Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of CH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.

Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require the use of radioactivity and usually rely upon subjective determination of the results. We have developed two competitive quantitative assays that use an enzyme-linked, amplicon hybridization assay (ELAHA) to detect the products of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved to be simple, rapid, and sensitive, avoiding the need for Southern hybridization, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rapidly detect proviral DNA from any retroviral vector carrying the common selective and marker genes neomycin phosphotransferase and green fluorescent protein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

Detection of Influenza type A virus by LightCycler RT-PCR

Using the Roche LightCycler we developed a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay using the Influenza A LightCycler RT-PCR (FA-LC-RTPCR) for the rapid detection of Influenza A. The assay was used to examine 178 nasopharyngeal aspirate (NPA) samples, from patients with clinically recognised respiratory tract infection, for the presence of Influenza A RNA. The results were then compared to a testing algorithm combining direct immunofluorescent assy (DFA) and a culture augmented DFA (CA-DFA) assay. In total, 76 (43%) specimens were positive and 98 (55%) specimens were negative by both the FA-LC-RTPCR and the DFA and CA-DFA algorithm. In addition, the FA-LC-RTPCR detected a further 4 (2%) positive specimens, which were confirmed by a conventional RT-PCR method. The high level of sensitivity and specificity, combined with the rapid turnaround time for results, makes the LC-RT-PCR assay suitable for the detection of Influenza A in clinical specimens.

Field evaluation of a sentinel mosquito (Diptera : culicidae) trap system to detect Japanese encephalitis in remote Australia

Incursions of Japanese encephalitis (JE) virus into northern Queensland are currently monitored using sentinel pigs. However, the maintenance of these pigs is expensive, and because pigs are the major amplifying hosts of the virus, they may contribute to JE transmission. Therefore, we evaluated a mosquito-based detection system to potentially replace the sentinel pigs. Single, inactivated JE-infected Culex annulirostris Skuse and C. sitiens Wiedemann were placed into pools of uninfected mosquitoes that were housed in a Mosquito Magnet Pro (MM) trap set under wet season field conditions in Cairns, Queensland for 0, 7, or 14 d. JE viral RNA was detected (cycling threshold [CT] = 40) in 11/ 12, 10/14, and 2/5 pools containing 200, 1,000, and 5,000 mosquitoes, respectively, using a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability to detect virus was not affected by the length of time pools were maintained under field conditions, although the CT score tended to increase with field exposure time. Furthermore, JE viral RNA was detected in three pools of 1,000 mosquitoes collected from Badu Island using a MM trap. These results indicated that a mosquito trap system employing self-powered traps, such as the MosquitoMagnet, and a real-time PCR system, could be used to monitor for JE in remote areas.

Decreased coumarin 7-hydroxylase activities and CYP2A6 expression levels in humans caused by genetic polymorphism in CYP2A6 promoter region (CYP2A6*9)

One of seven poor metabolizers of coumarin found in Thai subjects was previously genotyped as heterozygote for the CYP2A6*4 (whole deletion) and CYP2A6*9. Thus, we aimed to investigate the relationship between the genetic polymorphism in the TATA box of the CYP2A6 gene (CYP2A6*9), expression levels of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers. Levels of CYP2A6 mRNA were quantified by real-time quantitative reverse transcriptase-polymerase chain reaction. The mean expression levels of CYP2A6 mRNA in individuals with CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 58%, 71% and 21% of the individuals genotyped as CYP2A6*1/*1, respectively. The mean in-vitro coumarin 7-hydroxylase activities in subjects carrying CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 41%, 71% and 12%, respectively, compared to those of the subjects judged as wild-type. Vmax values for coumarin 7-hydroxylation in the liver microsomes from human subjects with genotypes of CYP2A6*1/*1, CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 0.58, 0.26, 0.44 and 0.13 nmol/min/nmol total P450, respectively. CYP2A6 protein levels in human liver microsomes with the CYP2A6*4 and the CYP2A6*9 alleles were markedly decreased. These results suggest that the genetic polymorphism in the promoter region of the CYP2A6 gene (CYP2A6*9) reduced the expression levels of CYP2A6 mRNA and protein in human livers, resulting in the decrease of coumarin 7-hydroxylase activities. Individuals judged as CYP2A6*4/*9 were expected to be poor metabolizers, having extremely low activity of CYP2A6.

