A naturally occurring recombinant isolate of Lettuce mosaic virus

LMV-Common and LMV-Most are two seed-borne types of Lettuce mosaic virus (LMV), genus Potyvirus. LMV-Most, but not LMV-Common, overcomes the resistance afforded to lettuce by two recessive genes, mo1(1) and mo1(2). An RT-PCR-based assay thought to be specific for LMV-Most also amplified LMV-Tn2, previously typified as LMV-Common. The sequence of selected regions along the genome indicated that LMV-Tn2 is a natural recombinant between LMV-Most and LMV-Common isolates, with a putative recombination site located within the P3 coding region. This is the first evidence of a naturally occurring LMV recombinant isolate.

Frequência de Lettuce big-vein associated virus e Mirafiori lettuce big-vein virus em Plantas de alface no Estado de São Paulo e transmissão através de extrato vegetal e solo

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

Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use. (C) 2004 Elsevier B.V. All rights reserved.

Tritrichomonas fetus extracellular products decrease progressive motility of bull sperm

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

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America. (c) 2007 Published by Elsevier B.V.

Effect of low-level laser irradiation on odontoblast-like cells

Low-level laser therapy (LLLT), also referred to as therapeutic laser, has been recommended for a wide array of clinical procedures, among which the treatment of dentinal hypersensitivity. However, the mechanism that guides this process remains unknown. Therefore, the objective of this study was to evaluate in vitro the effects of LLL irradiation on cell metabolism (MTT assay), alkaline phosphatase (ALP) expression and total protein synthesis. The expression of genes that encode for collagen type-1 (Col-1) and fibronectin (FN) was analyzed by RT-PCR. For such purposes, oclontoblast-like cell line (MDPC-23) was previously cultured in Petri dishes (15000 cells/cm(2)) and submitted to stress conditions during 12 h. Thereafter, 6 applications with a monochromatic near infrared radiation (GaAlAs) set at predetermined parameters were performed at 12-h intervals. Non-irradiated cells served as a control group. Neither the MTT values nor the total protein levels of the irradiated group differed significantly from those of the control group (Mann-Whitney test; p > 0.05). on the other hand, the irradiated cells showed a decrease in ALP activity (Mann-Whitney test; p < 0.05). RT-PCR results demonstrated a trend to a specific reduction in gene expression after cell irradiation, though not significant statistically (Mann-Whitney test; p > 0.05). It may be concluded that, under the tested conditions, the LLLT parameters used in the present study did not influence cell metabolism, but reduced slightly the expression of some specific proteins.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

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

Survey for Foot-and-mouth Disease in the Endangered Marsh Deer (Blastocerus dichotomus) from Marshlands of the Parana River Basin, Brazil

Habitat fragmentation and diseases have resulted in a decline of the marsh deer (Blastocerus (dichotomus) throughout its South American range. Our objectives were to determine whether marsh deer intended for translocation from a region of the Rio Parana Basin had been infected previously by foot-and-mouth disease virus (FMDV) and whether they were carrying virus We captured marsh deer from June to October 1998 and collected blood from 108 animals and esophageal-pharyngeal fluid from 53 Serum was tested for antibodies against three FMDV serotypes (O, A, and C) by liquid-phase-blocking sandwich enzyme-linked immunosorbent assay (ELISA) Esophageal-pharyngeal fluid was tested for FMDV RNA by reverse transcription polymerase chain reaction (RT-PCR) and inoculation into three successive baby hamster kidney (BHK-21) cell subcultures, followed by RT-PCR of cultures We detected low log(10) titers (range 1 0-1 5) to FM DV subtype A(24) Cruzeiro in 19 of 108 sampled marsh deer, but failed to isolate FMDV or detect FMDV RNA in any samples we conclude that marsh deer from our study site were unlikely to carry FMDV, however, as a preventive measure, the 19 animals with titers for FMDV were not sent to FMDV-free Brazilian states

Migratory route of Strongyloides venezuelensis in Lewis rats: Comparison of histological analyses and PCR

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

Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells

Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500 ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500 ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500 ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500 ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation. (C) 2011 Elsevier B.V. All rights reserved.

Opposite effects of bFGF and TGF-beta on collagen metabolism by human periodontal ligament fibroblasts

This study evaluated the effects of bFGF and TGF-beta, individually and combined, on cell proliferation and collagen metabolism. Primary human periodontal ligament cells were stimulated with two concentrations (I and 10 ng/ml) of each growth factor, both individually and combined. Proliferation was determined by a commercial biochemical assay. Real time RT-PCR determined gene expression of NMP-1 and -2, collagen types I and III, TIMP-1, -2 and -3. Autocrine effects on synthesis of bFGF and TGF-beta were evaluated by ELISA. Only TGF-beta, either isolated or associated with bFGF, significantly increased cell proliferation. TGF-beta had anabolic effects, increasing expression of type I and III collagen as well as of TIMPs, whereas bFGF had opposite effects. When bFGF and TGF-beta were associated, the anabolic effects prevailed. Synthesis of TGF-beta was induced only by the association of lower concentrations of the growth factors, whereas there was a dose-dependent production of bFGF. It is concluded that bFGF had a predominantly catabolic effect, and TGF-beta exerted an anabolic effect on hPDL cells. (c) 2007 Elsevier Ltd. All rights reserved.

Loss of imprinting and loss of heterozygosity on 11p15.5 in head and neck squamous cell carcinomas

Background. IGF2 and H19 are reciprocal imprinted genes with paternal and maternal monoallelic expression, respectively. This is interesting, because IGF2 is known as a growth factor, and H19 encodes a RNA with putative tumor suppressor action. Furthermore, IGF2 and H19 are linked genes located on chromosome 11p15.5, a common site of loss of heterozygosity in human cancers.Methods. We performed an allelic-typing assay using a PCR-RFLP-based method for identification of heterozygous Informative cases in head and neck squamous cell carcinomas. Tumoral total RNA was extracted from each of the heterozygotes and further studied by RT-PCR analysis.Results. We detected the expression of the IGF2 gene in 10 of 10 informative cases. Two cases exhibited LOI of the IGF2 gene as evidenced by biallelic expression, and in another case, LOH was coupled with monoallelic expression of this growth factor. LOI for the H19 gene was observed in 1 of 14 informative samples analyzed. In this case, we also detected parallel mono-allelic expression of the IGF2 gene. Down-regulation of the H19 gene was observed in 10 of 14 cases.Conclusion. These findings support the hypothesis that H19 may be a tumor suppressor gene involved In head and neck carcinogenesis. Furthermore, our data showed that genetic and epigenetic chances at 11p15.5 could lead to abnormal expression of imprinted genes in HNSCC. (C) 2001 John Wiley & Sons, Inc.

Progesterone upregulates GATA-1 on erythroid progenitors cells in liquid culture

Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects. (C) 2002 Elsevier B.V. (USA).

Leishmania chagasi infection in cats with dermatologic lesions from an endemic area of visceral leishmaniosis in Brazil

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

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5

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