16(323) and OPF10(1168)) by means of RAPD. These markers were successfully converted into SCARs (OPC16(726) and OPF10(984)) using two pairs of specific primers.Furthermore, inverse PCR (iPCR) methodology was successfully applied to elongate OPC16(323) marker in 159% (from 323 to 837 bp). Both markers are shown to be highly conserved (similarity ≥95%) among bovine zebu and taurine cattle; OPC16(323) is also highly similar to a bubaline Y-chromosome-specific sequence. The primers derived from the two Y-chromosome-specific conserved sequences described in this article showed 100% accuracy when used for identifying male and female bovine genomic DNA, thereby proving their potential usefulness for bovine embryo sexing.

Ultrastructural characterization of CD133+ stem cells bound to superparamagnetic nanoparticles: possible biotechnological applications.

CD133 antigen is an integral membrane glycoprotein that can bind with different cells. Originally, however, this cellular surface antigen was expressed in human stem cells and in various cellular progenitors of the haematopoietic system. Human cord blood has been described as an excellent source of CD133(+) haematopoietic progenitor cells with a large application potential. One of the main objectives of the present study is to describe for the first time the ultrastructural characteristics of CD133(+) stem cells using transmission electronic microscopy. Another objective of the manuscript is to demonstrate through transmission electronic microscopy the molecular image of magnetic nanoparticles connected to the stem cells of great biotechnological importance, as well as demonstrating the value of this finding for electronic paramagnetic resonance and its related nanobioscientific value. Ultrastructural results showed the monoclonal antibody anti-CD133 bound to the superparamagnetic nanoparticles by the presence of electrondense granules in cell membrane, as well as in the cytoplasm, revealing the ultrastructural characteristics of CD133(+) cells, exhibiting a round morphology with discrete cytoplasmic projections, having an active nucleus that follows this morphology. The cellular cytoplasm was filled up with mitochondrias, as well as microtubules and vesicles pinocitic, characterizing the process as being related to internalization of the magnetic nanoparticles that were endocyted by the cells in question. Electronic paramagnetic resonance analysis of the CD133(+) stem cells detected that the signal (spectrum) generated by the labelled cells comes from the superparamagnetic nanoparticles that are bound to them. These results strongly suggest that these CD133(+) cells can be used in nanobiotechnology applications, with benefits in different biomedical areas.

In vitro study of CD133 human stem cells labeled with superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) are applied in stem cell labeling because of their high magnetic susceptibility as compared with ordinary paramagnetic species, their low toxicity, and their ease of magnetic manipulation. The present work is the study of CD133+ stem cell labeling by SPIONs coupled to a specific antibody (AC133), resulting in the antigenic labeling of the CD133+ stem cell, and a method was developed for the quantification of the SPION content per cell, necessary for molecular imaging optimization. Flow cytometry analysis established the efficiency of the selection process and helped determine that the CD133 cells selected by chromatographic affinity express the transmembrane glycoprotein CD133. The presence of antibodies coupled to the SPION, expressed in the cell membrane, was observed by transmission electron microscopy. Quantification of the SPION concentration in the marked cells using the ferromagnetic resonance technique resulted in a value of 1.70 x 10(-13) mol iron (9.5 pg) or 7.0 x 10(6) nanoparticles per cell (the measurement was carried out in a volume of 2 muL containing about 6.16 x 10(5) pg iron, equivalent to 4.5 x 10(11) SPIONs).

Falha na sexagem por inibição do desenvolvimento de embriões bovinos produzidos in vitro com anticorpos anti H-Y / Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1) embriões inibidos em estádio de mórula (classificados como machos) e 2) embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas). O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3 por cento, 5 por cento ou 7 por cento, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05) nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.

In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1) embryos arrested at morula stage (classified as males); and 2) embryos that developed and formed a blastocoele (classified as female). The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3 percent, 5 percent or 7 percent, was identified by polimerase chain reaction. The results showed no difference (P>0.05) on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.

Nitrogen-fixing chemo-organotrophic bacteria isolated from cyanobacteria-deprived lichens and their ability to solubilize phosphate and to release amino acids and phytohormones.

AIMS: Cyanobacteria-deprived lichens of the species Canoparmelia caroliniana, Canoparmelia crozalsiana, Canoparmelia texana, Parmotrema sancti-angeli and Parmotrema tinctorum were screened for the presence of chemo-organotrophic nitrogen-fixing bacteria. METHODS AND RESULTS: Fifty-three lichen samples subjected to enrichment selection using a nitrogen-free minimal medium were positive for acetylene reduction. Seventeen isolates, able to fix nitrogen, belonged to Gamma-proteobacteria group and were identified as: Acinetobacter sp., Pantoea sp., Pseudomonas sp., Pseudomonas stutzeri, Serratia marcescens and Stenotrophomonas maltophilia, according to 16S rRNA gene sequences and biochemical tests. The excretion of amino acid and phytohormone and the ability of mineral phosphate solubilization were determined in 14 isolates. All isolates were able to release amino acids and 3-indoleacetic acid. About 64% of the isolates solubilized phosphates and 30% released ethylene. CONCLUSIONS: These data confirm sparse evidence from the literature on the occurrence of chemo-organotrophic nitrogen-fixing bacteria in cyanobacteria-deprived lichens; the isolates presented physiologic features which might benefit the host if they are expressed when the bacteria are harboured by lichens. SIGNIFICANCE AND IMPACT OF THE STUDY: Chemo-organotrophic nitrogen-fixing bacteria were isolated from a high percentage (72.6%) of cyanobacteria-deprived lichens. All isolates presented important physiological characteristics, some of which are being described here for the first time.

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset.

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals (n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.

Sexing of murine and bovine embryos by developmental arrest induced by high-titer H-Y antisera.

