Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naive control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naive ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

Identical sequence patterns in the ends of exons and introns of human protein-coding genes

Intron splicing is one of the most important steps involved in the maturation process of a pre-mRNA. Although the sequence profiles around the splice sites have been studied extensively, the levels of sequence identity between the exonic sequences preceding the donor sites and the intronic sequences preceding the acceptor sites has not been examined as thoroughly. In this study we investigated identity patterns between the last 15 nucleotides of the exonic sequence preceding the 5' splice site and the intronic sequence preceding the 3' splice site in a set of human protein-coding genes that do not exhibit intron retention. We found that almost 60% of consecutive exons and introns in human protein-coding genes share at least two identical nucleotides at their 3' ends and, on average, the sequence identity length is 2.47 nucleotides. Based on our findings we conclude that the 3' ends of exons and introns tend to have longer identical sequences within a gene than when being taken from different genes. Our results hold even if the pairs are non-consecutive in the transcription order. (C) 2012 Elsevier Ltd. All rights reserved.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Comparative study of reserve lipid accumulation during somatic and zygotic Acca sellowiana (O. Berg.) Burret embryogenesis

Somatic embryogenesis is an in vitro morphogenetic route in which isolated cells or a small group of somatic cells give rise to bipolar structures resembling zygotic embryos. Lipids, carbohydrates, and proteins are major compounds in plant and animal metabolism. Comparative analysis along different developmental stages of Acca sellowiana (Myrtaceae) zygotic and somatic embryos, revealed a progressive increase in levels of total lipids. A high degree of similarity could be found in the total lipids composition between A. sellowiana somatic and zygotic embryos. High lipid levels were found in zygotic embryos in the torpedo and cotyledonary stages, and these levels increased according to the progression in the developmental stages. Somatic embryos obtained through direct embryogenesis route showed higher levels of lipids than in indirect somatic embryogenesis. The compounds most frequently were linoleic acid (C18:2), palmitic (C16:0) and oleic (C18:1). These results indicate a high similarity degree of accumulation of total lipids, regardless of zygotic or somatic embryogenesis.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbata (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbata for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiol levels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.

Synchrotron X-ray diffraction characterization of healthy and fluorotic human dental enamel

With the introduction of fluoride as the main anticaries agent used in preventive dentistry, and perhaps an increase in fluoride in our food chain, dental fluorosis has become an increasing world-wide problem. Visible signs of fluorosis begin to become obvious on the enamel surface as opacities, implying some porosity in the tissue. The mechanisms that conduct the formation of fluorotic enamel are unknown, but should involve modifications in the basic physical-chemistry reactions of demineralization and remineralisation of the enamel of the teeth, which is the same reaction of formation of the enamel's hydroxyapatite (HAp) in the maturation phase. The increase of the amount of fluoride inside of the apatite will result in gradual increase of the lattice parameters. The aim of this work is to characterize the healthy and fluorotic enamel in human tooth using Synchrotron X-ray diffraction. All the scattering profile measurements were carried out at the X-ray diffraction beamline (XRD1) at the Brazilian Synchrotron Light Laboratory-LNLS, Campinas, Brazil. X-ray diffraction experiments were performed both in powder samples and polished surfaces. The powder samples were analyzed to obtain the characterization of a typical healthy enamel pattern. The polished surfaces were analyzed in specific areas that have been identified as fluorotic ones. X-ray diffraction data were obtained for all samples and these data were compared with the control samples and also with the literature data. (c) 2012 Elsevier Ltd. All rights reserved.

Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in Essential Thrombocythemia and Myelofibrosis

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34(+) hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters. Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34(+) cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34(+) cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly. Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

Proteomic analysis of papaya fruit ripening using 2DE-DIGE

Papayas have a very short green life as a result of their rapid pulp softening as well as their susceptibility to physical injury and mold growth. The ripening-related changes take place very quickly, and there is a continued interest in the reduction of postharvest losses. Proteins have a central role in biological processes, and differential proteomics enables the discrimination of proteins affected during papaya ripening. A comparative analysis of the proteomes of climacteric and pre-climacteric papayas was performed using 2DE-DIGE. Third seven proteins corresponding to spots with significant differences in abundance during ripening were submitted to MS analysis, and 27 proteins were identified and classified into six main categories related to the metabolic changes occurring during ripening. Proteins from the cell wall (alpha-galactosidase and invertase), ethylene biosynthesis (methionine synthase), climacteric respiratory burst, stress response, synthesis of carotenoid precursors (hydroxymethylbutenyl 4-diphosphate synthase, GcpE), and chromoplast differentiation (fibrillin) were identified. There was some correspondence between the identified proteins and the data from previous transcript profiling of papaya fruit, but new, accumulated proteins were identified, which reinforces the importance of differential proteomics as a tool to investigate ripening and provides potentially useful information for maintaining fruit quality and minimizing postharvest losses. (C) 2011 Elsevier B.V. All rights reserved.

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n = 45) received daily injections of alendronate (n = 27) or sterile saline solution as control (n = 18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.

