Amounts Of mRNA Transcripts Research Articles

Effect of triploidy on liver gene expression in coho salmon (Oncorhynchus kisutch) under different metabolic states

BackgroundTriploid coho salmon are excellent models for studying gene dosage and the effects of increased cell volume on gene expression. Triploids have an additional haploid genome in each cell and have fewer but larger cells than diploid coho salmon to accommodate the increased genome size. Studying gene expression in triploid coho salmon provides insight into how gene expression may have been affected after the salmonid-specific genome duplication which occurred some 90 MYA. Triploid coho salmon are sterile and consequently can live longer and grow larger than diploid congeners in many semelparous species (spawning only once) because they never reach maturity and post-spawning mortality is averted. Triploid fishes are also of interest to the commercial sector (larger fish are more valuable) and to fisheries management since sterile fish can potentially minimize negative impacts of escaped fish in the wild.ResultsThe vast majority of genes in liver tissue had similar expression levels between diploid and triploid coho salmon, indicating that the same amount of mRNA transcripts were being produced per gene copy (positive gene dosage effects) within a larger volume cell. Several genes related to nutrition and compensatory growth were differentially expressed between diploid and triploid salmon, indicating that some loci are sensitive to cell size and/or DNA content per cell. To examine how robust expression between ploidies is under different conditions, a genetic/metabolic modifier in the form of different doses of a growth hormone transgene was used to assess gene expression under conditions that the genome has not naturally experienced or adapted to. While many (up to 1400) genes were differentially expressed between non-transgenic and transgenic fish, relatively few genes were differentially expressed between diploids and triploids with similar doses of the transgene. These observations indicate that the small effect of ploidy on gene expression is robust to large changes in physiological state.ConclusionsThese findings are of interest from a gene regulatory perspective, but also valuable for understanding phenotypic effects in triploids, transgenics, and triploid transgenics that could affect their utility in culture conditions and their fitness and potential consequences of release into nature.

Abstract 4503: Cycloxygenase-2 and breast cancer prognosis: The role of gene polymorphisms in the regulation of COX-2 expression and their association with histopathological features

Abstract Introduction: Cycloxygenase-2 (COX-2) overexpression in breast cancer is associated with parameters of aggressiveness, including great tumor size, positive nodal status and lower survival. COX-2 is encoded by a polymorphic gene, PTGS2, with variant alleles in regulatory sites of the promoter and of the 3′-untranslated regions. Aim: To investigate the association between the haplotypes formed by the for most frequent PTGS2 SNPs (rs689465, rs689466, rs20417 and rs5275) and: 1) histopathological parameters with prognostic value on clinical outcome of breast cancer; 2) COX-2 expression in breast tumors. Methods: The study was conducted at the Brazilian National Cancer Institute with 606 women with non-metastatic breast cancer (Protocols #116/07 and #129/08). Recruitment occurred between Jan/ 2008 and Dec/2010 and all patients were genotyped using PCR-RFLP or Real Time-PCR. PTGS2 haplotypes were inferred using the computer software Haploview 4.2, and the individual histopathological profiles were obtained from data available in electronic medical records. The expression of PTGS2 gene in breast tumors was evaluated by the amounts of mRNA transcripts determined with Real Time RT-PCR and by the amounts of COX-2 determined with immunohistochemistry (IHC) in paraffin-embedded tissues. Results: A total of 8 PTGS2 haplotypes were inferred, whose distribution varied as a function of tumor size (P: 0.011), estrogen receptor (ER) status (P: 0.018) and HER2 status (P: 0.025). PTGS2 haplotype *7 (formed by rs689465G, rs689466A, rs20417C, rs5275T) was positively associated with higher tumor size (OR: 3.72, 95%CI: 1.19-11.22, P: 0.006), ER negativity (OR: 2.43, 95%CI: 0.97-5.98, P: 0.032), progesterone receptor (PR) negativity (OR: 2.58, 95%CI: 1.05-6.39, P: 0.02), and HER2 positivity (OR: 4.17, 95%CI:1.19-14.44, P: 0.007). The results also indicated positive associations between variant genotypes of rs689465 and ER negativity (OR: 1.59, 95%CI: 1.04-2.44, P: 0.02) or HER2 positivity (OR: 1.79, 95%CI: 1.00-3.18, P: 0.03), and between variant genotypes of rs20417 and ER negativity (OR: 1.75, 95%CI: 1.15-2.57, P:0. 005), PR negativity (OR: 1.56, 95%CI: 1.09-2.22, P: 0.01) or HER2 positivity (OR: 1.80, 95%CI: 1.04-3.13, P: 0.02). In contrast, variant genotypes of rs689466 were negatively associated with ER negativity (OR: 0.57, 95%CI: 0.33-0.98, P: 0.03). The analyses of PTGS2 expression in breast tumors are currently under course, with 166 tumor species submitted to IHC and 100 submitted to Real Time RT-PCR. Conclusion: Our results suggest that PTGS2 haplotype *7 may contribute to higher growth of untreated breast cancer. We expect that the results regarding the regulation of PTGS2 expression may help understanding the prognostic value of PTGS2 SNPs in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4503. doi:1538-7445.AM2012-4503

