RNase Protection Assay Research Articles

Quantitative nuclease protection assay (qNPA) for gene expression analysis on breast cancer core biopsies.

49 Background: qNPA employs in situ hybridization of detection probes to cross linked mRNA, making it ideal for formalin fixed paraffin embedded (FFPE) tissue. It has been shown to measure gene expression in archived lymphoid and lung cancer tissue. We assessed the feasibility of qNPA to measure differentially expressed genes in pretreatment FFPE core breast biopsies among pathologic responders (pR) and nonresponders (pNR) to preoperative chemotherapy. Methods: We included preoperative breast cancer patients treated at our institution from 2003-09 with FFPE core biopsies. mRNA expression of 170 genes, representing oncogenic pathways or associated with anthracycline and taxane response was measured by qNPA (HTG, Tucson, AZ). Data was normalized to 3 housekeeper genes and average of 3 biologic replicates reported. Seven genes below detection in > 50% samples were excluded. Expression values of 163 unique genes were analyzed for pR vs pNR with dChip software. Empirical FDR was estimated using 1,000 permutations of sample labels. Results: Treatment and response: Sample failure: 6/57 (10%). pR vs pNR did not separate on hierarchical clustering. FLJ12650 and IGFBP2 showed lower expression in pR vs pNR with fold changes of 4.09 and 2.40, respectively (p < 0.01; median FDR: 1/163). FLJ12650 was significant (p < 0.01, median FDR: 0/163) when patients receiving anthracycline ± taxane were analyzed (groups 1-3) and showed a trend (p < 0.05) in group 1 alone. Conclusions: qNPA for limited available FFPE tissue from core biopsies is feasible with acceptable sample failure rates. Small sample size and number of analyzed genes limited definitive conclusion about informative genes in our study. Our FLJ12650 results, a gene coding for membrane Na+/K+ ATPase interacting protein, are consistent with previous findings of overexpression in pNR to anthracyclines + taxanes (Hess et al, JCO 2006, Vol 24; 4236) using fresh tissue. Future qNPA validation of predictive markers, identified by whole transcriptome analysis in a homogenous cohort may provide more definitive results. [Table: see text]

Cerebrovascular connexin expression: effects of traumatic brain injury.

Traumatic brain injury (TBI) results in dysfunction of the cerebrovasculature. Gap junctions coordinate vasomotor responses and evidence suggests that they are involved in cerebrovascular dysfunction after TBI. Gap junctions are comprised of connexin proteins (Cxs), of which Cx37, Cx40, Cx43, and Cx45 are expressed in vascular tissue. This study tests the hypothesis that TBI alters Cx mRNA and protein expression in cerebral vascular smooth muscle and endothelial cells. Anesthetized (1.5% isoflurane) male Sprague-Dawley rats received sham or fluid-percussion TBI. Two, 6, and 24 h after, cerebral arteries were harvested, fresh-frozen for RNA isolation, or homogenized for Western blot analysis. Cerebral vascular endothelial and smooth muscle cells were selected from frozen sections using laser capture microdissection. RNA was quantified by ribonuclease protection assay. The mRNA for all four Cx genes showed greater expression in the smooth muscle layer compared to the endothelial layer. Smooth muscle Cx43 mRNA expression was reduced 2 h and endothelial Cx45 mRNA expression was reduced 24 h after injury. Western blot analysis revealed that Cx40 protein expression increased, while Cx45 protein expression decreased 24 h after injury. These studies revealed significant changes in the mRNA and protein expression of specific vascular Cxs after TBI. This is the first demonstration of cell type-related differential expression of Cx mRNA in cerebral arteries, and is a first step in evaluating the effects of TBI on gap junction communication in the cerebrovasculature.

Mfc1 Is a Novel Forespore Membrane Copper Transporter in Meiotic and Sporulating Cells

To gain insight in the molecular basis of copper homeostasis during meiosis, we have used DNA microarrays to analyze meiotic gene expression in the model yeast Schizosaccharomyces pombe. Profiling data identified a novel meiosis-specific gene, termed mfc1(+), that encodes a putative major facilitator superfamily-type transporter. Although Mfc1 does not exhibit any significant sequence homology with the copper permease Ctr4, it contains four putative copper-binding motifs that are typically found in members of the copper transporter family of copper transporters. Similarly to the ctr4(+) gene, the transcription of mfc1(+) was induced by low concentrations of copper. However, its temporal expression profile during meiosis was distinct to ctr4(+). Whereas Ctr4 was observed at the plasma membrane shortly after induction of meiosis, Mfc1 appeared later in precursor vesicles and, subsequently, at the forespore membrane of ascospores. Using the fluorescent copper-binding tracker Coppersensor-1 (CS1), labile cellular copper was primarily detected in the forespores in an mfc1(+)/mfc1(+) strain, whereas an mfc1Δ/mfc1Δ mutant exhibited an intracellular dispersed punctate distribution of labile copper ions. In addition, the copper amine oxidase Cao1, which localized primarily in the forespores of asci, was fully active in mfc1(+)/mfc1(+) cells, but its activity was drastically reduced in an mfc1Δ/mfc1Δ strain. Furthermore, our data showed that meiotic cells that express the mfc1(+) gene have a distinct developmental advantage over mfc1Δ/mfc1Δ mutant cells when copper is limiting. Taken together, the data reveal that Mfc1 serves to transport copper for accurate and timely meiotic differentiation under copper-limiting conditions.