Linear and nonlinear measures of fetal heart rate patterns evaluated on very short fetal magnetocardiograms

We analyzed the effectiveness of linear short- and long-term variability time domain parameters, an index of sympatho-vagal balance (SDNN/RMSSD) and entropy in differentiating fetal heart rate patterns (fHRPs) on the fetal heart rate (fHR) series of 5, 3 and 2 min duration reconstructed from 46 fetal magnetocardiograms. Gestational age (GA) varied from 21 to 38 weeks. FHRPs were classified based on the fHR standard deviation. In sleep states, we observed that vagal influence increased with GA, and entropy significantly increased (decreased) with GA (SDNN/RMSSD), demonstrating that a prevalence of vagal activity with autonomous nervous system maturation may be associated with increased sleep state complexity. In active wakefulness, we observed a significant negative (positive) correlation of short-term (long-term) variability parameters with SDNN/RMSSD. ANOVA statistics demonstrated that long-term irregularity and standard deviation of normal-to-normal beat intervals (SDNN) best differentiated among fHRPs. Our results confirm that short-and long-term variability parameters are useful to differentiate between quiet and active states, and that entropy improves the characterization of sleep states. All measures differentiated fHRPs more effectively on very short HR series, as a result of the fMCG high temporal resolution and of the intrinsic timescales of the events that originate the different fHRPs.

Evaluation of a Maxillary Sinus Floor Augmentation in the Presence of a Large Antral Pseudocyst

Sinus floor augmentation has been established as a predictable technique to overcome maxillary bone loss. Antral cystic lesions may lead to intrasurgical complications and should be accurately diagnosed. However, antral pseudocysts have recently been described not to be contraindicated for sinus-grafting procedures. The current article sought to report clinical, cone beam computed tomographic, and histologic results of a maxillary sinus floor augmentation, performed with piezoelectric surgery, in the presence of a large antral pseudocyst. Success of graft maturation was confirmed with histologic analysis, which also indicated the absence of inflammatory infiltration in the tissue evaluated. On the basis of our findings, it is possible to perform a predictable treatment based on sinus floor augmentation in the presence of antral pseudocysts. Graft maturation can also be achieved 6 months after sinus-lifting surgeries.

Gene expression during early somatic embryogenesis in Brazilian pine (Araucaria angustifolia (Bert) O. Ktze)

Brazilian pine (Araucaria angustifolia (Bert) O. Ktze) is the only native conifer species with economic importance in Brazil. Recently, due to intensive exploitation Brazilian pine was included in the official list of endangered Brazilian plants, under the "vulnerable" category. Biotechnology tools like somatic embryogenesis (SE) are potentially useful for mass clonal propagation and ex situ conservation strategies of commercial and endangered plant species. In spite of that, numerous obstacles still hamper the full application of SE technology for a wider range of species, including Brazilian pine. To enhance somatic embryogenesis in Brazilian pine and to gain a better understanding of the molecular events associated with somatic embryo development, we analyzed the steady-state transcript levels of genes known to regulate somatic embryogenesis using semiquantitative reverse transcription polymerase chain reaction (sqRT-PCR). These genes included Argonaute (AaAGO), Cup-shaped cotyledon1 (AaCUC), wushel-related WOX (AaWOX), a S-locus lectin protein kinase (AaLecK), Scarecrow- like (AaSCR), Vicilin 7S (AaVIC), Leafy Cotyledon 1 (AaLEC), and a Reversible glycosylated polypeptide (AaRGP). Expression patterns of these selected genes were investigated in embryogenic cultures undergoing different stages of embryogenesis, and all the way to maturation. Up-regulation of AaAGO, AaCUC, AaWOX, AaLecK, and AaVIC was observed during transition of somatic embryos from stage I to stage II. During the maintenance phase of somatic embryogenesis, expression of AaAGO and AaSCR, but not AaRPG and AaLEC genes was influenced by presence/ absence of plant growth regulators, both auxins and cytokinins. The results presented here provide new insights on the molecular mechanisms responsible for somatic embryo formation, and how selected genes may be used as molecular markers for Brazilian pine embryogenesis.

Lower numbers of natural killer T cells in HIV-1 and Mycobacterium leprae co-infected patients

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes that recognize antigens presented by CD1d and have attracted attention because of their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on peripheral blood mononuclear cells. We demonstrated that patients co-infected with HIV-1 and M.leprae have significantly lower NKT cell frequencies [median 0.022%, interquartile range (IQR): 0.0070.051] in the peripheral blood when compared with healthy subjects (median 0.077%, IQR: 0.0320.405, P < 0.01) or HIV-1 mono-infected patients (median 0.072%, IQR: 0.0300.160, P < 0.05). Also, more NKT cells from co-infected patients secreted interferon-? after stimulation with DimerX, when compared with leprosy mono-infected patients (P = 0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and M.leprae co-infected patients compared with HIV-1 mono-infected patients alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1-infected subjects, a concurrent mycobacterial infection probably hyper-activates and lowers circulating NKT cell numbers.

Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (54) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.