Reverse transcriptase-PCR Products Research Articles

Comparative Analysis of Salinity Responsive Candidate Gene Expression in Selected Sri Lankan Rice Varieties

Studying of expression dynamics of salt responsive candidate genes under different genetic background of rice is important to develop resilient salt tolerant rice varieties. The present study was focused to assess the comparative expression of salinity responsive genes which are involved in Na+/K+ homeostasis (OsNHX1), signal transduction pathway (OsMAPK5, OsCDPK7) and synthesis of osmoprotectant (OsTPS1). Comparative expression of selected genes were assessed in At354 (improved), Godawee (traditional) and Kuruwi Perunel (traditional), salinity tolerant varieties and Suwandel, a salinity susceptible traditional variety. The assessment was done before salinization (at 0 h) and after salinization at 24 h and 72 h using semi-quantitative Reverse Transcriptase PCR (RT-PCR). RT-PCR products were analysed using 1.5% agarose gel electrophoresis. Resulted gel profiles were assessed and relative level of gene expression was measured based on the intensity of the PCR product in terms of average pixel values. Relative level of gene expression in four varieties was plotted against the exposure time to salinity. Accordingly, marked variation in expression pattern of the selected genes was detected in four varieties over the tested in Godawee and Suwandel compared to At354 and Kuruwi Perunel. At 24 h, all four genes were up-regulated in At354 and Godawee while they were down-regulated in Suwandel. In Kuruwi Perunel, OsNHX1, OsMAPK5 and OsCDPK7 genes were up-regulated at 24 h while OsTPS1 was up-regulated at 72 h. At 72 h, continuous expression of all genes was observed in Kuruwi Perunel and Godawee, whereas expression was comparatively down-regulated in At354 approximately to the same level under non-stress condition. It could be suggested that At354 might have been adapted to the stress relatively earlier compared to Godawee and Kuruwi Perunel, where expressions of genes were less pronounced. Further experiments with various time period of salinity exposure should be carried out to make detailed inference regarding the expression dynamics in tested rice varieties.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein–Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV–Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Influence of 5-azacytidine and salicylic acid on demethylase gene expression in cell cultures of Vitis amurensis Rupr.

DNA methylation, especially cytosine methyl- ation, is known to play an important role in various developmental processes and defense mechanisms in plants and other organisms. The level and pattern of cytosine methylation are determined by both DNA methylation and demethylation machineries. DNA methylation is known to be established and maintained by DNA methyltransferases, whereas active DNA demethylation is performed by DNA glycosylases. In our previous study, the expression of methylatransferases in cell cultures of Vitis amurensis was studied. The purpose of the present work was to analyze demethylase (Dem) gene expression in the control (V2) and transformed with rolB gene (VB2) from Agrobacterium rhizogenes cell cultures of V. amurensis under the influence of 5-azacytidine (azaC) induced DNA demethylation and treatment with salicylic acid (SA), a plant stress phytohormone. The lowest total Dem expression was detected in the V. amurensis calli of the control V2 cell culture without treatment, while higher Dem expression was detected in the leaves of the 8-year-old V. amurensis plant. Treatment with azaC and SA significantly increased total Dem expression in the V. amurensis V2 and VB2 cell cultures 1.4-3.2 times. Using frequency analysis of reverse transcriptase PCR products obtained with degenerate primers and real-time PCR we analyzed expression of the three Dem transcripts: VaDem1, VaDem2, and VaDem3. The deduced amino acid sequence of VaDem1 is highly homologous to the V. vinifera Ros1-like gene (XM_002277365), VaDem2 to the VvDML3-like (XM_002270849); VaDem3 to the VvDemeter-like (XM_002267274). In the cDNA of the V. amurensis cell cultures the VaDem1 transcripts were more abundant than the VaDem2 and VaDem3 transcripts. Addition of azaC and SA significantly increased the VaDem1 and VaDem2 expression. The results indicate that the VaDem2 gene (a homologue of DML3 of A. thaliana) and the VaDem1 gene (a homologue of ROS1 of A. thaliana) are important in stress response and our data suggest that the VaDem2 and VaDem1 genes are important candidates for future research on the mechanisms of plant defence responses.