Potent anti-aging activity of Aruncus dioicus, a native plant of Ulleung-do, South Korea, in CCD-986sk fibroblasts via suppression of matrix metalloproteinases

Aruncus dioicus var. kamtschaticus HARA, also known as goat's beard, is a native plant in Ulleung-do, South Korea. It has been used as a remedy in skin care, detoxification, blood stanching, tonsillitis. High performance liquid chromatography with diode array detection was used for partial validation of bioactive chemicals in A. dioicus ethyl acetate (EtOAc) extract, and EtOAC extract was examined for its effect on ultraviolet (UV)-induced cell aging using CCD-986sk-human skin fibroblast cells. Cells were exposed to UV-B for 1min before extract treatment. An established viability assay was performed to test cell toxicity of A. dioicus at 5, 10, or 50μg/ml concentrations, and activities of matrix metalloproteinase (MMP) 1, 2, 3, phosphorylated-p38 (p-p38, an activated form of p38), p38, and c-fos transcription factors were evaluated. A. dioicus extract decreased the amount of mRNA transcripts and total proteins of MMP1, 2, 3 as well as p-p38 and c-fos. The c-fos expression was also confirmed by in vivo fluorescent staining of CCD-986sk cells after UV-B exposure followed by EtOAc extract treatment. The results showed that expression of skin aging related genes encoding MMP1, 2, and 3 was inhibited by reduced transcription factor expression of p-p38 and c-fos by A. dioicus EtOAc extract. The results suggest that A. dioicus extract can be used to reduce UV-B-induced skin aging and is a potential candidate for cosmedical materials.

Cystic fibrosis transmembrane conductance regulator expression in human spinal and sympathetic ganglia

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel protein, and mutations of its gene cause cystic fibrosis. CFTR is known to be expressed in epithelial cells of the respiratory, digestive and reproductive tracts. It is also present in rat neurons and heart ganglion cells. In humans, it is expressed in the hypothalamus, but has not been identified in other parts of the human nervous system. In this study, immunohistochemistry, double-staining immunofluorescence, in situ hybridization, nested reverse transcription–PCR and relative quantification of real-time PCR analysis were performed on spinal and sympathetic ganglia from seven human autopsies with no known nervous system disease. CFTR protein was expressed in most ganglion cells with no obvious difference in the amounts of mRNA transcript in ganglia of different sites. We conclude that CFTR protein and its mRNA were extensively expressed at relatively constant levels in human spinal and sympathetic ganglion cells, and may be important in physiological and pathological conditions. Moreover, CFTR in ganglia may be associated with pathophysiological changes seen in cystic fibrosis.

Role of Silent Gene Mutations in the Expression of Caprine Growth Hormone in Escherchia coli

This report describes the strategy for overexpression of caprine growth hormone (cGH) gene of beetal goat in E. coli through introducing silent mutations in the 5'-end of the coding sequence. The silent mutations introduced were aimed at minimizing translation-inhibiting secondary structures in the mRNA. Free energies of the resultant mRNAs were calculated from the ribosomal binding site of mRNA to +24 base using the Mfold web server. The construct with native sequence did not show any expression, whereas introduction of the silent mutations had strong influence on the expression levels. Some constructs (pETcGH2-7) showed 12-30% expression of total cell proteins while some others (pETcGH8-16) showed 30 to 53% of total cell protein. Any variation in the amount of mRNA transcript for the various constructs, as determined by quantitative PCR, was not enough to suggest that the variable level of the gene expression was due to variation in the transcription levels. It appears that the expression levels are not always correlated with free-energy values of the secondary structures in the 5'-end region of the mRNA; instead some key silent nucleotide alterations at certain sites of 5'-end of the sequence reorganize the secondary structure in such a way that it has positive impact on translation without considerably altering the free-energy values. An empirical approach for determining the optimum 5'-end substitutions for hyperexpression of a recombinant protein thus seems necessary.

The Queen of Dopamine

The queen bee controls the physiology and behavior of her fellow bees and essentially determines the workings of the entire society of insects. The queen exerts this influence by producing a cocktail of pheromones known as queen mandibular pheromone (QMP), but it has not been clear just how the mixture produces its effects. Beggs et al . noted that one component of QMP, homovanillyl alcohol (4-hydroxy-3-methoxyphenylethanol, or HVA) has a chemical structure similar to that of the neurotransmitter dopamine. The authors therefore tested the effects of the pheromone on dopaminergic function in bees. Exposure of newly emerged adult bees to QMP (a commercially available strip impregnated with nine queen-equivalents of pheromone mixture) for 2 days decreased the amount of mRNA transcript encoding one of the bee’s dopamine receptors. Cultured neurons from the mushroom body normally respond to dopamine with an increase in production of cAMP (adenosine 3′,5′-monophosphate), but neurons taken from flies exposed to QMP also showed a small decrease in production of cAMP. HVA produced responses similar to those evoked by dopamine. Total amounts of dopamine in the brain were reduced in flies exposed to HVA for 2 days. The authors propose that HVA in the QMP mixture may interact directly with dopamine receptors in the bee nervous system, perhaps decreasing the expression of dopamine receptors, thus altering the response of the neurons to endogenous dopamine. K. T. Beggs, K. A. Glendining, N. M. Marechal, V. Vergoz, I. Nakamura, K. N. Slessor, A. R. Mercer, Queen pheromone modulates brain dopamine function in worker honey bees. Proc. Natl. Acad. Sci. U.S.A. 104 , 2460-2464 (2007). [Abstract] [Full Text]

