Cardiac myocyte gene expression profiling during H2O2-induced apoptosis

High levels of oxidative stress promote cardiac myocyte death, though lower levels are potentially cytoprotective/anabolic. We examined the changes in gene expression in rat neonatal cardiac myocytes exposed to apoptotic (0.2 mM) or nontoxic (0.04 mM) concentrations of H2O2 (2, 4, or 24 h) using Affymetrix microarrays. Using U34B arrays, we identified a ubiquitously expressed, novel H2O2-responsive gene [putative peroxide-inducible transcript 1 (Perit1)], which generates two alternatively spliced transcripts. Using 230 2.0 arrays, H2O2 (0.04 mM) promoted significant changes in expression of only 32 genes, all of which were seen with 0.2 mM H2O2. We failed to detect any increase in the rate of protein synthesis in cardiac myocytes exposed to <0.1 mM H2O2, further suggesting that global, low concentrations of H2O2 are not anabolic in this system. H2O2 (0.2 mM) promoted significant (P < 0.05, >1.75-fold) changes in expression of 649 mRNAs and 187 RNAs corresponding to no established gene. Of the mRNAs, 114 encoded transcriptional regulators including Krüppel-like factors (Klfs). Quantitative PCR independently verified the changes in Klf expression. Thus, H2O2-induced cardiac myocyte apoptosis is associated with dynamic changes in gene expression. The expression of these genes and their protein products potentially influences the progression of the apoptotic response.

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR >= 11.01, p <= 0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.

Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

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

Gene expression profiling in leiomyosarcomas and undifferentiated pleomorphic sarcomas: SRC as a new diagnostic marker

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

Single-cell expression profiling reveals a dynamic state of cardiac precursor cells in the early mouse embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study.

Expression profiling and cross-species RNA interference (RNAi) of desiccation-induced transcripts in the anhydrobiotic nematode Aphelenchus avenae

Abstract Background Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance. Results To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species. Conclusions This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.

Global gene expression profiling of oral cavity cancers suggests molecular heterogeneity within anatomic subsites

Abstract Background Oral squamous cell carcinoma (OSCC) is a frequent neoplasm, which is usually aggressive and has unpredictable biological behavior and unfavorable prognosis. The comprehension of the molecular basis of this variability should lead to the development of targeted therapies as well as to improvements in specificity and sensitivity of diagnosis. Results Samples of primary OSCCs and their corresponding surgical margins were obtained from male patients during surgery and their gene expression profiles were screened using whole-genome microarray technology. Hierarchical clustering and Principal Components Analysis were used for data visualization and One-way Analysis of Variance was used to identify differentially expressed genes. Samples clustered mostly according to disease subsite, suggesting molecular heterogeneity within tumor stages. In order to corroborate our results, two publicly available datasets of microarray experiments were assessed. We found significant molecular differences between OSCC anatomic subsites concerning groups of genes presently or potentially important for drug development, including mRNA processing, cytoskeleton organization and biogenesis, metabolic process, cell cycle and apoptosis. Conclusion Our results corroborate literature data on molecular heterogeneity of OSCCs. Differences between disease subsites and among samples belonging to the same TNM class highlight the importance of gene expression-based classification and challenge the development of targeted therapies.

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.

Expression profiling in developing peach seed and mesocarp as affected by growth regulators

Jasmonates (JAs) and spermidine (Sd) influence fruit (and seed) development and ripening. In order to unravel their effects in peach fruit, at molecular level, field applications of methyl jasmonate (MJ) and propyl dihydrojasmonate (PDJ), and Sd were performed at an early developmental stage (late S1). At commercial harvest, JA-treated fruit were less ripe than controls. Realtime RT-PCR analyses confirmed a down-regulation of ethylene biosynthetic, perception and signaling genes, and flesh softening-related genes. The expression of cell wall-related genes, of a sugar-transporter and hormone-related transcript levels was also affected by JAs. Seeds from JA-treated fruit showed a shift in the expression of developmental marker genes suggesting that the developmental program was probably slowed down, in agreement with the contention that JAs divert resources from growth to defense. JAs also affected phenolic content and biosynthetic gene expression in the mesocarp. Levels of hydroxycinnamic acids, as well as those of flavan-3-ols, were enhanced, mainly by MJ, in S2. Transcript levels of phenylpropanoid pathway genes were up-regulated by MJ, in agreement with phenolic content. Sd-treated fruits at harvest showed reduced ethylene production and flesh softening. Sd induced a short-term and long-term response patterns in endogenous polyamines. At ripening the up-regulation of the ethylene biosynthetic genes was dramatically counteracted by Sd, leading to a down-regulation of softening-related genes. Hormone-related gene expression was also altered both in the short- and long-term. Gene expression analyses suggest that Sd interfered with fruit development/ripening by interacting with multiple hormonal pathways and that fruit developmental marker gene expression was shifted ahead in accord with a developmental slowing down. 24-Epibrassinolide was applied to Flaminia peaches under field conditions early (S1) or later (S3) during development. Preliminary results showed that, at harvest, treated fruit tended to be larger and less mature though quality parameters did not change relative to controls.

