Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension

Objective To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. Results A multilevel factorial design was used to quantify effects of temperature (33–37 C), fresh medium addition after the viral adsorption step (100–200 % with respect to the initial cell suspension volume before infection) and harvest time (8–40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml-1 ) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml-1 ). Conclusion These results establish the basis for designing bioprocess to produce RVGP.

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular mapping of the viral determinants of systemic wilting induced by a Lettuce mosaic virus (LMV) isolate in some lettuce cultivars

The isolate AF199 of Lettuce mosaic virus (LMV, genus Potyvirus) causes local lesions followed by systemic wilting and plant death in the lettuce cultivars Ithaca and Vanguard 75. Analysis of the phenotype of virus chimeras revealed that a region within the PI protein coding region (nucleotides 112-386 in the viral genome) and/or another one within the CI protein coding region (nucleoticles 5496-5855) are sufficient together to cause the lethal wilting in Ithaca, but not in Vanguard 75. This indicates that the determinants of this particular symptom are different in these two lettuce cultivars. The wilting phenotype was not directly correlated with differences in the deduced amino acid sequence of these two regions. Furthermore, transient expression of the LMV-AF 199 proteins, separately or in combination, did not induce local necrosis or any other visible reaction in the plants. Together, these results Suggest that the systemic wilting reaction might be Clue to RNA rather than protein sequences. (c) 2004 Elsevier B.V. All rights reserved.

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutations in the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups. Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. of these,14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAG-->TGG, X314W), and one splice acceptor site mutation (IVS1 - 2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1 - 2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.

Identificação e caracterização de promotores de genes de café (coffea arabica)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolamento e caracterização de promotores órgão-específicos a partir de informações do Banco FORESTs (Eucalyptus Genome Sequencing Project Consortium)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Semliki Forest Virus as a Vector: Pros and Cons for Its Use in Biopharmaceuticals Production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Short communication: Acute but transient increase in serum insulin reduces messenger RNA expression of hepatic enzymes associated with progesterone catabolism in dairy cows

The objective of this experiment was to evaluate the effects of glucose infusion on serum concentrations of glucose, insulin, and progesterone (P4), as well as mRNA expression of hepatic CYP2C19 and CYP3A4 in nonlactating, ovariectomized cows in adequate nutritional status. Eight Gir × Holstein cows were maintained on a low-quality Brachiaria brizantha pasture with reduced forage availability, but they individually received, on average, 3. kg/cow daily (as fed) of a corn-based concentrate from d -28 to 0 of the experiment. All cows had an intravaginal P4-releasing device inserted on d -14, which remained in cows until the end of the experiment (d 1). On d 0, cows were randomly assigned to receive, in a crossover design containing 2 periods of 24. h each (d 0 and 1), (1) an intravenous glucose infusion (GLUC; 0.5. g of glucose/kg of BW, over a 3-h period) or (2) an intravenous saline infusion (SAL; 0.9%, over a 3-h period). Cows were fasted for 12. h before infusions, and they remained fasted during infusion and sample collections. Blood samples were collected at 0, 3, and 6. h relative to the beginning of infusions. Liver biopsies were performed concurrently with blood collections at 0 and 3. h. After the last blood collection of period 1, cows received concentrate and returned to pasture. Cows gained BW (16.5 ± 3.6. kg) and BCS (0.08 ± 0.06) from d -28 to 0. Cows receiving GLUC had greater serum glucose and insulin concentrations at 3. h compared with SAL cohorts. No treatment effects were detected for serum P4 concentrations, although mRNA expression of CYP2C19 and CYP3A4 after the infusion period was reduced for cows in the GLUC treatment compared with their cohorts in the SAL treatment. In conclusion, hepatic CYP3A4 and CYP2C19 mRNA expression can be promptly modulated by glucose infusion followed by acute increases in circulating insulin, which provides novel insight into the physiological mechanisms associating nutrition and reproductive function in dairy cows. © 2013 American Dairy Science Association.

