Academic literature on the topic 'Gene epression'

Abstract Mantle cell lymphoma (MCL) is characterized by the translocation t(11;14)(q13;q32), which leads to the overexpression of cyclin D1. The cyclin D1-overexpression by itself is however not sufficient for lymphoma development. To identify other genes that are deregulated in MCL, we have performed integrated profiling of high-resolution array-based comparative genomic hybridization (array-CGH) with RNA-expression array analysis on the same set of MCL cases. DNA-alterations of MCL cases were assessed using a 3.6k BAC array with a resolution of 800 kb. On the same cases, RNA-expression array analysis was performed using the GeneChip® Human Genome U133 Plus 2.0 Array from Affymetrix. Integration of genomic-and transcription profiling data was performed using statistical tools. With array-CGH, we identified regions of chromosomal gain or loss and determined minimal common regions (mcr’s) by identifying the smallest region of overlap present in at least 36% of the cases. Integration of array-CGH and expression profiling was performed for genes located within the mcr’s and unsupervised clustering of gene-expression values within each mcr was performed. For several mcr’s, e.g. 1p22.1–31.1, 6q23.2–27 and 11q22.3–23.3, the clustering tree showed two groups, one with the chromosomal aberration and one without. Also, we observed that only a subset of genes located within a cytogenetic anomaly has a concomitant change in mRNA expression level. These genes are regulated by DNA-copy number (gene dosage). Amongst these, we identified several “hypothetical genes” and genes, which encode proteins involved in mitochondrial protein synthesis, the DNA damage repair pathway and the cAMP regulated pathway. This study shows the potential of the integrated profiling approach for identifying genes that are regulated by gene dosage (DNA-copy number). We anticipate that the genes we identified are important for MCL and it’s characteristic features like the low apoptosis rate and the chemotherapy-resistance.

Abstract There are several gene therapy approaches, which require transfer and co-expression of two transgenes within one target cell. To this end, we have created and tested two-gene expression HIV-1 based vectors, which encode enhanced green fluorescent protein (EGFP) and P144K mutant of canine O6-methylguanine-DNA-methyltransferase (MGMT) transgenes under either the phosphoglycerate kinase gene promoter (PGKp), or the elongation factor 1 alpha promoter (EF1a_p). Eight different configurations of the two transgene expression cassettes were created and tested within the same lentiviral backbone (see Figure). Individual VSV-G pseudotyped vectors stocks were produced and used for infection of canine thyroid adenocarcinoma (CTAC) cells at low multiplicity of infection (MOI = 0.1) to ensure 1 copy of proviral vector per transduced cell. The cells were harvested one week later and an aliquot was assayed for EGFP expression by flow cytometry. Another portion was subjected to selection with O6-benzylguanine (BG, 40 m M for 18 hrs) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 100 m M for 2 hrs) and kept in culture for additional two weeks to eliminate cells expressing insufficient MGMT. The percentage of GFP positive cells, prior to selection with BG and BCNU, ranged between 0.1 % to 6.6% for the dual-transgene expression cassette encoding HIV-1 vectors. Following selection with BG and BCNU, the percentage of GFP positive cells increased for all vectors with the exception of vector #2 PGEM. Two of the vectors (#1 PMEG and #8 EGMP) demonstrated over 80% GFP positivity after selection. The results of flow cytometry after selection were corroborated by fluorescence microscopy of individual BCNU-resistant colonies. GFP expression was readily detected in drug-resistant colonies transduced with vectors #1 PMEG or #8 EGMP. Weaker GFP expression was detected in drug resistant cells transduced with vectors #3 PMGE, #5 EMPG or #6 EGPM. No significant GFP expression was observed in drug-resistant colonies transduced with vectors #2 PGEM, #4 PGME or #7 EMGP. Drug resistance to BCNU (IC50 values) provided by each of the vectors, was also determined. The data showed that the IC50 values for #1 PMEG and #8 EGMP vectors were 2.4-fold and 4.4-fold, respectively, higher than for mock transduced control cells. The above results indicate that coordinated co-expression of two transgenes using independent expression cassettes is promoter, position and orientation dependent. The data also indicate that two potentially useful vectors (#1 PMEG and #8 EGMP) have been identified for evaluation, ex vivo and in vivo, in the canine model for co-expression of two transgenes.