Differential expression of the eukaryotic release factor 3 (eRF3/GSPT1) according to gastric cancer histological types

Background: There are now several lines of evidence to suggest that protein synthesis and translation factors are involved in the regulation of cell proliferation and cancer development. Aims: To investigate gene expression patterns of eukaryotic releasing factor 3 (eRF3) in gastric cancer. Methods: RNA was prepared from 25 gastric tumour biopsies and adjacent non-neoplastic mucosa. Real time TaqMan reverse transcription polymerase chain reaction (RT-PCR) was performed to measure the relative gene expression levels. DNA was isolated from tumour and normal tissues and gene dosage was determined by a quantitative real time PCR using SYBR Green dye. Results: Different histological types of gastric tumours were analysed and nine of the 25 tumours revealed eRF3/GSPT1 overexpression; moreover, eight of the 12 intestinal type carcinomas analysed overexpressed the gene, whereas eRF3/GSPT1 was overexpressed in only one of the 10 diffuse type carcinomas (Kruskal-Wallis Test; p , 0.05). No correlation was found between ploidy and transcript expression levels of eRF3/GSPT1. Overexpression of eRF3/GSPT1 was not associated with increased translation rates because the upregulation of eRF3/GSPT1 did not correlate with increased eRF1 levels. Conclusions: Overexpression of eRF3/GSPT1 in intestinal type gastric tumours may lead to an increase in the translation efficiency of specific oncogenic transcripts. Alternatively, eRF3/GSPT1 may be involved in tumorigenesis as a result of its non-translational roles, namely (dis)regulating the cell cycle, apoptosis, or transcription.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

Differential expression of GSPT1 GGCn alleles in cancer

The human eukaryotic release factor 3a (eRF3a), encoded by the G1 to S phase transition 1 gene (GSPT1; alias eRF3a), is upregulated in various human cancers. GSPT1 contains a GGCn polymorphism in exon 1, encoding a polyglycine expansion in the N-terminal of the protein. The longer allele, GGC12, was previously shown to be associated to cancer. The GGC12 allele was present in 2.2% of colorectal cancer patients but was absent in Crohn disease patients and in the control group. Real-time quantitative RT-PCR analysis showed that the GGC12 allele was present at up to 10-fold higher transcription levels than the GGC10 allele (P < 0.001). No GSPT1 amplifications were detected, and there was no correlation between the length of the alleles and methylation levels of the CpG sites inside the GGC expansion. Using flow cytometry, we compared the levels of apoptosis and proliferation rates between cell lines with different genotypes, but detected no significant differences. Finally, we used a cytokinesis-block micronucleus assay to evaluate the frequency of micronuclei in the same cell lines. Cell lines with the longer alleles had higher frequencies of micronuclei in binucleated cells, which is probably a result of defects in mitotic spindle formation. Altogether, these findings indicate that GSPT1 should be considered a potential proto-oncogene.

Modulation of translation factor's gene expression by histone deacetylase inhibitors in breast cancer cells

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

A feedback-based decentralised coordination model for distributed open real-time systems

Moving towards autonomous operation and management of increasingly complex open distributed real-time systems poses very significant challenges. This is particularly true when reaction to events must be done in a timely and predictable manner while guaranteeing Quality of Service (QoS) constraints imposed by users, the environment, or applications. In these scenarios, the system should be able to maintain a global feasible QoS level while allowing individual nodes to autonomously adapt under different constraints of resource availability and input quality. This paper shows how decentralised coordination of a group of autonomous interdependent nodes can emerge with little communication, based on the robust self-organising principles of feedback. Positive feedback is used to reinforce the selection of the new desired global service solution, while negative feedback discourages nodes to act in a greedy fashion as this adversely impacts on the provided service levels at neighbouring nodes. The proposed protocol is general enough to be used in a wide range of scenarios characterised by a high degree of openness and dynamism where coordination tasks need to be time dependent. As the reported results demonstrate, it requires less messages to be exchanged and it is faster to achieve a globally acceptable near-optimal solution than other available approaches.