Expression profiling und rationale Expressionsoptimierung am Beispiel eines prokaryontischen Wirt-,Vektor-Systems

Durch globale Expressionsprofil-Analysen auf Transkriptom-, Proteom- oder Metabolom-Ebene können biotechnologische Produktionsprozesse besser verstanden und die Erkenntnisse für die zielgerichtete, rationale Optimierung von Expressionssystemen genutzt werden. In der vorliegenden Arbeit wurde die Überexpression einer Glukose-Dehydrogenase (EC 1.1.5.2), die von der Roche Diagnostics GmbH für die diagnostische Anwendung optimiert worden war, in Escherichia coli untersucht. Die Enzymvariante unterscheidet sich in sieben ihrer 455 Aminosäuren vom Wildtyp-Enzym und wird im sonst isogenen Wirt-/Vektor-System in signifikant geringeren Mengen (Faktor 5) gebildet. Das prokaryontische Expressionssystem wurde auf Proteom-Ebene charakterisiert. Die 2-dimensionale differenzielle Gelelektrophorese (DIGE) wurde zuvor unter statistischen Aspekten untersucht. Unter Berücksichtigung von technischen und biologischen Variationen, falsch-positiven (α-) und falsch-negativen (β-) Fehlern sowie einem daraus abgeleiteten Versuchsdesign konnten Expressionsunterschiede als signifikant quantifiziert werden, wenn sie um den Faktor ≥ 1,4 differierten. Durch eine Hauptkomponenten-Analyse wurde gezeigt, dass die DIGE-Technologie für die Expressionsprofil-Analyse des Modellsystems geeignet ist. Der Expressionsstamm für die Enzymvariante zeichnete sich durch eine höhere Variabilität an Enzymen für den Zuckerabbau und die Nukleinsäure-Synthese aus. Im Expressionssystem für das Wildtyp-Enzym wurde eine unerwartet erhöhte Plasmidkopienzahl nachgewiesen. Als potenzieller Engpass in der Expression der rekombinanten Glukose-Dehydrogenase wurde die Löslichkeitsvermittlung identifiziert. Im Expressionsstamm für das Wildtyp-Enzym wurden viele Proteine für die Biogenese der äußeren Membran verstärkt exprimiert. Als Folge dessen wurde ein sog. envelope stress ausgelöst und die Zellen gingen in die stationäre Wuchsphase über. Die Ergebnisse der Proteomanalyse wurden weiterführend dazu genutzt, die Produktionsleistung für die Enzymvariante zu verbessern. Durch den Austausch des Replikationsursprungs im Expressionsvektor wurde die Plasmidkopienzahl erhöht und die zelluläre Expressionsleistung für die diagnostisch interessantere Enzymvariante um Faktor 7 - 9 gesteigert. Um die Löslichkeitsvermittlung während der Expression zu verbessern, wurde die Plasmidkopienzahl gesenkt und die Coexpression von Chaperonen initiiert. Die Ausbeuten aktiver Glukose-Dehydrogenase wurden durch die Renaturierung inaktiven Produkts aus dem optimierten Expressionssystem insgesamt um einen Faktor von 4,5 erhöht. Somit führte im Rahmen dieser Arbeit eine proteombasierte Expressionsprofil-Analyse zur zielgerichteten, rationalen Expressionsoptimierung eines prokaryontischen Modellsystems.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.