Functional and molecular characterization of Tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK, receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RTPCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin INK, receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK, receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK, receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

The transcriptome of Populus in elevated CO2

The consequences of increasing atmospheric carbon dioxide for long-term adaptation of forest ecosystems remain uncertain, with virtually no studies undertaken at the genetic level. A global analysis using cDNA microarrays was conducted following 6 yr exposure of Populus × euramericana (clone I-214) to elevated [CO2] in a FACE (free-air CO2 enrichment) experiment.• Gene expression was sensitive to elevated [CO2] but the response depended on the developmental age of the leaves, and < 50 transcripts differed significantly between different CO2 environments. For young leaves most differentially expressed genes were upregulated in elevated [CO2], while in semimature leaves most were downregulated in elevated [CO2].• For transcripts related only to the small subunit of Rubisco, upregulation in LPI 3 and downregulation in LPI 6 leaves in elevated CO2 was confirmed by anova. Similar patterns of gene expression for young leaves were also confirmed independently across year 3 and year 6 microarray data, and using real-time RT–PCR.• This study provides the first clues to the long-term genetic expression changes that may occur during long-term plant response to elevated CO2.

Detection of tobacco mosaic tobamovirus in cigarettes through RT-PCR

TMV was tried to recover from a variety of branded cigarettes and cigars. Tobacco from six different brands of cigarettes and cigar were processed and reverse transcriptase polymerase chain reaction was employed for the detection of TMV. RTPCR confirmed the presence of TMV in tobacco from one brand of cigarette and one brand of cigar. Bean plants (Phaseolus vulgaris) were inoculated manually with tobacco sap of cigarettes resulting in the production of localized disease lesions. Together, these results showed that tobacco used to make cigarettes and cigars can function as an effective disease vector, potentially aiding the movement of infectious TMV between countries. This is an important finding prompting a need to test smoking tobacco for other virus particles that infect tobacco plants and survive processing as well as considering biosecurity measures to limit virus transmission

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

Relevance of Budgerigars (Melopsittacus undulatus) in Experimental Epidemiology of Newcastle Disease.

This study was carried out to clarify the real role that was played by the budgerigars (Melopsittacus undulatus) in the epidemiological plan, under the perspective of its being an infection source of the Newcastle Disease Virus (NDV). For this, the study used Specific-Pathogen-Free chicks (SPF) that were housed with budgerigars that were inoculated with a pathogenic strain (velogenic viscerotropic) of NDV (EID5o =10815/0.1 mL) pathogenic to chickens, by the ocular-nasal via. Each group was composed by 10 SPF chicks and 5 budgerigars. After 5 days of the inoculation of the budgerigars with NDV, SPF chicks were put together with each group of budgerigars, so that there was a direct contact between both species. Cloaca) swabs and blood samples were collected in both species (budgerigars and SPF chicks) after 13 and 19 days post-challenge, respectively, for genome viral excretion by Reverse Transcription Polymerase Chain Reaction (RTPCR) and antibody's search by the inhibition of hemmaglutination test (HI). Budgerigars did not demonstrate any clinical signs of Newcastle Disease (ND). They were refractory to the clinical disease with the NDV. However, antibody titres from inhibition of Hemagglutination (HI) test were detected 9 and 21 days after challenge. Therefore, it was demonstrated the state of carrier of NDV in this species. In SPF chicks allocated with infected budgerigars, NDV genome was detected 13 and 19 days after challenge. Thus, the transmission of the pathogenic virus from the budgerigars to SPF chicks that were housed together was evident until 19 days of the experimental infection with this pathogen. This reveals the importance of the budgerigars from the epidemiological point of view as a potential source of infection of the NDV to commercial chickens that could be raised near this species.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Differential expression of myogenic regulatory factor MyoD in pacu skeletal muscle (Piaractus mesopotamicus Holmberg 1887: Serrasalminae, Characidae, Teleostei) during juvenile and adult growth phases

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

Atividade do micronutriente selênio na cinética de proliferação celular, citotoxicidade, indução de apoptose e expressão dos genes CASP9, BCL-XL e APC em células HT29

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção in vitro da hepatite C (VHC) em plaquetas provenientes de indivíduoas não infectados expostas ao vírus

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Resultado reagente próximo ao limiar de reaticidade: uma dificuldade no diagnóstico laboratorial da hepatite C

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Detecção e expressão dos genes supressores p53 E c-Myc em tumores palpebrais de cães

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

Comparação entre as técnicas de RT-PCR e inoculação intracerebral em camundongos para detecção do vírus da raiva em amostras mantidas por longos períodos em diferentes estados de conservação

Pós-graduação em Ciência Animal - FMVA

Detecção e diferenciação do vírus da bronquite infecciosa pela técnica de imunocaptura-RT-PCR E RFLP

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)