High Incidence of Primary Resistance to Imatinib in the Group of Ph-Negative BCR-ABL-positive CML Patients,

Abstract 3772 Background.Around 1–2% of the CML patients do not have t(9;22)(q34;q11) detectable by conventional cytogenetics, but carry BCR-ABL fusion revealed by fluorescence in situ hybridization (FISH) and/or reverse-transcriptase PCR (RT-PCR). Outcome of Imatinib (IM) treatment in this group of patients remains unclear. Aim. To evaluate IM treatment efficacy in Ph-negative BCR-ABL- positive CML patients in comparison with Ph-positive ones. Methods. Initial diagnostics including chromosome banding analysis (CBA) and RT-PCR was performed in 251 CML patients who received 400 or 600 mg IM once a day. This group consisted of 181 patients in early chronic phase (CP), 65 patients in late CP and 4 patients in accelerated phase (AP). CBA was done after 24 hours culture. G-banding was performed by a trypsin-Giemsa method. Karyotypes were described according to ISCN (2005). At least 20 metaphases for each sample were analyzed. FISH assay using Dual-Colour Dual-Fusion BCR-ABL Translocation Probe (Abbott, USA) was applied on at least 200 interphase nuclei (I-FISH) and on all available metaphases. CBA and FISH were performed at the time of diagnostics and every 6 months of IM treatment. Cytogenetic response (CyR), partial cytogenetic response (PCyR) and complete cytogenetic response (CCyR) in Ph-positive CML patients were evaluated by CBA in contrast to Ph-negative patients those were assessed by I-FISH only. In Ph-negative CML patients CCyR were assumed as less than 1% of BCR-ABL -positive nuclei (N. Testoni et al, Blood, 2009). Quantitative measurement of BCR-ABL/ABL transcripts ratio by real-time quantitative PCR (RQ-PCR) was done every 3–6 months. Detection of point mutations in the BCR-ABL tyrosine kinase domain (TKD) was performed by direct sequencing of RT-PCR products. According to European LeukemiaNet recommendations (M. Baccarani et al, JCO, 2009), failures were defined as no complete hematological response (CHR) at 3 months, no CyR at 6 months, less that PCyR at 12 mo, less than CCyR at 18 months, loss of CHR, CCyR or major molecular response (MMR) at any time during treatment, newly acquired BCR-ABL mutation poorly sensitive to IM (G250E, E255V/K, T315I), or progression to the AP and/or blast crisis (BC). In Ph-negative patients 6 months and 12 months time-points, when CyR and PCyR have to be estimated, were excluded from the analysis due to lack of direct correlation between percentage of BCR-ABL -positive nuclei and CBA data (N. Testoni et al, Blood, 2009). Failure-free survival (FFS) and overall survival (OS) were calculated separately for Ph-positive and Ph-negative patients’ groups. Results. In respect to t(9;22)(q34;q11) detection by CBA at the time of initial diagnosis all CML patients were divided into 2 groups: Ph-positive (n=244) and Ph-negative (n=7). In spite of presence of normal karyotype, all patients in the second group harboured BCR-ABL fusion gene revealed by FISH and RT-PCR. Groups were not significantly different in age at diagnosis, sex, Sokal risk group distribution, stage of disease at the time of IM treatment beginning, types of BCR-ABL fusion transcripts. Median time of follow-up was 43 months (range 26–66) in Ph-negative group and 36 months (range 18–105) in Ph-positive. Treatment results are shown in table. Although 6 of 7 Ph-negative CML patients achieved CHR by 3 months, only 1 of them was in CCyR by 18 mo, that was significantly lower than in Ph-positive group (p<0.001). Moreover, Ph-negative patients had high percentage of BCR-ABL -positive nuclei in BM both at 12 months (median 35% (range 1–60%)) and at 18 months of IM therapy (median 49% (range 0–92%)). Two Ph-negative CML patients progressed to AP and died subsequently. None of Ph-negative patients had BCR-ABL mutations, duplication or amplification. FFS in Ph-negative CML patients treated by IM was significantly lower than in 244 Ph-positive ones 0.14±0.13 vs 0.62±0.03 (p=0.007), while OS was comparable: 0.70±0.15 vs 0.85±0.02, (p=0.47), respectively. Conclusions. In our series treatment outcomes in Ph-negative CML patients who received IM at a dose of 400 or 600 mg once daily were significantly worse in comparison with Ph-positive ones. However, dose escalation or switching to second-generation TKI prevented further disease progression or CML-related deaths. Resistance in the observed group seems to have BCR-ABL-independent mechanisms. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High allele frequency of the p.Q258X mutation and identification of a novel mis-splicing mutation in the STAR gene in Korean patients with congenital lipoid adrenal hyperplasia

