Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Lipopolysaccharide-induced Stem Cell Factor Messenger RNA Expression and Production in Odontoblast-like Cells

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.

Temporal analysis of prostaglandin F2 alpha receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2 alpha receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium

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

Estrogen induced desensitization of c -{\it fos\/} messenger RNA expression {\it in vivo\/}

In this thesis, we investigated the regulation of the nuclear proto-oncogene, c-fos by estrogen in vivo. In the uterus, estrogen causes a rapid, dramatic and transient induction of c-fos mRNA and this occurs by transcriptional activation. We have discovered a previously unrecognized regulatory mechanism by which fos becomes desensitized to estrogen following the transient induction. We investigated three aspects of this desensitization: (1) the kinetics and general characteristics of the phenomenon; (2) the molecular mechanism of the desensitization; and (3) the relationship of desensitization to estrogen stimulated DNA synthesis. The desensitization occurs between 3-24 hours after initial hormonal stimulation and is reversible within 72 hours. The desensitization is not species specific, in that it occurs in both the rat and mouse. The desensitization also occurs in at least two estrogen responsive tissues, the uterus and vagina. The desensitization is not unique to c-fos, since both c-myc and c-jun show similar patterns of desensitization. However, the desensitization is not observed with creatine kinase B (CKB), indicating that not all estrogen inducible genes become desensitized. In the second general area, we determined the desensitization is at the transcriptional level. The desensitization is homologous, but not heterologous, since estrogen induction does not desensitize c-fos to other agents. Other studies show that the desensitization is not due to the lack of functional estrogen receptors. Taken together, these findings suggest that the desensitization occurs at the level of the estrogen responsive element. In the third major area, we demonstrated that the desensitization appears to be related to estrogen induced DNA synthesis. Support for this suggestion comes from the observation that short acting estrogens which induce fos, but not DNA synthesis, do not produce desensitization. ^

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

Interleukin 18 messenger RNA and proIL-18 protein expression in chorioamniotic membranes from pregnant women with preterm prelabor rupture of membranes

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

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated.2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii.3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h.4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly, the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion, high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Tamoxifen down-regulates CaMKII messenger RNA levels in normal human breast tissue

Tamoxifen was proven to reduce the incidence of breast cancer by 49% in women at increased risk of the disease in the Breast Cancer Prevention Trial. In order to identify potential candidates to explain the preventive effect induced by tamoxifen on breast cancer, normal breast tissue obtained from 42 fibroadenoma patients, randomly assigned to receive placebo or tamoxifen, was analyzed by the reverse Northern blot and RT-PCR techniques. The cDNA fragments used on Northern blot membranes were generated by the Human Cancer Genome Project funded by the Ludwig Institute for Cancer Research and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil). Total RNA was obtained from normal breast tissue from patients with clinical, cytological and ultrasound diagnosis of fibroadenoma. After a 50-day treatment with tamoxifen (10 or 20 mg/day) or placebo, normal breast tissue adjacent to the tumor was collected during lumpectomy with local anesthesia. One differentially expressed gene, Calcium/calmodulin-dependent protein kinase II (CaMKII), was found to be down-regulated during TAM treatment. CaMKII is an ubiquitous serine/threonine protein kinase that has been implicated in the diverse effects of hormones utilizing Ca2+ as a second messenger as well as in c-fos activation. These results indicate that the down-regulation of CaMKII induced by TAM might represent alternative or additional mechanisms of the action of this drug on cell cycle control and response to hormones in normal human breast tissue.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Investigation of a unique short open reading frame within the 3' untranslated region of the canine distemper virus matrix messenger RNA

Increasing evidence suggest that the long "untranslated" region (UTR) between the matrix (M) and the fusion (F) proteins of morbilliviruses has a functional role. In canine distemper virus (CDV), the F 5' UTR was recently shown to code for a long F signal peptide (Fsp). Subsequently, it was reported that the M/F UTRs combined with the long Fsp were synergistically regulating the F mRNA and protein expression, thereby modulating virulence. Unique to CDV, a short putative open reading frame (ORF) has been identified within the wild-type CDV-M 3' UTR (termed M2). Here, we investigated whether M2 was expressed from the genome of the virulent and demyelinating A75/17-CDV strain. An expression plasmid encoding the M2 ORF tagged both at its N-terminal (HA) and C-terminal domains (RFP), was first constructed. Then, a recombinant virus with its putative M2 ORF replaced by HA-M2-RFP was successfully recovered from cDNA (termed recA75/17(green)-HA-M2-RFP). M2 expression in cells transfected or infected with these mutants was studied by immunoprecipitation, immunofluorescence, immunoblot and flow cytometry analyses. Although fluorescence was readily detected in HA-M2-RFP-transfected cells, absence of red fluorescence emission in several recA75/17(green)-HA-M2-RFP-infected cell types suggested lack of M2 biosynthesis, which was confirmed by the other techniques. Consistent with these data, no functional role of the short polypeptide was revealed by infecting various cell types with HA-M2-RFP over-expressing or M2-knockout recombinant viruses. Thus, in sharp contrast to the CDV-F 5' UTR reported to translate a long Fsp, our data provided evidence that the CDV-M 3' UTR does not express any polypeptides.

Messenger RNA half-life measurements in mammalian cells.

The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.