Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation.

OBJECTIVE: To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation. METHODS: Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip(®) Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip(®) Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry. RESULTS: We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group. CONCLUSION: Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

A fast and efficient polymerase chain reaction-based method for the preparation of in situ hybridization probes.

AIMS: In situ hybridization (ISH) is the method of choice for analysis of the local distribution of gene expression in tissue samples at the cellular level. In this study we present a rapid and efficient protocol for the generation of labelled cRNA probes. METHODS AND RESULTS: The protocol is based on the preparation of DNA in vitro transcription templates using polymerase chain reaction (PCR), using primers that include RNA polymerase promoter sequences and size-based purification of PCR fragments containing the target gene-specific cDNA and promoter elements for T7 and SP6 RNA polymerase. The optimized purification protocols ensure high transcription efficiency and target specificity of the labelled cRNA. The cRNA hybridization probes obtained are compatible with established in situ hybridization protocols. CONCLUSIONS: Purified PCR fragment-based in vitro transcription enables preparation of in situ hybridization probes which allow the rapid detection of gene expression distribution in tissue slices from any gene of interest.

Generation and analysis of transcriptomics data.

Transcript profiling ("Transcriptomics") is a widely used technique that obtains information on the abundance of multiple mRNA transcripts within a biological sample simultaneously. Therefore, when a number of such samples are analysed, as in a scientific experiment, large and complex data sets are gene-rated. Here, we describe the use of one method commonly used to generate transcriptomics data, namely the use of Affymetrix GeneChip microarrays. Data generated in transcriptomics experiments can be analysed using a multitude of approaches, but a common goal is to identify those transcripts whose abundance is altered by the experimental conditions, or which differ between sets of samples. Here, we describe a simple approach, the calculation of the volcano score, which identifies transcripts with altered abundance, taking into account both the magnitude of the alteration and its statistical significance.

Strand-specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA.

Real-time RT-PCR is used to quantify individual influenza viral RNAs. However, conventional real-time RT-PCR, using strand-specific primers, has been shown to produce not only the anticipated strand-specific products, but also substantial amounts of non-strand-specific products, indicating lack of specificity. Therefore, in this study, a novel strand-specific real-time RT-PCR method was established to quantify the three types of influenza viral RNA (vRNA, cRNA, and mRNA) separately. This method is based on reverse transcription using tagged primers to add a 'tag' sequence at the 5' end and the hot-start method. Real-time PCR using the 'tag' portion as the forward primer and a segment-specific reverse primer ensured the specificity for quantifying the three types of RNA. Using this method, specific target RNA was detected at 100-100,000-folds higher level than other types of RNA. This method was also used to evaluate the vRNA, cRNA, and mRNA levels of segments 5 and 6 in MDCK cells infected with influenza A virus at different time point post-infections. The cRNA level was 1/10 to 1/100 lower than that of the vRNA and mRNA. Moreover, different dynamics of vRNA, cRNA, and mRNA synthesis were observed; the copy number of the vRNA gradually increased throughout the infection, the cRNA increased and then plateaued, while the mRNA increased and then decreased. This novel method thus provides data critical for understanding the influenza virus life cycle, including transcription, replication, and genome incorporation into virions.

A simple, sensitive, specific detection of mixed infection of multiple plant viruses using macroarray and microtube hybridization.

A simple, sensitive and specific method using a cDNA macroarray to detect multiple viruses was devised. The method is used in plants such as potato and lily, which need a reliable routine diagnosis for mixed infection. The biotinylated cRNA targets were prepared using an in vitro transcription-based system that was designed especially to eliminate nonspecific hybridizations. The macroarray hybridization was carried out using a convenient, cost-effective "microtube hybridization" (MTH) system. By this method, lily viruses including Cucumber mosaic virus, Lily symptomless virus, Lily mottle virus, and Plantago asiatica mosaic virus were detected successfully from leaves or roots of lily bulbs.

Improved high-throughput ultrafiltration process enables cRNA purification for gene expression profiling.

Ultrafiltration of nucleic acids has been used for a wide variety of applications, including sequence reaction purification and amplicon cleanup prior to spotting onto microarrays. Here we describe a novel process, using ultrafiltration, that purifies cRNA products for sensitive downstream applications. Initial attempts at this high-throughput purification for cRNA resulted in low sensitivity when compared against an industry standard (silica-based bind, wash, and elute purification). We modified the ultrafiltration process to include a proteinase K preincubation and a phosphate buffer wash that, when combined, increased sensitivity and signal-to-noise ratio in microarray applications. The protocol that we have developed eliminates the use of chaotropic salts (such as guanidinium thiocyanate) that are typically used in silica binding purification methods. The data demonstrate good performance for sensitive RNA applications using well-defined metrics, and thus the technique might be useful for a broader range of nucleic acid purifications.

Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1.

Interleukin-1 is a proinflammatory and immunomodulatory cytokine that plays a crucial role in inflammatory diseases of the skin, including bacterial infections, bullous diseases, UV damage, and especially psoriasis. To characterize the molecular effects of IL-1 in epidermis, we defined the transcriptional changes in human epidermal keratinocytes 1, 4, 24, and 48 h after treatment with IL-1alpha. IL-1 significantly regulated 388 genes, including genes associated with proteolysis, adhesion, signal transduction, proliferation, and epidermal differentiation. IL-1 induces many genes that have antimicrobial function. Secreted cytokines, chemokines, growth factors, and their receptors are the prominent targets of IL-1 regulation, including IL-8, IL-19, elafin, C3, and S100A proteins, which implicate IL-1 in the pathogenesis of inflammatory diseases. IL-1 induced not only proliferation-associated genes but also differentiation marker genes such as transglutaminase-1 and involucrin, which suggests that IL-1 plays an important role in the aberrant proliferation and differentiation seen in psoriasis. Correlation of IL-1 regulated genes with the TNFalpha and IFNgamma regulated ones showed more similarities between IL-1 and TNFalpha than IL-1 and IFNgamma, whereas Oncostatin-M (OsM) affected a largely unrelated set of genes. IL-1 regulates many genes previously shown to be specifically over-expressed in psoriasis. In summary, IL-1 regulates a characteristic set of genes that define its specific contribution to inflammation and aberrant differentiation in skin diseases.

Genome-scale transcript analyses in human neutrophils.

Transcriptome analyses of single- and multicellular organisms have changed fundamental understanding of biological and pathological processes across multiple scientific disciplines. Over the past 5 yr, studies of polymorphonuclear leukocyte (or neutrophil) (PMN) gene expression on a global scale have provided new insight into the molecular processes that promote resolution of infections in humans. Herein, we present methods to analyze gene expression in human neutrophils using Affymetrix oligonucleotide microarrays, which include isolation of high-quality RNA, generation and labeling of cRNA, and GeneChip hybridization and scanning. Notably, the procedures utilize commercially available reagents and materials and thus represent a standardized approach for evaluating PMN transcript levels.

Evaluation of two methods for generating cRNA for microarray experiments from nanogram amounts of total RNA.

Several methods have been developed for amplification of RNA, making it possible to use cDNA microarrays for analysis of samples limited in amount of total RNA. The most widely used amplification protocol, the Eberwine method, amplifies RNA in a linear manner through in vitro transcription (IVT). However, when starting material is limited to nanogram amounts of total RNA, several rounds of amplification are necessary, making this method both expensive and labor-intensive. Amplification by PCR is robust and is able to amplify extremely limiting material. However, it is possible that the nonlinear nature of PCR could result in reduced reproducibility of the amplification compared with IVT. We have evaluated two methods that use a combination of PCR and IVT for amplification of nanogram amounts of total RNA. We have compared microarray results obtained by these methods with results obtained by two established methods: indirect labeling of 20 microg total RNA and Eberwine amplification of 1 microg total RNA. Starting from as little as 5 ng of total RNA, both methods yielded results in concordance with the Eberwine method.

Gene expression profiling of primary tumor cell populations using laser capture microdissection, RNA transcript amplification, and GeneChip microarrays.

Gene expression profiling from microdissected cell populations is a powerful approach to explore molecular processes involved in development and solid tumor biology. In this chapter, we detail robust and validated methods for tissue preparation and isolation of high-quality RNA from microdissected cell populations. A protocol is also provided for linear transcript amplification using as little as 10 ng of total RNA to produce labeled cRNA targets for hybridization to GeneChip high-density oligonucleotide microarrays. Particular emphasis is placed on troubleshooting each technical step in the protocol and measures of quality assurance for both RNA isolation and resulting microarray data.

Activation of NR1a/NR2B receptors by soluble factors from APP-stimulated monocyte-derived macrophages: implications for the pathogenesis of Alzheimer's disease.

Amyloid-beta peptide (Abeta), the major component of amyloid plaques, can activate brain mononuclear phagocytes (MP; macrophages and microglia), leading to their secretion of neurotoxins. Recent studies strongly suggest that MP-mediated neurotoxicity plays an important role in the pathogenesis of Alzheimer's disease (AD). To further explore this notion, human monocyte-derived macrophages (MDM) were stimulated with naturally secreted alpha-processing soluble amyloid precursor protein/p3 (alphaAPPs/p3) or beta-processing APP/Abeta (betaAPPs/Abeta). MDM conditioned media (MCM) was recovered and tested for its ability to activate recombinant N-methyl-d-aspartate (NMDA) receptor subtype NR1a/NR2B expressed in Xenopus oocytes. Pressure ejection of alphaAPPs/p3- and betaAPPs/Abeta-stimulated MCM produced inward currents of 59.5 +/- 8.9 nA (mean +/- S.E.M., n = 31) and 111.1 +/- 21.0 nA (n = 42) in NR1a/NR2B-expressing oocytes, respectively. The MCM-induced currents were concentration dependent and blocked by 50 microM of the NMDA receptor antagonist 2-amino-5-phosphnovalerate, but not by a non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). The alphaAPPs/p3- and betaAPPs/Abeta-stimulated MCM placed in non-injected oocytes failed to generate inward current. These results demonstrate that APPs/Abeta-stimulated MCM directly activate NMDA receptor subtypes relevant in the pathogenesis of AD.

Mitogenic actions of neuropeptide Y in vascular smooth muscle cells: synergetic interactions with the beta-adrenergic system.

Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smooth muscle cell (VSMC) growth, but which of its Y1-Y5 receptors are involved remains unclear. In quiescent rat VSMCs, NPY receptor mRNAs were undetectable (reverse transcription-polymerase chain reaction), but Y1, Y2, and Y5 expression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold [3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nM concentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low affinity peak by 50% and fully blocked NPY's response when combined. In VSMCs lacking NPY receptors and responsiveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCs with low or no NPY responsiveness, pre-exposure to beta-adrenergic receptor agonist (isoproterenol), forskolin, or dibutyryl cAMP augmented NPY's mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenol only). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is low in nonproliferating cells, amplification of NPY's mitogenic responses requires upregulation of at least the Y1 receptor by NPY itself or beta-adrenergic, cAMP-dependent activation.

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines.

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Differential effects of general anesthetics on G protein-coupled inwardly rectifying and other potassium channels.

BACKGROUND: General anesthetics differentially affect various families of potassium channels, and some potassium channels are suggested to be potential targets for anesthetics and alcohols. METHODS: The voltage-gated (ERG1, ELK1, and KCNQ2/3) and inwardly rectifying (GIRK1/2, GIRK1/4, GIRK2, IRK1, and ROMK1) potassium channels were expressed in Xenopus oocytes. Effects of volatile agents [halothane, isoflurane, enflurane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and the structurally related nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane)], as well as intravenous (pentobarbital, propofol, etomidate, alphaxalone, ketamine), and gaseous (nitrous oxide) anesthetics and alcohols (ethanol and hexanol) on channel function were studied using a two-electrode voltage clamp. RESULTS: ERG1, ELK1, and KCNQ2/3 channels were either inhibited slightly or unaffected by concentrations corresponding to twice the minimum alveolar concentrations or twice the anesthetic EC50 of volatile and intravenous anesthetics and alcohols. In contrast, G protein-coupled inwardly rectifying potassium (GIRK) channels were inhibited by volatile anesthetics but not by intravenous anesthetics. The neuronal-type GIRK1/2 channels were inhibited by 2 minimum alveolar concentrations of halothane or F3 by 45 and 81%, respectively, whereas the cardiac-type GIRK1/4 channels were inhibited only by F3. Conversely, IRK1 and ROMK1 channels were completely resistant to all anesthetics tested. Current responses of GIRK2 channels activated by mu-opioid receptors were also inhibited by halothane. Nitrous oxide (approximately 0.6 atmosphere) slightly but selectively potentiated GIRK channels. Results of chimeric and multiple amino acid mutations suggest that the region containing the transmembrane domains, but not the pore-forming domain, may be involved in determining differences in anesthetic sensitivity between GIRK and IRK channels. CONCLUSIONS: G protein-coupled inwardly rectifying potassium channels, especially those composed of GIRK2 subunits, were inhibited by clinical concentrations of volatile anesthetics. This action may be related to some side effects of these agents.

Reduced synthesis of inflammatory cytokines by a free radical scavenger after hemorrhagic shock in rats.

OBJECTIVES: Intestinal ischemia/reperfusion during hemorrhage and resuscitation may be a major trigger for cytokine expression. To assess whether free radicals produced on tissue reperfusion may play a role in the inflammatory response after hemorrhage, we tested the effect of a free radical scavenger on the production of inflammatory cytokines in a rat model of hemorrhagic shock. DESIGN: A prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Hemorrhage was induced in anesthetized rats. by bleeding the animal to achieve a mean arterial blood pressure of 40 mm Hg for 60 mins. Resuscitation was then induced by reinjecting shed blood followed by NaCl 0.9% to maintain arterial blood pressure within control values. Treated rats received the free radical scavenger N-2-mercaptopropionyl glycine (MPG; 20mg/kg iv bolus 30 mins before resuscitation followed by 20 mg/kg/hr). MEASUREMENTS AND MAIN RESULTS: MPG reduced the volume of saline necessary to restore blood pressure during resuscitation (untreated 85+/-6; MPG 35+/-5 mL/kg; p < .05). As compared with untreated rats, MPG markedly reduced the systemic and mesenteric plasma concentrations of tumor necrosis factor (TNF)-alpha (as measured by ELISA) and interleukin (IL)-6 (as measured by bioassay), assessed at the end of resuscitation. MPG also reduced TNF-alpha and IL-6 mRNA expression (as measured by reverse transcriptase-polymerase chain reaction) assessed in peritoneal macrophages isolated from shock rats. Finally, in vitro experiments showed that MPG also markedly reduced the mRNA expression and release of TNF-alpha and IL-6 in peritoneal macrophages isolated from normal rats and subjected to hypoxia and reoxygenation. CONCLUSION: Reactive oxygen species contribute to the production of proinflammatory cytokines during posthemorrhage resuscitation. Free radicals scavengers may be a useful treatment in the prevention of the systemic inflammatory response that occurs in shock states.