Steroidogenic acute regulatory (STAR) protein plays a crucial role in steroidogenesis, and mutations in the STAR gene cause congenital lipoid adrenal hyperplasia (CLAH). This study investigated the STAR mutation spectrum and functionally analyzed a novel STAR mutation in Korean patients with CLAH. Mutation analysis of STAR was carried out in 25 unrelated Korean CLAH patients. A region of STAR comprising exons 4-7 was cloned from human genomic DNA into an expression vector, followed by site-directed mutagenesis and transient expression in COS7 cells. The splicing pattern was analyzed by in vitro transcription, and each transcript was functionally characterized by measuring pregnenolone production in COS7 cells cotransfected with the cholesterol side chain cleavage system. Mutation p.Q258X was identified in 46 of 50 alleles (92%); mutation c.653C>T was detected in two alleles (4%); and mutations p.R182H and c.745-6_810del were found in one allele (2%). Reverse transcriptase-PCR products amplified from a patient heterozygous for compound c.653C>T and c.745-6_810del mutation revealed multiple alternatively spliced mRNAs. In vitro expression analysis of a minigene consisting of exons 4-7 containing the c.653C>T yielded two transcripts in which exon 6 or exons 5 and 6 were skipped. The encoded proteins exhibited defective pregnenolone-producing ability. The c.745-6_810del mutation led to full and partial intron retention. p.Q258X is the most common STAR mutation in Korea. A previously reported c.653C>T variant was found to cause aberrant splicing at the mRNA level, resulting in perturbation of STAR function. The c.745-6_810del mutation also resulted in aberrant splicing.

Comprehensive Analysis of the MYB-NFIB Gene Fusion in Salivary Adenoid Cystic Carcinoma: Incidence, Variability, and Clinicopathologic Significance

The objectives of this study were to determine the incidence of the MYB-NFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathologic significance of the fusion, and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. We did an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcriptase-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both the MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (P = 0.03) with patients older than 50 years of age. No correlation with other clinicopathologic markers, factors, and survival was found. We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC.

Differential RNA expression of KIR alleles

Allelic polymorphisms dramatically influence the phenotype of human killer immunoglobulin-like receptors (KIR) by modifying their expression in cell surfaces. It is unclear though to what extent this involves transcriptional or post-transcriptional mechanisms, as quantitative RNA expression of KIR alleles has not been systematically compared. We measured RNA transcript abundance of common KIR alleles by real-time quantitative reverse transcriptase PCR (RT-PCR) in 85 PBL samples that were allele-typed in parallel. Allele type showed little influence on transcript abundance for a given KIR gene, except for: (1) KIR2DL5B*002, which consistently showed undetectable transcripts levels; (2) truncated KIR2DS4 alleles, associated with lowered expression levels; and (3) alleles of KIR2DL4 with a single-base deletion, associated with higher expression than average. Lowered levels of truncated KIR2DS4 transcripts were confirmed by dot blot of RT-PCR products, indicating imbalanced allelic RNA expression in heterozygote genotypes containing these alleles. Imbalanced expression of truncated KIR2DS4 alleles was corroborated in family samples. Gene copy number of KIR2DL1, KIR2DL3 and KIR3DL1 influenced RNA expression, genotypes with a single copy expressing on average lower transcript amounts than those with two copies. The data show that for a given KIR gene, the common allele types found in our population express comparable RNA levels, except truncated or null alleles. Thus, variation of KIR expression on cell surfaces more likely involves post-transcriptional mechanisms.

Functional Analysis of the Synonymous R385R Mutation in the Low-Density Lipoprotein Receptor Gene

Familial hypercholesterolemia is caused by mutations in the low-density lipoprotein receptor (LDLR) gene. The synonymous mutation R385R has been shown to introduce a cryptic splice site in exon 9. The aims of this study were to establish to what extent the cryptic splice site is selected ahead of the normal splice site and to determine if the aberrant transcript is degraded by nonsense-mediated mRNA decay. The relative amount of the aberrant transcript was determined by real-time PCR and found to vary from 25% to 45% in heterozygous familial hypercholesterolemia individuals. Epstein-Barr virus-transformed lymphocytes were established from one heterozygous patient, and treatment of these cells with cycloheximide increased the amount of aberrant transcript, indicating that the aberrant transcripts are degraded by nonsense-mediated mRNA decay. Cloning of reverse transcriptase-PCR products from one of the heterozygous patients and introduction of the R385R mutation into a minigene reporter construct revealed an almost exclusive use of the cryptic splice site in the mutated allele. Thus, the synonymous mutation R385R converts the mutated allele to a null allele unable to produce functional mRNA.

Synthesis, Structure, and Activities of an Oral Mucosal α-Defensin from Rhesus Macaque

The oral cavity is an environment challenged by a large variety of pathogens. Consequently, the antimicrobial peptides expressed in that environment are interesting as they evolved to defend against a broad spectrum of bacteria and fungi. Here we report the discovery of new alpha-defensins from rhesus macaque oral mucosa and determine the first alpha-defensin structure from that species. The new peptides were identified by sequencing of reverse transcriptase-PCR products obtained from oral mucosal tissues, disclosing three mucosal alpha-defensins, termed rhesus macaque oral alpha-defensins (ROADs). The peptide corresponding to fully processed ROAD-1 was synthesized, subjected to folding/oxidation conditions, and purified. ROAD-1 was active against Staphylococcus aureus, Escherichia coli, and Candida albicans in a concentration-dependent manner. We determined the structure of ROAD-1 using NMR spectroscopy and find that the synthetic peptide adopts the canonical disulfide pairing and alpha-defensin fold. The antimicrobial mechanism of defensins has been correlated with their ability to disrupt and permeabilize the cell envelope, activities that depend on the surface features of the folded peptide. Although ROAD-1 maintains the defensin fold, the oral defensin displays distinct surface features when compared with other alpha-defensin structures.

MRD Monitoring in AML Patients with MLL-MLLT4 by Quantification of Fusion Gene Transcripts and Genomic Chromosomal Breakpoint Sequences

Statement. The MLL-MLLT4 (former MLL-AF6) fusion gene (FG) is relatively rare genetic abnormality, predominantly found in AML. It averages 3–5% among other MLL rearrangements. Here we present data of MRD monitoring in 2 patients with AML carrying an MLL-MLLT4 rearrangement by 2 approaches: usingFG transcript at RNA/cDNA level in comparison withFG at genomic DNA level.Materials and methods. Patients (pts) were diagnosed according French-American-Britain (FAB) classification. Initial diagnostics included cytomorphology, immunophenotyping of blast cells, cytogenetics, FISH and reverse transcriptase PCR (RT-PCR). RT-PCR products were directly sequenced afterwards. In both cases identification of genomic chromosomal breakpoint sequences within MLL and MLLT4 genes was done by long-distance inverse PCR (LDI-PCR). MRD quantification in genomic DNA was performed using patient-specific primers and probes by real-time quantitative PCR (RQ-PCR). 500 ng of DNA was used per reaction. Standard curve was received by serial 10-fold dilutions of pts' DNA into the DNA isolated from pooled lymphocytes of ten healthy donors. b-actin was used as DNA quality and quantity control. Detection of FG transcript kinetics during treatment was performed by RQ-PCR according to “Europe Against Cancer” recommendations for normalization by using control gene ABL (Gabert J. et al Leukemia, 2003, 17) and for using 10-fold dilutions of plasmids carrying MLL-MLLT4 fragment as source of standard curve. MRD value for cDNA targets were estimated as previously described (Beillard E. et al Leukemia, 2003, 17). Each sample was run in triplicates. According to the treatment design, time-points for MRD estimation were scheduled before each block of treatment. Totally 5 samples were evaluated in each patient (initial and 4 follow-ups). FLT3-ITD status was estimated at the time of diagnosis. Informed consent was obtained in both cases.Patients' characteristics, treatment and clinical outcomeCase # 1Case #2Age5813SexMaleMaleInitial WBC*106/ml5.594.9ImmunophenotypeCD34+CD117+HLA-DR+ CD11c+CD13+CD33+CD65+CD34+CD117+CD13+ CD33+CD45+MPO+Cytogenetics46, XY, del(5)(q?), der(5)t(5;6;11) (q22;q15q27;q23), der(6)t(5;6) (q22;q15), del der(11)46, XY, t(6;11)(q27;q23)FISH with LSI MLLMLL deletionMLL splitRT-PCRMLL-MLLT4 positiveMLL-MLLT4 positiveMLL-MLLT4 FG transcriptexon 9-exon 2exon 9-exon 2Localization of genomic chromosomal breakpoint within MLL and MLLT4intron 9-intron 1intron 9-intron 1FLT3-ITDNegativeNegativeInduction treatment7+3AIEConsolidation therapy2× HAM 2× HDAC1× HAM 1× FLAG 1× HAEMaintenance−+Duration of therapy, months87Achievement of CR++OS, months87EFS, months65Current statusAlive in CRAlive in CRResults. Despite of achievement of CR, MLL-MLLT4 FG transcripts were detected in every sample tested after induction and consolidation chemotherapy by RQ-PCR. MRD value in case #1 in cDNA was fluctuated significantly within 2 log. Although in case #2 there was successive reduction from 260% at the beginning of treatment till 0.7% before maintenance therapy (after HAE block). Limited dilution series of a MLL-MLLT4-positive RNA into RNA of ten healthy donors showed a sensitivity limitation of 1E–05. For quantification of genomic chromosomal breakpoint sequences b-actin was amplified in each well. Deviation between Ct values of b-actin in different wells did not exceed ±2.0. In case # 1 the standard curve of the RQ-PCR assay for MLL-MLLT4 FG had slope of −3.19. Correlation coefficient was 0.987. Quantitative range of this assay was 1E–04 and sensitivity 1E–5. It was also observed a considerable variation of MRD levels in genomic DNA during treatment, like it was observed in MRD monitoring by FG transcripts. Fluctuations run up to 2.5 log. In case #2 the standard curve of the RQ-PCR assay for MLL-MLLT4 had a slope of −3.63 with correlation coefficient 0.992. Quantitative range of this assay was reached 1E–04 with sensitivity 1E–05. MRD level in this patient constantly decreased.Conclusions. The same tendency has been shown in each patient: fluctuations of MRD levels (2.5 log in case #1) and successive reduction (case #2). Results received at RNA/cDNA level and in genomic DNA cannot substitute each other, but they can be used as additives. It has been demonstrated that quantification of MLL-MLLT4 FG in genomic DNA is precise and suitable for MRD monitoring.