Murine and bovine embryos at the late morula stage were cultured in medium containing high-titer rat H-Y antisera. After 12h of incubation, embryos blocked at the late morulae stage were classified as males and those at the blastocyst stage were classified as females. Sexing of murine embryos by PCR and cytogenetics revealed that 83% of the embryos classified as males and 82% of those classified as females had their sex correctly predicted (P < 0.05). Bovine embryos were transferred to recipient females. Pregnancy rates were 71.4% (10/14) for embryos classified as males and 68.8% (11/16) for embryos classified as females. The sex was correctly predicted for 80% (8/10) of the embryos classified as males and for 81.8% (9/11) of those classified as females (overall accuracy, 80.9%, P < 0.05). Therefore, the induction of developmental arrest by high-titer male-specific antisera was an efficient strategy for non-invasive embryo sexing. The procedure was straightforward and has considerable commercial potential for sexing bovine embryos.

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos.

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%.

Persistence of Müllerian remnants in complete androgen insensitivity syndrome.

One of the unusual findings in androgen insensitivity syndrome (AIS) is the persistence of Mullerian derivatives. Several hypotheses have been advanced to explain such persistence: the coincidental occurrence of mutations affecting the androgen receptor (AR) and the synthesis and/or action of anti-Müllerian hormone (AMH); the loss of AMH paracrine action due to early testicular descent; the exposure to drugs such as diethylstilbestrol. We describe a patient with complete AIS for whom surgical and laboratory findings rule out all these hypotheses. She has a missense mutation on the AR gene but no mutations were detected on the genes coding for AMH and AMH receptor. The gonads were found very close to the Mullerian structures (enough to exert a paracrine action), gonadal tissue stained positively for AMH, and yet Mullerian derivatives were present and well developed. These findings indicate the possibility of interactions between the androgen receptor and AMH action.

A polymorphism in endostatin, an angiogenesis inhibitor, predisposes for the development of prostatic adenocarcinoma.

We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.

Genetic differences between Escherichia coli O26 strains isolated in Brazil and in other countries.

Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup -- encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains -- was evaluated through random amplified polymorphic DNA (RAPD) analysis. Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters. RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H(-) serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data. The epidemiological significance of these data is discussed.

High serum endostatin levels in Down syndrome: implications for improved treatment and prevention of solid tumours.

We report here a comparison of serum endostatin levels in Down syndrome patients to normal control subjects. We analysed serum samples from 35 patients with Down syndrome and 54 normal control subjects and found that although serum levels of endostatin vary widely in a normal human population, serum endostatin levels are significantly elevated in patients with Down syndrome. This result may explain the relative decrease in incidence of various solid tissue tumours observed in Down syndrome, given the role of endostatin as a potent inhibitor of tumour-induced angiogenesis in both human and animal models. Based upon these data, we propose that an increase of about one-third of normal endostatin serum levels may represent an effective therapeutic dose to significantly inhibit many solid tumours.

X;Y translocation revealed by chromosome microdissection and FISH in fertile XY females in the Brazilian rodent Akodon montensis.

In a Brazilian population of the neotropical rodent Akodon montensis we found five sex-reversed XY females. These animals were cytogenetically analyzed by chromosome painting using species-specific DNA probes from the Y chromosome, generated by chromosomal microdissection and subsequent use of the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The results showed a chromosome complement with an apparently normal Y chromosome and an X chromosome carrying a translocation that encompasses a large portion of the Y chromosome (seemingly the entire Y). Ovarian histology suggested that these females are fertile. Amplification of the SRY HMG box sequence by PCR shows that at least one copy of the Sry gene is present in the A. montensis XY females. Based on our findings, we suggest that the breakpoint of the X;Y translocation probably altered an X-linked sex-determining locus (or loci), blocking testicular organogenesis in the XY females. Further studies are necessary to determine the precise location and role of this putative sex-determining chromosomal region. Genetic mechanisms of XY sex reversal in A. montensis populations are discussed.

Ullrich-Turner syndrome: relevance of searching for Y chromosome fragments.

Forty consecutive patients with Ullrich-Turner syndrome (UTS) were followed-up and investigated for the presence of Y chromosome fragments in their genomes. We used the polymerase chain reaction (PCR) to detect SRY (sex-determining region on the Y chromosome) and the sequence-tagged sites (STS) sY57, sY59, sY85, sY94, sY124 and sY157--which correspond to regions 3C (sY57 and 59), 5C, 5G, 5P, and 6F, respectively, of the Y chromosome--searching for Y fragments that could bear the putative locus (loci) for gonadoblastoma (GBY). It has been shown that the presence of GBY greatly increases the risk of dysgenic gonads to undergo malignant transformation. Among our 40 patients, we found Y-derived sequences--including SRY and the region spanning from sY57 to sY94--in two. These two patients had a marker chromosome detected by conventional cytogenetic analysis (45,X/46,X + mar). Their gonads were excised and found to be streaks. In one of the patients, we found foci of primitive sex cords (amidst the gonadal stroma), oviducts and Wolffian remnants. Fluorescence in situ hybridization (FISH) did not show Y chromosome material in her gonad-derived fibroblasts. The other girl had hyperplastic Leydig cells in the gonadal stroma, oviducts and Wolffian remnants, with signs of epididymal differentiation. PCR assays performed on DNA extracted from paraffin-embedded gonadal tissue were negative for SRY sequences in both patients. These findings show that all UTS patients should be examined for Y chromosome material, and that positive cases should have their dysgenic gonads excised due to the high risk of malignancy.

The same mutation affecting the splicing of WT1 gene is present on Frasier syndrome patients with or without Wilms' tumor.

Denys-Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA-Arg codon to a TGA-stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C-->T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS.