Relative Changes In mRNA Levels Research Articles

Analysis of Expression of Genes CLN3, GABBR1, and WFS1 in Patients with Parkinson’s Disease

Parkinson’s disease (PD) is one of the most common neurodegenerative pathologies. This disorder is associated with death of predominantly dopaminergic neurons. A characteristic feature of PD is a long latent period of disease development, which makes it impossible to investigate and diagnose PD at the earliest clinical stages. One of the approaches to solving this problem is the examination of the peripheral blood of untreated PD patients at the early clinical stages. Changes in relative mRNA levels of genes in peripheral blood can be considered as potential biomarkers at the early stages of PD. In this study, we analyzed changes in relative mRNA levels of genes CLN3, GABBR1, and WFS1 in peripheral blood in treated and untreated PD patients and in comparison groups using RT-qPCR with TaqMan probes. All the known functions of the proteins of the studied genes, as well as the identified associations of these genes with various neurological diseases, may indicate their possible involvement in PD pathogenesis. However, no statistically significant changes in mRNA levels in PD patients relative to the control were shown for any gene. No significant changes in relative mRNA levels in the neurological control group were shown either. So, the data presented in this paper suggest that genes CLN3, GABBR1, and WFS1 do not contribute to the pathogenesis of the disease, at least at the mRNA level, at the early symptomatic stages in patients with PD and, therefore, cannot be considered as biomarkers of the early stages of PD.

The ZntA-like NpunR4017 plays a key role in maintaining homeostatic levels of zinc in Nostoc punctiforme.

Analysis of cellular response to zinc exposure provides insights into how organisms maintain homeostatic levels of zinc that are essential, while avoiding potentially toxic cytosolic levels. Using the cyanobacterium Nostoc punctiforme as a model, qRT-PCR analyses established a profile of the changes in relative mRNA levels of the ZntA-like zinc efflux transporter NpunR4017 in response to extracellular zinc. In cells treated with 18μM of zinc for 1h, NpunR4017 mRNA levels increased by up to 1300% above basal levels. The accumulation and retention of radiolabelled (65)Zn by NpunR4107-deficient and overexpressing strains were compared to wild-type levels. Disruption of NpunR4017 resulted in a significant increase in zinc accumulation up to 24% greater than the wild type, while cells overexpressing NpunR4107 accumulated 22% less than the wild type. Accumulation of (65)Zn in ZntA(-) Escherichia coli overexpressing NpunR4017 was reduced by up to 21%, indicating the capacity for NpunR4017 to compensate for the loss of ZntA. These findings establish the newly identified NpunR4017 as a zinc efflux transporter and a key transporter for maintaining zinc homeostasis in N. punctiforme.

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

BackgroundKisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation.MethodsFemale fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay.ResultsBoth kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms.ConclusionThe results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.

Effect of rosemary polyphenols on human colon cancer cells: transcriptomic profiling and functional enrichment analysis

In this work, the effect of rosemary extracts rich on polyphenols obtained using pressurized fluids was investigated on the gene expression of human SW480 and HT29 colon cancer cells. The application of transcriptomic profiling and functional enrichment analysis was done via two computational approaches, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. These two approaches were used for functional enrichment analysis as a previous step for a reliable interpretation of the data obtained from microarray analysis. Reverse transcription quantitative-PCR was used to confirm relative changes in mRNA levels of selected genes from microarrays. The selection of genes was based on their expression change, adjusted p value, and known biological function. According to genome-wide transcriptomics analysis, rosemary polyphenols altered the expression of~4% of the genes covered by the Affymetrix Human Gene 1.0ST chip in both colon cancer cells. However, only~18% of the differentially expressed genes were common to both cell lines, indicating markedly different expression profiles in response to the treatment. Differences in induction of G2/M arrest observed by rosemary polyphenols in the two colon adenocarcinoma cell lines suggest that the extract may be differentially effective against tumors with specific mutational pattern. From our results, it is also concluded that rosemary polyphenols induced a low degree of apoptosis indicating that other multiple signaling pathways may contribute to colon cancer cell death.

Genetic responses in a plant‐endophyte interplay

A plant can respond to the threat of a pathogen through resistance defenses or tolerance. Resistance has been widely studied in many host pathosystems but little is known about genetic events in tolerance. In this study a recently developed model for a tolerant tomato‐fungal (Verticillium dahliae) wilt pathosystem was used to examine changes in gene expression during both a tolerant interaction and in competition with a susceptible infection. Relative changes in mRNA levels were measured using a customized TVR (tomato‐Verticillium response) DNA chip and actual changes in cellular proteins were assessed using two dimensional gel electophoresis and MS‐based protein identification. Heat maps were used to identify responding groups of proteins and individual groups were examined further for common functions and other relationships including correspondence between mRNA and protein changes. In addition to elevated levels of defense gene responses to pathogen colonization, suppression or blocked responses were evident in mixed infections. These included some transcription factors and PR proteins such as class IV chitinases and beta glucanases known to target fungal spores and mycelium. Taken together the results reveal intriguing but complex molecular changes with potential agricultural benefits. Supported by NSERC (R.N.N. and J.R.), NIH, NHLBI (A.K.) and a Canadian Commonwealth Scholarship (H.O.S.).

D469del-COMP Retention in Chondrocytes Stimulates Caspase-Independent Necroptosis

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1β generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

BACH2 Directly Regulates Expression of Foxp3 in UCB CD4+ T-Cells

Introduction: We have previously reported low constitutive nuclear factor of activated T cells (NFAT1) protein expression in unstimulated umbilical cord blood (UCB) CD4+ T-cells, and delayed up-regulation of NFAT1 expression during primary stimulation compared to adult primary CD4+ T-cells. Recent data has shown NFAT1 regulating Foxp3, an observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, and our work demonstrating BACH2 directly regulates IL2 expression in the absence of NFAT1 protein. We sought to determine if BACH2 plays a role in regulating Foxp3 expression in UCB CD4+ T-cells. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein mafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. We hypothesized that BACH2 may directly regulate Foxp3 transcription by binding to putative NFAT1/AP1 binding sites within the Foxp3 proximal promoter. These sites may be vacant due to an absence of NFAT1 protein in resting UCB CD4+ T-cells. This hypothesis was tested by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells and then examining the level of Foxp3 expression at both the mRNA and protein level. In addition chromatin immunoprecipitation (ChIP) was performed in order to identify if BACH2 was binding to regions proximal to the Foxp3 gene. Finally Foxp3 driven luciferase plasmids were constructed and transiently transfected into primary UCB CD4+ T-cells with and without BACH2 siRNA and the relative expression determined.Methods: UCB T-cells were purified using autoMACs system. After overnight culture, T-cells were transfected with BACH2 siRNA using Amaxa nucleofector system. Both siRNA treated and control cells were incubated in media for 24 hours, and then stimulated using anti-CD3 and anti-CD28 antibodies. Aliquots of cells were collected at 24 hours and 30 hours post-stimulation for protein and total RNA isolation. The relative changes in mRNA levels for BACH2, FoxP3, were determined by Applied Biosystems Taqman real time RT-PCR system. Western blots were run to confirm results seen in the qRT-PCR. ChIP using BACH2 antibody, and PCR was used to determine putative BACH2 binding in DNA region surround exon 1 of Foxp3.Results: Loss of BACH2 expression correlates with reduced expression of (40-fold) FoxP3, mRNA in CD4+ UCB T-cells. Western blot performed on BACH2 siRNA treated, and anti-CD3/CD28 antibody stimulated UCB CD4+ T-cells; confirmed the observed loss of Foxp3 in unstimulated UCB CD4+ T-cells. Analysis of ChIP data from unstimulated UCB CD4+ T-cells showed that BACH2 and smafK bound to 2 regions flanking exon 1 of the Foxp3 gene, while results from unstimulated adult PB CD4+ T-cells showed BACH2 and smafK co-precipitated with only one region flanking exon 1 of Foxp3.Conclusions: NFAT1 protein has recently been shown to be required for the activation of Foxp3 in CD4+ T-cells, yet in UCB CD4+ T-cells NFAT1 protein expression is significantly attenuated. However, Foxp3 expression is unchanged in UCB CD4+ T-cells compared to adult PB CD4+ T-cells. UCB CD4+ T-cells BACH2 binds to two site surrounding exon 1 of Foxp3 gene, promoting the expression of Foxp3 mRNA in the absence of NFAT1 protein expression. These results suggest that expression of BACH2 in UCB CD4+ T-cells may underlie UCB immune tolerance in the unrelated allogeneic setting via regulation of FoxP3 impacting Treg development.

Transient Receptor Potential Vanilloid 1 Mediates Hyperalgesia and Is Up-Regulated in Rats With Chronic Pancreatitis

The neurobiologic basis of pancreatic hyperalgesia in chronic pancreatitis (CP) is understood poorly and there is a need to identify novel therapeutic targets. Our aim was to study the role of the transient receptor potential vanilloid 1 (TRPV1), a key integrator of noxious stimuli, in the pathogenesis of pancreatic pain in a rat model of CP. CP was induced in rats by intraductal injection of trinitrobenzene sulfonic acid. TRPV1 currents in pancreas-specific DRG neurons were measured using perforated patch-clamp techniques. Reverse-transcription polymerase chain reaction was used to measure mRNA expression of TRPV1 in these neurons after laser capture microdissection. Immunofluorescence and Western blot analysis, using TRPV1-specific antibodies, also were performed. Pancreatic hyperalgesia was assessed by rat's nocifensive behavior to electrical stimulation of the pancreas. CP was associated with a 4-fold increase in capsaicin-induced current density (P < .02), along with an increase in the proportion of pancreas-specific DRG neurons that responded to capsaicin (52.9% in controls vs 79.0% in CP; P < .05). CP also was associated with a significant increase in TRPV1 expression both at the messenger RNA and protein level in whole thoracic DRGs and pancreas-specific sensory neurons. Systemic administration of the TRPV1 antagonist SB-366791 markedly reduced both visceral pain behavior and referred somatic hyperalgesia in rats with CP, but not in control animals. TRPV1 up-regulation and sensitization is a specific molecular mechanism contributing to hyperalgesia in CP and represents a useful target for treating pancreatic hyperalgesia caused by inflammation.

Role of cytosolic malate dehydrogenase in oocyte maturation and embryo development

To elucidate the function of cytosolic malate dehydrogenase (Mor2) in oocyte maturation and embryo development using RNA interference (RNAi). Experimental animal study. Research unit of university. Female 4-week-old (C57/BL6) mice. Isolation of immature germinal vesicle (GV) oocytes or fertilized pronucleus (PN) embryos, microinjection of Mor2 double-stranded RNA (dsRNA), and reverse transcription and polymerase chain reaction (RT-PCR) analysis to investigate Mor2-specific messenger RNA (mRNA) knockdown. Relative changes in mRNA levels after microinjection of Mor2 dsRNA and in rates of oocyte maturation and preimplantation embryo development. Mor2 mRNA mostly was knocked down in germinal vesicle- and metaphase I (MI)-arrested oocytes, compared with metaphase II (MII)-developed oocytes, after microinjection of Mor2 dsRNA and in vitro culture for 16 hours. In vitro oocyte maturation was significantly decreased (34%), compared with noninjected (73.4%) and buffer-injected (67.5%) control groups. The rate of blastocyst development (48.1%) was lower in the Mor2 dsRNA-injected group than in buffer-injected control (88.2%). In the present study, the function of Mor2 was analyzed with the aid of RNAi. On the basis of the data obtained, we propose that Mor2 is an essential factor for oocyte maturation and embryo development in mouse.

Does mRNA level of microsomal carnitine palmitoyltransferase predict yield of peripheral blood stem cell apheresis?

Transplantation of autologous hematopoietic stem cells is a well established therapeutic procedure. Despite advances in efficacy of the stem cell mobilization and apheresis process until now a predictive factor for the expected stem cell yield before initiation of mobilization therapy could not be identified. The main objective of our study was to evaluate alterations in enzymes involved in fatty acid metabolism on the level of gene expression in mononuclear cells, as changes in relative mRNA levels of these enzymes could represent the hematopoietic regenerative potential. Data of 23 consecutive patients with different lymphoid malignancies undergoing stem cell mobilization were analyzed. Our results show that mRNA levels of microsomal carnitine palmitoyltransferase in peripheral blood mononuclear cells quantified before application of mobilization therapy correlate positively with the amount of CD34 positive cells in peripheral blood before first apheresis, in the first apheresis product and in the total harvest outcome. The association of enzymes involved in fatty acid metabolism with hematoopoiesis was further confirmed in healthy subjects on altitude-adaptation training and in proliferating or differentiating HL60 cells. This gives evidence for a possible predictive value of such analyzes though further data of a larger sample are to be collected to confirm our observations.

Modulation of the expression of vascular endothelial growth factor in human fibroblasts

To examine the up-regulation of vascular endothelial growth factor (VEGF) expression by hypoxia, a crucial event leading to neovascularization, as the reduction in VEGF expression may facilitate minimization of adhesion development. Prospective experimental study. University medical center. Five patients with adhesions undergoing laparotomy with excision of adhesions and normal peritoneum. Adhesion and normal peritoneal fibroblasts were treated with dichloroacetic acid (DCA) or NS-398 (a cyclooxygenase-2 [COX-2] inhibitor) for 24 to 48 hours. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) to quantify relative changes in mRNA levels of VEGF from each treatment. In both normal peritoneal and adhesion fibroblasts, VEGF mRNA was present with statistically significantly higher levels in adhesion fibroblasts (32%). The DCA treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (20%) and normal peritoneal (18%) fibroblasts. The NS-398 treatment resulted in a statistically significant decrease in VEGF mRNA levels in adhesion (25%) and normal peritoneal (16%) fibroblasts. Stimulation of aerobic metabolism by DCA or inhibition of COX-2 by NS-398 reduces VEGF expression. Angiogenesis, which is an integral component in the development of dense vascular adhesions, may be reduced by either COX-2 inhibitors or stimulation of aerobic metabolism by DCA.