The use of the cMyc epitope tag can be problematic for protein detection in Legionella pneumophila

In various recent experiments with respect to protein secretion by L. pneumophila, protein detection based on the cMyc-tag seemed to be problematic. For one specific protein, we studied the reason for this problem in more detail by comparing protein expression in the presence and absence of the cMyc-tag. We demonstrated a decrease in the amount of mRNA transcript when the cMyc coding sequence is present. We can conclude that this epitope tag is not a preferential tag for protein detection in L. pneumophila.

Insulin-like growth factor-binding proteins in R3230AC mammary tumors of intact and diabetic rats.

The insulin-responsive R3230AC mammary tumor possesses type I and type II insulin-like growth factor (IGF) receptors, and membrane preparations display affinity cross-linking of 125I-labeled IGF-I to IGF-binding proteins (IGFBPs). To identify the IGFBPs produced, Northern blotting analysis of poly(A)+ RNA extracts from tumor tissue was performed. Although transcripts of IGFBP-1 were not detected, intense bands were obtained at 1.7 and 6 kilobases (kb) when hybridized with radiolabeled IGFBP-2 and IGFBP-5 cDNA probes, respectively. A 2.6-kb band and a 2.4-kb weaker band were observed after hybridization with IGFBP-3 and IGFBP-4 probes, respectively. When IGFBP-6 cDNA was used, two bands were seen: a higher mol wt band at 6.3 kb and a smaller one at 1.3 kb. Tumors from streptozotocin-induced diabetic rats displayed an increase in the expression of IGFBP-2, and insulin treatment for 3 days normalized the IGFBP-2 mRNA levels. Tumors from diabetic rats displayed no change in IGFBP-3, -4, -5, and -6 mRNA levels from tumors of normoglycemic rats. However, tumors from insulin-treated rats showed significantly higher levels of mRNA for IGFBP-4 and IGFBP-5 than tumors from normoglycemic or diabetic rats. A similar, but less pronounced, pattern of changes in IGFBP-3 mRNA was seen, whereas levels of IGFBP-6 mRNA were unchanged throughout. To identify the cell type producing the mRNAs for these IGFBPs, in situ hybridization of tissue sections was used. Procedures were established that localized three of the five IGFBPs expressed in this tumor tissue. This technique showed that IGFBP-3 mRNA transcripts were observed mainly in endothelial cells of tumor vasculature, although they were also detected in stromal cells, IGFBP-4 was present mainly in tumor stroma cells, and IGFBP-5 mRNA was expressed predominantly in the epithelial cells of this tumor. Expression of IGFBP-5 mRNA transcripts was significantly diminished in primary and long term cultured R3230AC cells grown in alpha-Minimum Essential Medium and 10% fetal bovine serum. Tumors arising from injection of long term cultured cells that were injected into isologous rats contained high amounts of mRNA transcripts for IGFBP-5, suggesting the presence, in vivo, of positive regulators for the expression of this BP. Tumor cells cultured in the presence of insulin displayed a 2.2- to 2.5-fold increase in the expression of IGFBP-5. These findings imply a role for insulin as a regulator of the expression of IGFBP-5 in the R3230AC adenocarcinoma.

Alpha-fetoprotein gene expression in human lymphoblastoid cells and in PHA-stimulated normal T-lymphocytes

Alpha-fetoprotein (AFP) is mainly synthesized by the fetal liver, the yolk sac and, to a much lower extent, by a few non-hepatic fetal tissues (i.e. kidney, pancreas, lung). This property is considered to be lost in mature quiescent cells of the adult. In the present we have studied the expression of AFP mRNA sequences in phytohemagglutinin (PHA)-stimulated normal human T-lymphocytes and in several human lymphoma cell lines. The amount of mRNA transcripts detected in quiescent T-lymphocytes by dot and Northern blot analysis was very low. It increased rapidly after PHA-activation, reached a maximum at 72 hours (six fold the level observed for quiescent T-lymphocytes) and decreased thereafter. The lymphoma cell lines Daudi, Raji, Rh6 et CEM, all expressed elevated levels of AFP mRNA. The transcripts had the size expected for human AFP, suggesting that they were functional and probably translated into protein. The possible role of AFP synthesis in lymphocyte blastogenesis and in lymphoma growth is discussed in relation with the strong binding affinity of this protein for polyunsaturated fatty acids.