Transcription Analysis of stx1, marA, and eaeA Genes in Escherichia coli O157:H7 Treated with Sodium Benzoate

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37°C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate–treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

Structure and Function of the Smallest Vertebrate Telomerase RNA from Teleost Fish

Telomerase extends chromosome ends by copying a short template sequence within its intrinsic RNA component. Telomerase RNA (TR) from different groups of species varies dramatically in sequence and size. We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate TRs from five teleost fishes. The teleost TRs (312-348 nucleotides) are significantly smaller than the cartilaginous fish TRs (478-559 nucleotides) and tetrapod TRs. This remarkable length reduction of teleost fish TRs correlates positively with the genome size, reflecting an unusual structural plasticity of TR during evolution. The teleost TR consists of a compact three-domain structure, lacking most of the sequences in regions that are variable in other vertebrate TR structures. The medaka and fugu TRs, when assembled with their telomerase reverse transcriptase (TERT) protein counterparts, reconstituted active and processive telomerase enzymes. Titration analysis of individual RNA domains suggests that the efficient assembly of the telomerase complex is influenced more by the telomerase reverse transcriptase (TERT) binding of the CR4-CR5 domain than the pseudoknot domain of TR. The remarkably small teleost fish TR further expands our understanding about the evolutionary divergence of vertebrate TR.

An improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR

A new rapid (less than 6 h from insect-to-results) high-throughput assay that is sensitive and specific for detecting BTV RNA in Culicoides biting midges is reported. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using specialized beads in a homogenization buffer compatible with cell culture and RNA extraction. A portion of homogenate (10%) from each specimen was retained for confirmatory infectious virus isolation, while the remaining 90% was used for RNA extraction. The RNA was used in a single step reverse transcriptase PCR (RT-PCR) reaction with infrared (IR)-dye-labeled primers. The RT-PCR products were visualized in agarose gels with an infrared scanner. The adaptation of IR-dye-labeled primers in combination with a one step RT-PCR resulted in a detection limit of 0.5 pfu of purified BTV RNA. All 24 serotypes of BTV prototype strains and none of the 8 serotypes of the closely related epizootic hemorrhagic disease virus (EHDV) prototype strains were detected.