Abstract Porcine epidemic diarrhea virus (PEDV) infection can occur in all ages of pigs, but neonatal piglets are the most susceptible and sensitive to the virus. PEDV infection can cause intestinal dysfunction, severe diarrhea and even death in piglets. This study determined the effects of PEDV infection on the absorptive function and gene expression of nutrient transporters in the small intestine of piglets by microarray assay. Sixteen 7-day-old healthy piglets fed with milk replacer and were randomly allocated to one of two treatments (the Control and PEDV groups). After a 5-day adaptation period, piglets (n = 8) were orally administrated with either sterile saline or PEDV (Yunnan province strain) solution at 104.5TCID50 (50% tissue culture infectious dose) per piglet. On day 5 of the trial, D-xylose (0.1 g/kg BW) was orally administrated to all piglets. One hour later, jugular vein blood samples were collected, and then all piglets were killed to obtain the intestinal samples. Compared with the control, PEDV infection increased (P < 0.05) incidence of diarrhea, plasma DAO activity and iFABP level, while decreased (P < 0.05) plasma D-xylose concentration of piglets. Moreover, PEDV infection altered the amino acids profiles (P < 0.05), and decreased (P < 0.05) the gene expression of AQPs (AQP4, AQP8 and AQP10), amino acids transporters (y+LAT1, b0’+AT, and PepT1), molecules associated with lipid transport and metabolism (LPL, SLC27A2, and ACSL3), and glucose transport and metabolism (GLUT2 and INSR). However, PEDV infection enhanced (P < 0.05) the gene epression of PCK1, ASS1, SGLT1, and CFTR in the jejunum of piglets. Collectively, these comprehensive results indicate that PEDV infection impairs intestinal absorptive function and inhibits the expression of genes associated with nutrient transport and metabolism in piglets.

Abstract Introduction: Prostaglandins (PGs) are paracrine mediators thought to be important during pregnancy and labor. Synthesis of PGs is complex with three rate limiting steps, the second of which (PTGS2/cyclooxygenase) is a target for managing preterm labor, but this treatment is not always effective. It is suggested that alternate steps in the PG pathway may play distinct roles during labor and hence the potential as alternate targets for labor management. The first step of PG synthesis involves the action of phospholipase A2 (PLA2) enzymes, of which over a dozen isotypes are known. However, PLA2 expression and role in the decidua (maternal-fetal interface) is unknown. We hypothesize that distinct PLA2 isotypes are present within the decidua and play a role in term and preterm labor. To this end, we conducted an expression profile of PLA2 subtypes and related PG genes within human decidua and determined differential expression patterns with labor at term and preterm. Methods: Decidual samples from term (40wks) and preterm (26-37wks) Cesarean deliveries, were used to assess PG gene expression by RNAseq and qRT-PCR (n=7 preterm non-labor, n=11 preterm labor, n=31 term non-labor, n=31 term labor). Analyses were conducted using student’s t-test and one-way ANOVA to compare labor (non-labor vs labor) or with gestation (preterm vs term) and adjusted for multiple testing using the Benjamini-Hochberg method. Results: RNAseq analysis identified 30 highly expressed PG genes in decidua, of which 12 had not been previously reported in the uterus, including 7 PLA2 isotypes. Confirming previous work, expression of PTGS2 was higher with labor (p=0.011). In contrast, we demonstrate that PLA2 isotype expression is lower with labor, with further differences between term and preterm samples. With term labor, expression of PLA2 isotype 2D was lower compared to non-labour (p=0.047). Meanwhile, preterm labor was associated with lower expression of PLA2 isotype 16 (p=0.025) and 4C (p=0.026), compared to preterm non-labour. Epression of PLA2 subtypes 2A, 15 and 4A late was highest in late labor (p=0.016, 0.004, 0.03, respectively). Conclusion: The presence of multiple PLA2 subtypes within the decidua suggests the potential for fine tuning PG synthesis during pregnancy. Expression of PLA2 isotypes 16 and 4C was uniquely associated with preterm labor, suggesting these isotypes may play a role in the pathogenesis of preterm labor. Further investigation of the functional role of PLA2 isotypes may provide insight to a novel mechanism for preterm labor and identify potential targets for labor management.

