RT-PCR Analysis Of mRNA Expression Research Articles

A Comparative Study of the Effects of Electronic Cigarette and Traditional Cigarette on the Pulmonary Functions of C57BL/6 Male Mice.

Electronic cigarettes (E-cigs) are in a controversial state. Although E-cig aerosol generally contains fewer harmful substances than smoke from burned traditional cigarettes, aerosol along with other compounds of the E-cigs may also affect lung functions and promote the development of lung-related diseases. We investigated the effects of E-cig on the pulmonary functions of male C57BL/6 mice and reveal the potential underlying mechanisms. A total of 60 male C57BL/6 mice were randomly divided into four groups. They were exposed to fresh-air, traditional cigarette smoke, E-cig vapor with 12 mg/mL of nicotine, and E-cig with no nicotine for 8 weeks. Lung functions were evaluated by using quantitative analysis of the whole body plethysmograph, FlexiVent system, lung tissue histological and morphometric analysis, and RT-PCR analysis of mRNA expression of inflammation-related genes. In addition, the effects of nicotine and acrolein on the survival rate and DNA damage were investigated using cultured human alveolar basal epithelial cells. Exposure to E-cig vapor led to significant changes in lung functions and structures including the rupture of the alveolar cavity and enlarged alveolar space. The pathological changes were also accompanied by increased expression of interleukin-6 and tumor necrosis factor-α. The findings of the present study indicate that the safety of E-cig should be further evaluated. Some people currently believe that using nicotine-free E-cigs is a safe way to smoke. However, our research shows that E-cigs can cause lung damage regardless of whether they contain nicotine.

Expression of Components of the Serotonergic System in Folliculogenesis and Preimplantation Development in Mice

The functions of serotonin include the growth and development regulation of female germ cells as well as early embryo development. RT-PCR analysis of mRNA expression of the genes of the enzymes for synthesis and degradation and transporters and receptors of serotonin during folliculogenesis and preimplantation development of mice was performed to discover the particular mechanisms of these functions. The mRNA of tryptophan hydroxylase tph1 and monoaminoxidase maoa; membrane transporter sert and vesicular transporter vmat2; and serotonin receptors htr1b, htr1d, htr2a, htr5b, and htr7 were revealed in granulosa cells. The expression of mRNA of the aromatic amino acid decarboxylase ddc and the htr2b receptor additionally appears in the yellow body. The expression of mRNA of the genes of the tph2, ddc, and maoa enzymes; the sert, vmat1, and vmat2 transporters; and quite a number of receptors is observed during the preimplantation development, and it is transitory in most of them. The expression of all components and its dynamics suggest that the serotonergic signaling system is functionally active in mouse folliculogenesis and preimplantation development.

Physalin F from Physalis minima L. triggers apoptosis-based cytotoxic mechanism in T-47D cells through the activation caspase-3- and c-myc-dependent pathways

Ethnopharmacological relevancePhysalin F (a secosteroid derivative), is well recognized as a potent anticancer compound from Physalis minima L., a plant that is traditionally used to treat cancer. However, the exact molecular anticancer mechanism remains to be elucidated. Aim of the studyWe have recently reported the apoptosis-based cytotoxic effect of the chloroform extract of this plant. Here, we investigated the cytotoxicity and possible cell death mechanism elicited by the active constituent, physalin F on human breast T-47D carcinoma. Materials and methodsCytotoxic-guided fractionation of the chloroform extract of Physalis minima has led to the isolation of physalin F. The cytotoxicity activity was assayed using MTS assay. The effect of the compound to induce apoptosis was determined by biochemical and morphological observations through DeadEnd Colorimetric and annexin V assays, respectively, and RT-PCR analysis of mRNA expression of the apoptotic-associated genes. ResultsCytotoxicity screening of physalin F displayed a remarkable dose-dependant inhibitory effect on T-47D cells with lower EC50 value (3.60μg/ml) than the crude extract. mRNA expression analysis revealed the co-regulation of c-myc- and caspase-3-apoptotic genes in the treated cells with the peak expression at 9 and 12h of treatment, respectively. This apoptotic mechanism is reconfirmed by DNA fragmentation and phosphatidylserine externalization. ConclusionThese findings indicate that physalin F may potentially act as a chemopreventive and/or chemotherapeutic agent by triggering apoptosis mechanism via the activation of caspase-3 and c-myc pathways in T-47D cells.

Methotrexate Chemotherapy Promotes Osteoclast Formation in the Long Bone of Rats via Increased Pro-Inflammatory Cytokines and Enhanced NF-κB Activation

Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW(264.7) cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB.

Effects of aging, dietary restriction and glucocorticoid treatment on housekeeping gene expression in rat cortex and hippocampus—Evaluation by real time RT-PCR

Accurate normalization is the prerequisite for obtaining reliable results in the quantification of gene expression. Using TaqMan Real Time RT-PCR, we carried out an extensive evaluation of five most commonly used endogenous controls, gapdh, beta-actin, 18S rRNA, hprt and cypB, for their presumed stability of expression, in rat cortex and hippocampus, during aging, under dietary restriction and dexamethasone treatment. Valid reference genes (HKGs) were identified using GeNorm and NormFinder software packages and by direct comparison of Ct values. Analysis revealed gapdh and beta-actin as the most stable HKGs for all treatments analyzed, combined or separately, in the cortex, while in the hippocampus gapdh/hprt and beta-actin/hprt are the combination of choice for the single or combined effects of dietary restriction/dexamethasone, respectively. All treatments significantly influenced expression of 18S rRNA and cypB in both structures. In addition, we used gapdh and normalization factor, calculated by GeNorm, to compare the expression of alpha-syn in the cortex. Our results demonstrate the importance of the right choice of HKG and suggest the appropriate endogenous control to be used for TaqMan RT-PCR analysis of mRNA expression in rat cortex and hippocampus for selected experimental paradigms.