Relationship between RNA splicing and exon editing near intron junctions in wheat mitochondria

We have compared the RNA editing status of wheat mitochondrial unspliced precursor transcripts with spliced RNAs, focusing in particular on exon editing sites located very close to intron/exon junctions. Using direct sequencing of reverse transcriptase–PCR products to assess C‐to‐U editing in various RNA populations for the respiratory chain genes nad2, nad4, nad5, nad7, and cox2, we observed that candidate exon sites immediately upstream or downstream of introns remained unedited in the presence of the adjacent intron, whereas sites further away were typically partially (or completely) edited in precursor molecules. The ‘late’ editing of exon sites adjacent to splice junctions is consistent with access of the editing machinery being sterically hindered by the intron or alternatively by an editing recognition element being created by the exon/exon structure. When we examined RNA isolated from the embryo‐to‐seedling stages of wheat development, we found that fully spliced messenger RNAs showed editing at the expected sites in all stages, including dormant seeds. In contrast, we observed that cox2 precursors were less completely edited in RNA populations from 0 to 24 h postimbibition embryos than in 6‐day‐old seedlings not only at splice junctions but also at other exon sites, consistent with more efficient coupling between editing and transcription during later stages of development.

Association of reversible inactivation of the maize transposable element Ds with tissue-specific processing of the 35S:TPase transcript in carrot (Daucus carota L.)

SummaryAn Ac/Ds-based two-element transposon tagging system has been introduced into carrot. F1 progeny containing both the 35S-Ac-transposase gene (35S:TPase) and the Ds element were derived from crosses between 35S:TPase- and Dsbearing parents. While excision of Ds was not detected in any F1 plants carrying both 35S:TPase and the Ds element, calli initiated from these F1 plants had the Ds element excised, indicating Ds transposition. Reverse transcriptase-PCR analysis revealed that the 35S:TPase gene was expressed in both F1 plants and calli, and that introns 1, 2, and 3 were spliced correctly. Although intron 4 was also spliced correctly in calli, incorrectly spliced intron 4 was detected in F1 plants. Sequence analysis of incorrectly spliced reverse transcriptase-PCR products demonstrated the presence of a cryptic intron donor site within intron 4 of the 35S:TPase transcript. This probably competed with the proposed intron donor site during maturation of the major 35S:TPase transcript. These results suggested that the major transcript of 35S:TPase was incorrectly processed and, consequently, that the Ds element was reversibly inactivated in the somatic tissues of carrot plants, whereas this inactive Ds element was remobilised during tissue culture, where the 35S:TPase transcript was spliced correctly. These observations point to an important role for tissue-specific 35S:TPase transcript processing for successful transposition of Ds in carrot. Therefore, successful processing of the 35S:TPase transcript in carrot callus may indicate strategies to increase Ac transposition in other tissues.

Hydrogen Peroxide Stimulates Proliferation and Migration of Human Prostate Cancer Cells through Activation of Activator Protein-1 and Up-regulation of the Heparin Affin Regulatory Peptide Gene

It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration- and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HP-induced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5'-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.

High Glucose-suppressed Endothelin-1 Ca2+ Signaling via NADPH Oxidase and Diacylglycerol-sensitive Protein Kinase C Isozymes in Mesangial Cells