Genotoxicity, cytotoxicity and gene expression in patients undergoing elective surgery under isoflurane anaesthesia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Signaling pathways associated with the expression of inflammatory mediators activated during the course of two models of experimental periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1 beta and TNF-alpha in immortalized periodontal ligament fibroblasts

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1 beta (5 ng/mL) and TNF-alpha (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1 induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1 beta stimulation. Mmp-13 mRNA expression induced by TNF-alpha was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Behavioural Analysis of a Nonlinear Mechanical System Using Transient Trajectories

This work aims at a better comprehension of the features of the solution surface of a dynamical system presenting a numerical procedure based on transient trajectories. For a given set of initial conditions an analysis is made, similar to that of a return map, looking for the new configuration of this set in the first Poincaré sections. The mentioned set of I.C. will result in a curve that can be fitted by a polynomial, i.e. an analytical expression that will be called initial function in the undamped case and transient function in the damped situation. Thus, it is possible to identify using analytical methods the main stable regions of the phase portrait without a long computational time, making easier a global comprehension of the nonlinear dynamics and the corresponding stability analysis of its solutions. This strategy allows foreseeing the dynamic behavior of the system close to the region of fundamental resonance, providing a better visualization of the structure of its phase portrait. The application chosen to present this methodology is a mechanical pendulum driven through a crankshaft that moves horizontally its suspension point.

p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts

Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.

Morphology, sex ratio and gene expression of Day 14 in vivo and in vitro bovine embryos

The present study was designed to compare Day 14 bovine embryos that were produced entirely in vitro using the post-hatching development (PHD) system with in vivo-derived embryos without or with transient PHD culture from Day 7 to Day 14. Embryos on Day 14 were used for sex determination and gene expression analysis of PLAC8, KRT8, CD9, SLC2A1, SLC2A3, PGK1, HSF1, MNSOD, HSP70 and IFNT using real-time quantitative (q) polymerase chain reaction (PCR). First, Day 7 in vivo-and in vitro-produced embryos were subjected to the PHD system. A higher rate of survival was observed for in vitro embryos on Day 14. Comparing Day 14 embryos produced completely in vivo or completely in vitro revealed that the mean size of the former group was greater than that of the latter (10.29±1.83 vs 2.68±0.33mm, respectively). Expression of the HSP70 and SLC2A1 genes was down-and upregulated, respectively, in the in vitro embryos. The present study shows that in vitro embryos cultured in the PHD system are smaller than in vivo embryos and that of the 10 genes analysed, only two were differentially expressed between the two groups. These findings indicate that, owing to the poor survival rate, the PHD system is not reliable for evaluation of in vitro embryo quality. © 2013 CSIRO.

SOCS3 expression correlates with severity of inflammation, expression of proinflammatory cytokines, and activation of STAT3 and p38 MAPK in LPS-induced inflammation in vivo

SOCS3 is an inducible endogenous negative regulator of JAK/STAT pathway, which is relevant in inflammatory conditions. We used a model of LPS-induced periodontal disease in rats to correlate SOCS3 expression with the inflammatory status. In vitro we used a murine macrophage cell line to assess the physical interaction between SOCS3 and STAT3 by coimmunoprecipitation. 30 ug of LPS from Escherichia coli were injected in the gingival tissues on the palatal aspect of first molars of the animals 3x/week for up to 4 weeks. Control animals were injected with the vehicle (PBS). The rats were sacrificed at 7, 15, and 30 days. Inflammation and gene expression were assessed by stereometric analysis, immunohistochemistry, RT-qPCR, and western blot. LPS injections increased inflammation, paralleled by an upregulation of SOCS3, of the proinflammatory cytokines IL-1β, IL-6, and TNF-and increased phosphorylation of STAT3 and p38 MAPK. SOCS3 expression accompanied the severity of inflammation and the expression of proinflammatory cytokines, as well as the activation status of STAT3 and p38 MAPK. LPS stimulation in a macrophage cell line in vitro induced transient STAT3 activation, which was inversely correlated with a dynamic physical interaction with SOCS3, suggesting that this may be a mechanism for SOCS3 regulatory function. © 2013 João Antônio Chaves de Souza et al.