碩士
國立陽明大學
生物化學研究所
87
Treatment of ET-1 or 8-bromo-cAMP on 3T3-L1 adipocytes resulted in time-dependent expression of GLUT1 mRNA, while simultaneously addition of ET-1 and 8-bromo-cAMP had an synergistic effect on GLUT1 mRNA accumulation and dramatically stabilized GLUT1 mRNA. This synergistism was blocked by BQ-123 or staurosporine and unaffected under pretreatment of BQ-788 or PD98059. ET-1 alone-induced GLUT1 mRNA accumulation seemed irrelevant to GLUT1 mRNA stability and was diminished by staurosporine, PD98059, and the removal of ET-1 after 30 minutes incubation, which had no effect on synergistism. According to these results, we conclude that: a) ET-1 alone can induce GLUT1 mRNA accumulation through PKC and MAPK pathway without changing its halflife. b) The synergistim between ET-1 and 8-bromo-cAMP on GLUT1 mRNA expression needs the signal from ETAR, which may then go through PKC pathway and finally stabilize GLUT1 mRNA.

Alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic bronchial epithelial cells (a process termed ‘efferocytosis’) and bacteria. These defects may contribute to COPD pathogenesis in several ways. Secondary necrosis of uncleared apoptotic material may result in chronic airways inflammation and perpetuation of COPD disease. A reduced alveolar macrophage phagocytic host response to bacteria, especially non-typeable H influenzae (NTHi), may contribute to neutrophilic inflammation and NTHi colonization of the lower airway. However, the exact mechanism that leads to the phagocytic dysfunction is still unknown. The sphingosine 1-phosphate (S1P) signalling system is known to regulate macrophage function. Experiments described in Chapter 2 of the thesis therefore applied a novel approach of measuring all S1P signalling system components in alveolar macrophages from COPD patients and healthy controls. Several components of the S1P system, in particular relative mRNA levels for sphingosine kinases SPHK1 and S1P receptor S1PR5, were dysregulated in COPD and were strongly correlated with efferocytosis, suggesting a potential link to the defective alveolar macrophage phagocytic ability in COPD. Oxidative stress and inflammation have been shown to contribute to many COPD characteristics, such as uncontrolled activation of cell signalling pathways, increased airway epithelial cell apoptosis, and defective alveolar macrophage phagocytic ability. Chapter 3 describes the effect of two models of oxidative stress and inflammation, cigarette smoke (potential oxidative conditions) and lipopolysaccharide (LPS) (potential inflammatory conditions) on components of S1P signalling and on efferocytosis and phagocytosis of NTHi, using a human macrophage cell line in vitro. Cigarette smoke and LPS increased the mRNA expression of SPHK1 and S1PR5 in macrophages, extending the results in Chapter 2 and further supporting the potential link between the S1P signalling system and macrophage phagocytic ability. Cigarette smoke decreased the capacity of macrophages to phagocytose apoptotic cells and bacteria. However, LPS reduced phagocytosis of bacteria only. Treatment option for oxidative stress is anti-oxidants and thymoquinone (TQ) is anti-oxidant/anti-inflammatory agent that has been shown to modulate macrophage inflammatory responses and has successfully been trialled in human clinical studies. Chapter 3 further reports that TQ per se had a pro-phagocytic effect on macrophage phagocytic ability. TQ also rescued macrophages from the negative effects of cigarette smoke, and to lesser extent LPS, on macrophage efferocytosis and the mRNA expression respectively. In addition, TQ demonstrated a pro-survival effect on bronchial epithelial cells treated with cigarette smoke. The effects on relative mRNA expression of SPHK1 and S1PR5 in the cell line were mirrored using acutely isolated alveolar macrophages from COPD patients. COPD patients are at increased risk for developing lung cancer and there is strong evidence that pulmonary macrophage dysfunction plays an important role in the pathogenesis of both diseases. DNA methylation has been shown to be modified in COPD and lung cancer. However, it unknown whether the change in mRNA expression of the S1P system (Chapter 2) are controlled by epigenetic modifications such as DNA methylation, and whether DNA methylation regulates macrophage efferocytosis. Data presented in Chapter 4 connect epigenetic modulation, mRNA expression and macrophage function. The results indicate that DNA methylation potentially regulates macrophage efferocytosis and is negatively correlated with the mRNA expression of S1P system components, in particular the S1PR5 receptor, suggesting epigenetic for COPD such as anti-oxidants or epigenome modifying agents.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015.