High glucose (HG) is the underlying factor contributing to long term complications of diabetes mellitus. The molecular mechanisms transforming the glomerular mesangial cell phenotype to cause nephropathy including diacylglycerol-sensitive protein kinase C (PKC) are still being defined. Reactive oxygen species (ROS) have been postulated as a unifying mechanism for HG-induced complications. We hypothesized that in HG an interaction between ROS generation, from NADPH oxidase, and PKC suppresses mesangial Ca2+ signaling in response to endothelin-1 (ET-1). In primary rat mesangial cells, growth-arrested (48 h) in 5.6 mM (NG) or 30 mm (HG) glucose, the total cell peak [Ca2+]i response to ET-1 (50 nM) was 630 +/- 102 nM in NG and was reduced to 159 +/- 15 nM in HG, measured by confocal imaging. Inhibition of PKC with phorbol ester down-regulation in HG normalized the ET-1-stimulated [Ca2+]i response to 541 +/- 74 nM. Conversely, an inhibitory peptide specific for PKC-zeta did not alter Ca2+ signaling in HG. Furthermore, overexpression of conventional PKC-beta or novel PKC-delta in NG diminished the [Ca2+]i response to ET-1, reflecting the condition observed in HG. Likewise, catalase or p47phox antisense oligonucleotide normalized the [Ca2+]i response to ET-1 in HG to 521 +/- 58 nM and 514 +/- 48 nM, respectively. Pretreatment with carbonyl cyanide m-chlorophenylhydrazone or rotenone did not restore Ca2+ signaling in HG. Detection of increased intracellular ROS in HG by dichlorofluorescein was inhibited by catalase, diphenyleneiodonium, or p47phox antisense oligonucleotide. HG increased p47phox mRNA by 1.7 +/- 0.1-fold as measured by reverse transcriptase-PCR. In NG, H2O2 increased membrane-enriched PKC-beta and -delta, suggesting activation of these isozymes. HG-enhanced immunoreactivity of PKC-delta visualized by confocal imaging was attenuated by diphenyleneiodium chloride. Thus, mesangial cell [Ca2+]i signaling in response to ET-1 in HG is attenuated through an interaction mechanism between NADPH oxidase ROS production and diacylglycerol-sensitive PKC.

Development of a rapid method using nucleic acid sequence-based amplification for the detection of astrovirus

We have developed a rapid method to detect astrovirus in fecal specimens utilizing nucleic acid sequence-based amplification (NASBA) and several detection methodologies, including a sandwich hybridization assay based on DNA-tagged liposomes (liposome-strip detection assay). RNA was extracted from 65 stool specimens that were positive for astrovirus by enzyme immunoassay and was amplified by both NASBA and reverse transcriptase PCR (RT-PCR). Also extracted and amplified were 19 specimens containing rotavirus, 20 specimens containing norovirus, five specimens containing adenovirus, 15 water negative control specimens, and eight specimens containing astrovirus reference strains. NASBA products were detected by electrochemiluminescence detection (ECL) and by liposome-strip detection; RT-PCR products were detected by ethidium bromide staining following gel electrophoresis and by liquid hybridization assay (LHA). There was no significant difference in the detection rates of NASBA- and RT-PCR-based assays, with one exception in which the NASBA/ECL assay detected astrovirus in eight specimens that tested negative by the RT-PCR/LHA assay. These results suggest that these NASBA-based detection methods have detection rates that are as good as or better than those of RT-PCR-based methods. Both NASBA and liposome-strip detection may be useful for field studies and environmental testing because these methods are rapid and do not require specialized equipment.

Increased expression of C1-inhibitor mRNA in patients with hereditary angioedema treated with Danazol

The attenuated androgen Danazol can partially reverse the biochemical defect and prevent angioedema in patients with inherited C1-inhibitor (C1-INH) deficiency (hereditary angioedema, HAE). Though its clinical effectiveness is independent from significant increase of C1-INH plasma levels, its mechanism of action remains unknown. Since angioedema is a local phenomenon, it could be controlled by restoring tissue levels of C1-INH. We measured the expression of C1-INH mRNA in peripheral blood mononuclear cells (PBMCs) of 13 patients with HAE type 1 (seven untreated and asymptomatic, and six on Danazol at the minimal effective dose) and of eight normal controls. mRNA levels were quantitated by computerized optical densitometry of reverse transcriptase-PCR products, normalized for the amount of glyceraldehyde-3-phosphate-dehydrogenase and expressed as percent of normal pooled RNAs. Each determination represented the mean of three separate experiments. Measurement of C1-INH mRNA in two patients before and after 1 month of Danazol 400 mg per day demonstrated a post-treatment increase of 15 and 21%, respectively. When HAE patients and controls were analyzed as groups, C1-INH mRNA levels of patients untreated and asymptomatic (median 73%, range 65–78) were significantly lower ( P=0.001) compared to controls (median 101%, range 87–121) and to patients on Danazol (median 91%, range 82–96); the difference among the last two groups was not statistically significant. Our data demonstrate that minimal effective doses of Danazol increase the expression of C1-INH mRNA in PBMC of HAE patients even in the absence of a significant increase of C1-INH plasma levels.

Expression of chlorocatechol 1,2-dioxygenase and chlorocatechol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples.

In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H(2)O(2). Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from Pseudomonas chlororaphis RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene cbzE from Pseudomonas putida GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.