Implicações imunobiológicas da infecção a herpesvirus em tumores malignos : pesquisa e análise quantitativa de herpesvirus humanos no astrocitoma pilocítico

RESUMO: As infecções virais podem contribuir para o desenvolvimento do cancro, estando vários tumores malignos associados aos Herpesvirus (HHV). O vírus de Epstein-Barr (EBV) e o Herpesvirus 8, dois Herpesvirus, foram reconhecidos como agentes etiológicos de várias neoplasias. O astrocitoma pilocítico do cerebelo é um dos tumores cerebrais mais frequentes na criança, adolescentes e jovens adultos e a proliferação astrocitária ocorre geralmente após vários tipos de agressão, nomeadamente a infecção viral. Para investigar esta eventual interligação, estudámos 35 astrocitomas pilocíticos, pesquisando a presença dos 8 Herpesvirus. Neste estudo, foram utilizadas 10 amostras de biópsias do cerebelo de doentes que faleceram por doenças não relacionadas com infecção ou patologia tumoral. A maioria dos astrocitomas (33) eram tumores de baixa malignidade. As amostras foram analisadas por PCR (Polymerase Chain Reaction) quantitativa em tempo real (qPCR), com amplificação do gene da DNA polimerase viral. Treze astrocitomas e 7 controles revelaram pequenas quantidades de DNA viral (1-100 cópias/100ng DNA) de todos os Herpesvirus, com excepção do HHV6 A e B que estava ausente nas amostras. O EBV foi identificado em 9 dos 35 astrocitomas (26%) e em 7 dos 10 controles (70%) estando muito mais presente nos controles. As amostras positivas para o EBV foram também analisadas por imunohistoquímica, não tendo sido imunoreactivas para os anticorpos utilizados. A PCR com CODEHOP (consensus-degenerated hybrid oligonucleotide primers) foi utilizada para investigar a presença de um eventual Herpesvirus novo nestas amostras. Não foi identificada nenhuma sequência indicativa de um novo HHV por este método. 24. Em conclusão, os dados apontam para a presença de Herpesvirus, com particular relevância para o EBV, em tecido de cerebelo normal e em tumores cerebrais, embora em níveis demasiado baixos para poderem ser responsabilizados pela indução tumoral. A presença de sequências de DNA de Herpesvirus, nomeadamente do EBV, no Sistema Nervoso Central vem enriquecer a discussão sobre o significado da infecção viral na oncogénese humana, particularmente na neuro-oncogénese. ABSTRACT: Viral infections can contribute to the development of human cancer. Several human malignancies are linked with Human Herpesviruses (HHVs). Epstein-Barr virus and HHV8, two hHerpesvirus, have been recognized as etiologic agents of several neoplasms. Pilocytic astrocytoma of the cerebellum is one of the most common brain tumour in children, adolescents and young adults and astrocytary proliferation generally occurs after several types of injury, namely viral infection. To further explore this association, we have searched the tissue from 35 pilocytic astrocytoma, for all the 8 HHV. In this study, ten brain biopsies (cerebellum) from patients who died of unrelated diseases were used as controls. Most of the astrocytomas (33) were of low grade malignity. Samples were assessed by Real-time quantitative Polymerase Chain Reaction (q PCR) amplification of viral DNA polymerase gene. Thirteen astrocytoma and 7 controls showed low viral DNA levels (1-100 copies/100ng DNA) for all HHVs, with the exception of HHV6 that was absent. EBV was identified in 9 of the 35 astrocytoma (26 %) and in 7 of the 10 controls (70%) being more present in controls. EBV positive samples were also assessed by Immunohistochemistry (IHC) but none showed immunoreactivity for the antibodies used. PCR with consensus-degenerated hybrid oligonucleotide primers (CODEHOP) were also used to look for novel HHVs in these samples and no sequence indicative of a new HHV was detected. 26 Altogether the data indicate the presence of HHVs, with relevance for EBV in normal cerebellum tissue and also in brain tumours but at too low levels to be considered responsible for tumour induction. The presence of HHV DNA sequences, particularly EBV, in the studied brain tumours and control samples, further enriches the discussion about the relevance of viral infection in human oncogenesis, particularly neuro-oncogenesis.RÉSUMÉ: Les infections virales peuvent contribuer au développement du cancer. Les vírus de type Herpès sont associés à plusieurs néoplasies. Il est par exemple établi que les vírus Epstein-Barr et « human Herpesvirus 8 » (HHV-8) sont responsables de plusieurs tumeurs malignes. L´astrocytome pilocitique du cervelet est l’une des tumeurs les plus fréquentes chez les enfants, adolescents et adultes jeunes. En général la prolifération des astrocytes se produit en réponse à une agression. Posant l’hypothèse d’une agression d’origine virale, nous avons recherché la présence des 8 vírus Herpès dans les tissus de 35 astrocytomes. Dans cette étude, 10 échantillons de biopsie de cervelet de patients décédés suite à d’autres pathologies, ont été utilisés comme contrôles. La majorité des astrocytomes étaient de très basse malignité. Les échantillons ont été étudiés par PCR quantitative en temps réel, en amplifiant le gène de l’ADN-polymérase virale. Treize astrocytomes sur 35 (37%) et 7 contrôles sur 10 (70%) ont été trouvés positifs pour tous les HHV sauf l´HHV6, toujours avec un nombre de copies de polymérase virale bas (< 100 copies/100 ng d’ADN). Notamment l’EBV a été identifié 7 fois dans les contrôles (70%) et 9 fois dans les astrocytomes (26%). Les échantillons positifs pour l`EBV ont aussi été étudiés par immuno-histochimie. Aucun signal n’a été observé avec les anticorps utilisés. Enfin, une technique de PCR avec oligonucléotides dégénérés (CODEHOP ou consensus degenerated hybrid oligonucleotide primers) a été utilisée pour rechercher la présence d´un éventuel nouveau vírus Herpès dans les échantillons d’astrocytome. Aucun nouveau vírus n’a été identifié. 28 En résumé, nous avons établi la présence de vírus Herpès, en particulier l´EBV, dans le cervelet normal et dans les tumeurs du cerveau. Les quantités d’ADN viral retrouvées sont faibles et ne permettent pas d’attribuer à ces vírus la responsabilité de l’induction des tumeurs. Cependant, la présence d’ADN de vírus Herpès dans le cerveau sain ou pathologique vient enrichir la discussion sur le signification de l´infection virale dans les processus d´oncogenèse en général, et dans la neuroonco-genèse en particulier.

First isolation of dengue 3 in Brazil from an imported case

The authors report the isolation of dengue 3 virus for the first time in Brazil. The patient, resident in Limeira-SP, traveled to Nicaragua on May 16th, 1998, where he stayed for two months. Starting on August 14th he had fever, headache, myalgia, arthralgia, retro-orbital pain and diarrhea. He returned to Brazil on August 16th and was hospitalized in the next day. The patient had full recovery and was discharged on August 20th. The virus was isolated in C6/36 cell culture inoculated with serum collected on the 6th day after the onset of the symptoms. The serotype 3 was identified by indirect immunofluorescence assays performed with type-specific monoclonal antibodies. This serotype was further confirmed by polymerase chain reaction analysis. The introduction of a new dengue serotype in a susceptible population is a real threat for the occurrence of severe forms of the disease. The isolation and identification of dengue virus are important in order to monitoring the serotypes circulating in Brazil and to take the measures necessary to prevent and control an epidemic.