Regulation of Differentiation and Proliferation of Marrow Hematogenesis Stem Cells by Notch1 Signaling System from Patients with Aplastic Anemia and Chronic Myelogenous Leukemia.

Notch genes encode evolutionarily conserved transmembrane receptors that regulate cell fate determination. Notch activation promotes proliferation and inhibits differentiation of bone marrow stem cells. Our research was to study the differential expression of Notch1 on bone marrow mononuclear cells (BMMNC) from chronic aplastic anemia (AA) and chronic myelogenous leukemia (CML) patients. We demonstrated that Notch signaling is inactivated in AA patients and activated in CML patients. In CML patients, Notch1 furthers stimulation by its ligand Jagged1, resulting in a strong increase of tumor cell growth. Therefore, we suggest that inactivated Notch signaling plays a pivotal role in the pathogenesis of AA and that activated Notch signaling plays a key role in the pathogenesis of CML. We used the antibody to inactivate Notch1 signaling in mice, and then to detect the hematopoietic state of these mice.Methods: 5ml marrow were abstracted from chronic AA patient s, CML patients in chronic phase and normal people and the BMMNC was separated from the marrow. RNA was abstracted from these cells. Real Time Quantitative PCR was used to investigate the expression of Notch1 on BMMNCs in 30 AA patients, 20 normal controls and 30 CML patients, and the changes of their expression after chemotherapy. Notch1 protein of these BMMNC was also detected through Western Blots. We injected monoclonal anti-Notch1 and isotonic Na chloride as controls into the tail vein of mice, to detect the hematopoietic state through bone marrow slides and flow cytometry.Results: Real Time PCR: Expression of Notch1 on BMMNCs from AA patients was lower than that of controls (P<0.05). The expression of Notch1 on BMMNCs from CML patients was higher than that of controls(P<0.05). Notch1 on the BMMNCs from AA patients was expressed higher after chemotherapy (P<0.05). Notch1 on the BMMNCs from CML patients was expressed lower after chemotherapy(P<0.05). Western Blots: Notch1 receptors were highly expressed in BMMNCs of CML patient. BMMNCs of AA patients expressed low amounts of Notch1. Jagged1 induces Notch signaling in cultured cells; We performed RT-PCR analysis of mRNA expression of Hes-1, a member of the hairy enhancer of split family of transcriptional repressors that are direct transcriptional targets of activated Notch. The result was that the expression of Hes-1 of CML was much higher than AA. The growth rate of CML BMMNCs was also much higher than AA BMMNCs. Compared with controls, the hematopoietic state of mice treated with anti-Notch1 had an obvious low hematopoietic ability.Conclusions: The expression of Notch1 on BMMNCs from AA patients and normal controls was different, which may account for the occurrence of AA. The expression of Notch1 of AA patients was improved after effective chemotherapy, which may be one of the reason of recovery of hematopoietic function. The expression of Notch1 on BMMNCs from CML patients was much higher than that of controls, which decreased after chemotherapy and may be one of the factors accounting for occurrence of CML. BMMNCs cocultured with Jagged-1 expressing cell lines can enhance the growth rate, which indicate that Notch1 can improve proliferation of leukemic cells. The mice treated with anti-Notch1 had lower hematopoietic ability manifesting that Notch1 signaling is essential to maintain normal hematopoietic function.

Over-expression of AhR (aryl hydrocarbon receptor) induces neural differentiation of Neuro2a cells: neurotoxicology study

BackgroundDioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron.MethodsThe influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Rα. The activation of exogenous AhR in N2a-Rα cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Rα based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH).ResultsN2a-Rα cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Rα cells exhibited two significant functional features. Morphologically, N2a-Rα cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Rα cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Rα cells.ConclusionThe excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death.

RT-PCR analysis of mRNA expression of natriuretic peptide family and their receptors in rat inner ear

To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B, and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors.

Prolactin and prolactin receptors are expressed and functioning in human prostate.

Prolactin is widely expressed in different tissues, and it is presumed to have both local and systemic actions. In males it is known to influence reproductive functions but the significance and mechanisms of prolactin action in male accessory reproductive tissues are poorly understood. Here we show that prolactin acts as a direct growth and differentiation factor for human prostate, as measured by changes in DNA synthesis and epithelial morphology of organ cultures. Furthermore, we report the expression in human prostate of a short prolactin receptor form in addition to the long form, based upon ligand cross-linking studies and RT-PCR analysis of mRNA expression. The highest density of prolactin receptors was detected in the secretory epithelial cells by immunohistochemistry. Finally, we report that prolactin is locally produced in human prostate epithelium, as evidenced by marked prolactin immunoreactivity in a significant portion of prostate epithelial cells, with parallel expression of prolactin mRNA in human prostate. Collectively, these data provide significant support for the existence of an autocrine/paracrine loop of prolactin in the human prostate and may shed new light on the involvement of prolactin in the etiology and progression of neoplastic growth of the prostate.