Modulation of the expression of tissue plasminogen activator and its inhibitor by hypoxia in human peritoneal and adhesion fibroblasts

Objective To determine whether normal peritoneal and adhesion fibroblasts express tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-I) and whether their expression is regulated by oxygen. Design Prospective experimental study. Setting University medical center. Patient(s) Cultures of human fibroblasts established from peritoneal and adhesion tissues. Intervention(s) Hypoxia treatment of the primary cultured fibroblasts. Main outcome measure(s) We have used the multiplex reverse transcription polymerase chain reaction (RT-PCR) technique to determine the effect of hypoxia on the expression of tPA and PAI-I in normal peritoneal (NPF) and adhesion (ADF) fibroblasts. Cultures of NPF and ADF were exposed to hypoxia (2% O 2) for 24 hours. RNA was extracted from cells and subjected to multiplex RT-PCR to quantitate relative changes in mRNA levels of tPA and PAI-I in response to hypoxia treatment. Result(s) Basal tPA mRNA levels are present in both NPF and ADF and were 45% higher in NPF than ADF. Hypoxia decreased tPA in both NPF and ADF by 74% and 95%, respectively. Basal PAI-I mRNA levels were 64% higher in ADF than in NPF. Hypoxia increased PAI-I mRNA levels by 67% and 53% in NPF and ADF, respectively. Conclusion(s) Plasminogen activator activity (PAA) of the peritoneum does not solely reside in the mesothelial cells, as previously identified, but also exists within fibroblasts, thus providing the potential to resolve postoperative fibrinous collections even at sites at which the mesothelial cells have been injured, removed, or destroyed. Furthermore, PAA in fibroblasts is regulated by oxygen; creation of a hypoxic state markedly attenuates PAA, thereby leading to adhesion development.

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.

Leukemia inhibitory factor, interleukin-6, and their receptors are expressed transiently in the olfactory mucosa after target ablation.

Removal of the synaptic targets of olfactory receptor neurons by olfactory bulb ablation results in apoptosis of olfactory receptor neurons and up-regulation of proliferation of their progenitors. This study focuses on the expression of the neuropoietic cytokines leukemia inhibitory factor (LIF) and its receptor (LIFR) and interleukin 6 (IL-6) and its receptor (IL-6R) in intercellular signaling pathways in the olfactory mucosa after target ablation. Olfactory bulbectomy (OBX) resulted in several transient, early-onset, temporally integrated events that were detected immunohistochemically. Macrophages infiltrated the olfactory epithelium (OE) by 16 hours post-OBX. LIF expression was up-regulated transiently at 2 days post-OBX, when up-regulated expression of LIFR also was detected on globose basal cells (GBCs), a subpopulation of which are immediate progenitors of olfactory receptor neurons. GBC proliferation peaked at 3--4 days post-OBX. In the olfactory nerve (ON), LIF-positive and IL-6-positive macrophage infiltration was followed by the transient up-regulation of expression of LIFR, IL-6, and IL-6R in ensheathing cells by 3 days post-OBX. The mRNAs for LIF/LIFR, IL-6/IL-6R, and their common signal-transduction molecule, gp130, in olfactory-nasal mucosa from control mice and from 3-day post-OBX mice were detected with reverse transcriptase-polymerase chain reaction (RT-PCR). Analysis of Northern blot and relative quantitative RT-PCR demonstrated similar temporal patterns of changes in relative mRNA levels for both LIF and IL-6, which were up-regulated by 16 hours post-OBX and peaked at 2--3 days post-OBX. These data indicate that LIF from infiltrating macrophages acts as a mitogen for GBCs and that LIF from infiltrating macrophages and IL-6 from infiltrating macrophages and ensheathing cells act as repair factors in the ON.

Transcriptional regulation of plasma protein synthesis during inflammation.

The regulation of the synthesis of plasma proteins in rat liver during the acute phase response was studied by measuring gene transcription activities in a cell-free nuclear transcription system. The transcription activities for the genes of major acute phase alpha 1-protein, the beta-chain of fibrinogen, transferrin, alpha 1-acid glycoprotein, and alpha 2-macroglobulin increased, reaching a maximum level between 18 and 36 h after inducing an acute inflammation. The transcription activities for the genes of alpha 2u-globulin, albumin, and transthyretin (formerly called prealbumin) decreased, reaching a minimum level after 12 to 24 h. The extent of the relative changes in transcription activities was similar to that of the relative changes in mRNA levels for major acute phase alpha 1-protein, the beta-chain of fibrinogen, transferrin, alpha 2u-globulin, albumin, and transthyretin. This is consistent with the assumption that the principal mechanism of the regulation of the synthesis of these proteins operates at the level of transcription. In contrast, the relative changes in transcription activities for alpha 1-acid glycoprotein and alpha 2-macroglobulin were far smaller than the changes of their mRNA levels, suggesting that, in addition to transcriptional changes, other mechanism(s) are involved in the regulation of the synthesis of these proteins.