Suppressor of Cytokine Signaling-1 (SOCS1) Inhibits Lymphocyte Recruitment into the Retina and Protects SOCS1 Transgenic Rats and Mice from Ocular Inflammation

Suppressors of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine signals and defective expression of SOCS1 and SOCS3 has been reported in a number of human diseases. The purpose of this study was to investigate the role of SOCS1 in intraocular inflammatory diseases (uveitis) and whether SOCS1 expression is defective in patients with ocular inflammatory diseases. Blood from patients with scleritis or healthy human volunteers was analyzed for SOCS expression by RNase protection assay and RT-PCR. The authors generated SOCS1 transgenic rats and mice (SOCS1-Tg), induced experimental autoimmune uveoretinitis (EAU) by active immunization with interphotoreceptor retinal binding protein or adoptive transfer of uveitogenic T cells, and investigated effects of SOCS1 overexpression on EAU. SOCS1-mediated protection of retinal cells from apoptosis was assessed by annexin V staining. Induction of cytokine-induced SH2 protein was comparable between patients and volunteers, whereas 80% of lymphocytes from patients with scleritis failed to induce SOCS1 in response to IL-2. Compared with wild-type littermates, SOCS1-Tg rats/mice developed less severe EAU. Constitutive overexpression of SOCS1 in retina inhibited expression of chemokines (CCL17, CCL20, CXCL9, CXCL10), reduced Th17/Th1 expansion, and inhibited recruitment of inflammatory cells into the retina. The authors also show that SOCS1 protected retinal cells from staurosporine as well as H₂O₂-induced apoptosis. Defective expression of SOCS1 in patients with scleritis, taken together with SOCS1-mediated protection of neuroretinal cells from apoptosis, suggest that SOCS1 has neuroprotective function in the retina, implying that administration of SOCS1 mimetic peptides may be useful in treating uveitis or scleritis.

HIV-1 Infection Suppresses Expression of Host Cell Cycle-Associated Gene PDS5A

Objective: To unravel the interplay between HIV-1 and its host cell, the effect of HIV-1 infection on cellular gene expression was investigated. Methods: HIV-1_SF33-infected and uninfected H9 T cells were screened by differential display and RNase protection assay. The finding (PDS5A) was confirmed in HIV-1_Lai-infected P4-CCR5 HeLa cells, which were also examined after PDS5A siRNA knockdown in regard to HIV-1 replication by quantitative RT-PCR, p24 ELISA and LTR-driven β-galactosidase expression. The PDS5A knockdown effect on cellular gene expressions was studied by microarray analysis. PDS5A tissue expression was determined by Northern blotting. Results: Regulator of cohesion maintenance, homolog A (PDS5A) was found to be down-regulated by HIV-1. When PDS5A was suppressed by siRNA, HIV-1 replication was unaffected. PDS5A was found to be highly expressed in skeletal muscle tissue, and to lesser degrees in pancreas, heart, placenta, lung, kidney, liver and brain. Microarray analysis of PDS5A knockdown revealed 91 differential gene products over-representing cell cycle, transport and protein stability regulation, including 4 genes (PP2A, RANTES, PCAF, TCF7L2) previously reported to interact with HIV-1. Conclusion: The data show a downregulation of proliferation-associated host gene PDS5A and suggest a role of PDS5A in HIV-1-induced cellular pathogenesis but not viral replication.

Surfactant protein A modulates the lipopolysaccharide-induced inflammatory response related to preterm birth

Surfactant protein A (SP-A) functions in homeostasis of lung surfactant and in innate immunity. SP-A is secreted by the fetal lung into amniotic fluid. Additionally it has been detected in gestational tissues. We propose that SP-A influences intrauterine inflammation that is commonly associated with preterm birth, the main underlying cause of neonatal mortality and morbidity. We used our previously established mouse model of LPS-induced preterm birth of live-born pups to investigate the role of SP-A in preterm birth. Mice overexpressing rat SP-A (rSP-A) under the control of human SP-C promoter were used. Cytokine concentrations in maternal and fetal serum and in amniotic fluid and mRNA levels of several inflammatory mediators in lungs and in intrauterine tissues were quantified using Cytometric Bead Array and RNase Protection Assay, respectively. Higher levels of SP-A mRNA were observed in fetal lungs and intrauterine tissues of rSP-A mice compared with wild-type. Using Western blot we detected excess of SP-A protein in fetal lung and in amniotic fluid of rSP-A animals. Despite some differences in the basal levels of TNF-α and IL-10 between rSP-A and wild-type animals, there were no differences in the duration of pregnancy. However, the levels of TNF-α, IL-10 and some other inflammatory mediators in intrauterine tissues and in amniotic fluid differed significantly between the mouse lines after maternal LPS given at 17dpc. We conclude that SP-A modulates the levels of intrauterine inflammatory mediators involved in preterm birth and may contribute to inflammatory processes related to spontaneous preterm labor.

Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis

BackgroundRovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.Methodology/Principal FindingsIn this study, we characterized CpxR phosphorylation (CpxR∼P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR∼P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR∼P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate.Conclusions/SignificanceCpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR∼P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis.

Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii

The 23S rRNA gene of Coxiella burnetii, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in Coxiella's biology, we determined the intrinsic stability time periods (in vitro half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA. In vivo half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an Escherichia coli model. Intron RNAs were quantified in synchronous cultures of C. burnetii and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the Coxiella intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The in vitro efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant Coxiella RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator in vivo.

The Pisum sativum psp54 gene requires ABI3 and a chromatin remodeller to switch from a poised to a transcriptionally active state.

Aspects of transcriptional regulation in plants, such as the order in which transcriptional factors and the preinitiation complex are assembled, are obscure because studies carried out under conditions in which native chromatin structure is preserved are still few in comparison with those carried out under other conditions. In vivo chromatin immunoprecipitation (ChIP) experiments were used here to study the regulation of Pisum sativum psp54, which codes for the precursor of a chromatin-associated protein in dry seeds. Antibodies against PsSNF5, a component of the SWI/SNF remodelling complex, and against the transcriptional factor Pisum sativum abscisic acid insensitive 3 (PsABI3) were raised and used for ChIP experiments, which showed that both factors are bound to the psp54 promoter only when the gene is actively expressed during seed maturation and germination. However, RNA polymerase II appeared to be bound to the inactive promoter, which was poised for transcription, before the assembly of factors. Micrococcal nuclease protection assays showed that chromatin conformation at the proximal psp54 promoter changes in shifting from the active to inactive state. The changes in the promoter chromatin of psp54 are discussed. Stalled polymerase is described for the first time at the promoter of a non-heat-shock plant gene.

Both piRNA and siRNA pathways are silencing transcripts of the suffix element in the Drosophila melanogaster germline and somatic cells.

In the Drosophila melanogaster germline, the piRNA pathway silences retrotransposons as well as other transcribed repetitive elements. Suffix is an unusual short retroelement that was identified both as an actively transcribed repetitive element and also as an element at the 3′ ends of the Drosophila non-LTR F element. The copies of suffix that are F element-independent are far more actively transcribed than their counterparts on the F element. We studied the patterns of small RNAs targeting both strands of suffix in Drosophila ovaries using an RNase protection assay and the analysis of the corresponding RNA sequences from the libraries of total small RNAs. Our results indicate that suffix sense and antisense transcripts are targeted mainly by 23–29 nucleotides in length piRNAs and also by 21 nucleotides in length siRNAs. Suffix sense transcripts actively form longer RNA species, corresponding either to partial digestion products of the RNAi and Piwi pathways or to another RNA silencing mechanism. Both sense and antisense suffix transcripts accumulated in the ovaries of homozygous spn-E, piwi and aub mutants. These results provide evidence that suffix sense and antisense transcripts in the germ line and soma are targeted by both RNAi and Piwi pathways and that a Dicer-independent pathway of biogenesis of siRNAs could exist in Drosophila cells.

Hybridization kinetics analysis of an oligonucleotide microarray for microRNA detection

MicroRNA (miRNA) microarrays have been successfully used for profiling miRNA expression in many physiological processes such as development, differentiation, oncogenesis, and other disease processes. Detecting miRNA by miRNA microarray is actually based on nucleic acid hybridization between target molecules and their corresponding complementary probes. Due to the small size and high degree of similarity among miRNA sequences, the hybridization condition must be carefully optimized to get specific and reliable signals. Previously, we reported a microarray platform to detect miRNA expression. In this study, we evaluated the sensitivity and specificity of our microarray platform. After systematic analysis, we determined an optimized hybridization condition with high sensitivity and specificity for miRNA detection. Our results would be helpful for other hybridization-based miRNA detection methods, such as northern blot and nuclease protection assay.

Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of full-length capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5' splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection.

Structure of Long Human Telomeric RNA (TERRA): G-Quadruplexes Formed by Four and Eight UUAGGG Repeats Are Stable Building Blocks

The discovery of long RNA transcripts of telomeric repeats (TERRA) and their potential to form G-quadruplexes stimulated studies on the possible arrangements of G-quadruplexes along TERRA. Here we performed ribonuclease protection assay to investigate the structures formed by long human TERRA. We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded propeller-type conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e., 5'-5', 3'-3', 3'-5', and 5'-3', demonstrated stacking feasibility for all but the 5'-3' arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5'-5' stacking conformation. We also put forward other possible stacking arrangements that involve more than one linker connecting the two G-quadruplex subunits. On the basis of these results, we propose a "beads-on-a-string"-like arrangement along human TERRA, whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively.

Accurate Classification of Diffuse Large B-Cell Lymphoma into Germinal Center and Activated B-Cell Subtypes Using a Nuclease Protection Assay on Formalin-Fixed, Paraffin-Embedded Tissues

Classification of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin (COO) subtypes based on gene expression profiles has well-established prognostic value. These subtypes, termed germinal center B cell (GCB) and activated B cell (ABC) also have different genetic alterations and overexpression of different pathways that may serve as therapeutic targets. Thus, accurate classification is essential for analysis of clinical trial results and planning new trials by using targeted agents. The current standard for COO classification uses gene expression profiling (GEP) of snap frozen tissues, and a Bayesian predictor algorithm. However, this is generally not feasible. In this study, we investigated whether the qNPA technique could be used for accurate classification of COO by using formalin-fixed, paraffin-embedded (FFPE) tissues. We analyzed expression levels of 14 genes in 121 cases of R-CHOP-treated DLBCL that had previously undergone GEP by using the Affymetrix U133 Plus 2.0 microarray and had matching FFPE blocks. Results were evaluated by using the previously published algorithm with a leave-one-out cross-validation approach. These results were compared with COO classification based on frozen tissue GEP profiles. For each case, a probability statistic was generated indicating the likelihood that the classification by using qNPA was accurate. When data were dichotomized into GCB or non-GCB, overall accuracy was 92%. The qNPA technique accurately categorized DLBCL into GCB and ABC subtypes, as defined by GEP. This approach is quantifiable, applicable to FFPE tissues with no technical failures, and has potential for significant impact on DLBCL research and clinical trial development.

Identification of the in vitro target of an iron-responsive AraC-like protein from Neisseria meningitidis that is in a regulatory cascade with Fur

In this study we characterized a genetic locus that is predicted to encode one of the three AraC-like regulators of Neisseria meningitidis, a homologue of MpeR of Neisseria gonorrhoeae which is specific to the pathogenic Neisseria species. Previous microarray studies have suggested that this gene is a member of the Fur regulon. In strain MC58, it is a pseudogene (annotated as two ORFs, NMB1879 and NMB1878) containing a frameshift mutation which we show is common to all strains tested belonging to the ST-32 hypervirulent clonal complex. Using primer extension and S1 nuclease protection assays, we mapped two promoters in the upstream intergenic region: the mpeR promoter and the NMB1880 promoter. The latter promoter drives transcription of the divergent upstream locus, which is predicted to encode a high-affinity iron uptake system. We demonstrated that both promoters are induced during iron limitation and that this regulation is also mediated by the Fur regulator. DNA-binding studies with the purified MpeR protein revealed that it binds to a region directly upstream of the NMB1880 divergent promoter, suggesting a role in its regulation. Mutants of N. meningitidis strains lacking MpeR or overexpressing MpeR showed no significant differences in expression of the P(NMB1880) promoter, nor did global transcriptional profiling of an MpeR knockout identify any deregulated genes, suggesting that the MpeR protein is inactive under the conditions used in these experiments. The presence of MpeR in a regulatory cascade downstream of the Fur master iron regulator implicates it as being expressed in the iron-limiting environment of the host, where it may in turn regulate a group of genes, including the divergent iron transport locus, in response to signals important for infection.

Quantitative nuclease protection assay (qNPA) for gene expression analysis on breast cancer core biopsies.

Quantitative nuclease protection assay (qNPA) for gene expression analysis on breast cancer core biopsies.

Stress and cocaine interact to modulate Arc/Arg3.1 expression in rat brain

The interaction between stress and drugs of abuse is a critical component of drug addiction, but the underlying molecular mechanisms remain elusive. Arc/Arg3.1 is an effector immediate early gene that may represent a bridge connecting short- and long-term neuronal modifications associated with exposure to stress and drugs of abuse. This research aims to study the modulation of Arc/Arg3.1 expression as a marker of neuronal changes associated with exposure to stress and cocaine. Rats exposed to either single or repeated stress sessions were subjected to a single intraperitoneal injection of cocaine hydrochloride (10 mg/kg) and sacrificed 2 h later. RNase protection assay was used to determine changes in Arc/Arg3.1 gene expression in different brain regions. We found significant stress-cocaine interactions in the prefrontal cortex (p < 0.001) and hypothalamus (p < 0.05). In the prefrontal cortex, acute stress potentiated cocaine-induced Arc/Arg3.1 mRNA elevation, whereas prolonged stress attenuated the response to cocaine. In the hypothalamus, although markedly reduced by acute stress, Arc/Arg3.1 gene expression was still increased by cocaine. No interaction was observed following repeated stress. Notably, cocaine-induced Arc/Arg3.1 mRNA levels were not influenced by stress in striatum and hippocampus. In our experimental model, stress interacted with cocaine to alter Arc/Arg3.1 expression in a regionally selective fashion and in a way that depended on whether stress was acute or repeated. These results point to Arc/Arg3.1 as a potential molecular target modulated by stress to alter cellular sensitivity to cocaine.

Quantification methods for Alexandrium catenella, a toxic dinoflagellate: Comparison of competitive enzyme-linked immunosorbent assay and sandwich hybridization integrated with a nuclease protection assay

Alexandrium catenella (Whedon et Kofoid) Balech, a toxic dinoflagellate, is a bloom-forming planktonic species in cold water coastal regions. It produces strong paralytic shellfish poisoning (PSP) toxins which are transmitted via tainted shellfish. These toxins can affect humans, other mammals, fish and birds. In this study, polyclonal antiserum against A. catenella was produced, and a competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect A. catenella. The antiserum against A. catenella showed good specificity, the linear detection range was relatively large, between 38 and 600,000 cells. In addition, specific probes were designed to target the small subunit ribosomal RNA (SSU rRNA) of A. catenella, and quantitative sandwich hybridization integrated with a nuclease protection assay (NPA-SH) was established in order to identify and quantify A. catenella. The NPA-SH assay did not show good specificity as well as cELISA, by which A. catenella and A. tamarense could not be distinguished. Samples in different cell growth phases were analyzed with cELISA and NPA-SH. The results showed that the cell concentration calculated by cELISA was very similar with microscopy, while that of NPA-SH was sometimes higher than that of microscopy, especially in log phase. Comparing the two methods, both assays allow rapid identification of A. catenella without time-consuming microscopy when multiple sites need to be considered in routine monitoring. Meanwhile, cELISA was more specific and accurate in detection of A. catenella than NPA-SH.

Feed-Forward Regulation of Microbisporicin Biosynthesis in Microbispora corallina

Lantibiotics are ribosomally synthesized, posttranslationally modified peptide antibiotics. Microbisporicin is a potent lantibiotic produced by the actinomycete Microbispora corallina and contains unique chlorinated tryptophan and dihydroxyproline residues. The biosynthetic gene cluster for microbisporicin encodes several putative regulatory proteins, including, uniquely, an extracytoplasmic function (ECF) σ factor, σ(MibX), a likely cognate anti-σ factor, MibW, and a potential helix-turn-helix DNA binding protein, MibR. Here we examine the roles of these proteins in regulating microbisporicin biosynthesis. S1 nuclease protection assays were used to determine transcriptional start sites in the microbisporicin gene cluster and confirmed the presence of the likely ECF sigma factor -10 and -35 sequences in five out of six promoters. In contrast, the promoter of mibA, encoding the microbisporicin prepropeptide, has a typical Streptomyces vegetative sigma factor consensus sequence. The ECF sigma factor σ(MibX) was shown to interact with the putative anti-sigma factor MibW in Escherichia coli using bacterial two-hybrid analysis. σ(MibX) autoregulates its own expression but does not directly regulate expression of mibA. On the basis of quantitative reverse transcriptase PCR (qRT-PCR) data, we propose a model for the biosynthesis of microbisporicin in which MibR functions as an essential master regulator and the ECF sigma factor/anti-sigma factor pair, σ(MibX)/MibW, induces feed-forward biosynthesis of microbisporicin and producer immunity.