Quantitative Real-time Reverse Transcriptase PCR Research Articles

Gammaherpesvirus modulation of mouse adenovirus type 1 pathogenesis

Immune function is likely to be shaped by multiple infections over time. Infection with one pathogen can confer cross-protection against heterologous pathogens. We tested the hypothesis that latent murine gammaherpesvirus 68 (γHV68) infection modulates host inflammatory responses and susceptibility to mouse adenovirus type 1 (MAV-1). Mice were infected intranasally (i.n.) with γHV68. 21 days later, they were infected i.n. with MAV-1. We assessed cytokine and chemokine expression by quantitative reverse transcriptase real-time PCR, cellular inflammation by histology, and viral loads by quantitative real-time PCR. Previous γHV68 infection led to persistently upregulated IFN-γ in lungs and spleen and persistently upregulated CCL2 and CCL5 in the lungs. Previous γHV68 infection amplified MAV-1-induced CCL5 upregulation and cellular inflammation in the lungs. Previous γHV68 infection was associated with lower MAV-1 viral loads in the spleen but not the lung. There was no significant effect of previous γHV68 on IFN-γ expression or MAV-1 viral loads when the interval between infections was increased to 44 days. In summary, previous γHV68 infection modulated lung inflammatory responses and decreased susceptibility to a heterologous virus in an organ- and time-dependent manner.

Expression profiling and mapping of defence response genes associated with the barley–Pyrenophora teres incompatible interaction

Barley net- and spot-form of net blotch disease are caused by two formae of the hemibiotrophic fungus Pyrenophora teres (P. t. f. teres and P. t. f. maculata). In the present study, suppression subtractive hybridization (SSH) was used in combination with quantitative real-time reverse transcriptase PCR to identify and profile the expression of defence response (DR) genes in the early stages of both barley-P. teres incompatible and compatible interactions. From a pool of 307 unique gene transcripts identified by SSH, 45 candidate DR genes were selected for temporal expression profiling in infected leaf epidermis. Differential expression profiles were observed for 28 of the selected candidates, which were grouped into clusters depending on their expression profiles within the first 48 h after inoculation. The expression profiles characteristic of each gene cluster were very similar in both barley-P. t. f. teres and barley-P. t. f. maculata interactions, indicating that resistance to both pathogens could be mediated by induction of the same group of DR genes. Chromosomal map locations for 21 DR genes were identified using four doubled-haploid mapping populations. The mapped DR genes were distributed across all seven barley chromosomes, with at least one gene mapping to within 15 cM of another on chromosomes 1H, 2H, 5H and 7H. Additionally, some DR genes appeared to co-localize with loci harbouring known resistance genes or quantitative trait loci for net blotch resistance on chromosomes 6H and 7H, as well as loci associated with resistance to other barley diseases. The DR genes are discussed with respect to their map locations and potential functional role in contributing to net blotch disease resistance.

Cloning and functional identification of C-4 methyl sterol oxidase genes from the penicillin-producing fungusPenicillium chrysogenum

Two C-4 methyl sterol oxidase genes (Pcerg25A and Pcerg25B) that are involved in ergosterol biosynthesis have been cloned from the penicillin-producing fungus Penicillium chrysogenum. cDNAs of both Pcerg25A and Pcerg25B have an ORF 885 bp in length, encoding a peptide of 295 residues. The deduced amino acid sequences of PcErg25A and PcErg25B show 86% identity, and have high identities to the characterized C-4 methyl sterol oxidases from Candida albicans and Saccharomyces cerevisiae. The function of Pcerg25A and Pcerg25B was identified by complementation of a yeast erg25-deficient strain. Pcerg25A is located in the DNA region containing the penicillin gene cluster, and thus its copy number is dependent on the patterns of the cluster region. Up to eight copies of Pcerg25A were found in the high-productivity strain NCPC 10086. By contrast, Pcerg25B was present in just a single copy in all tested P. chrysogenum genomes. Differences in the transcript level of either Pcerg25A or Pcerg25B were observed in different P. chrysogenum strains by real-time quantitative reverse transcriptase PCR analysis.

Identification of plant defence genes in canola using Arabidopsis cDNA microarrays

We report the identification of novel defence genes in canola by using a cDNA microarray from Arabidopsis. We examined changes that occur in the abundance of transcripts corresponding to 2375 Arabidopsis expressed sequence tags (selected for defence gene identification) following inoculation of canola plants with the fungal necrotrophic leaf pathogen, Alternaria brassicicola. Microarray data obtained from this cross-hybridisation experiment were compared to expression profiles previously obtained from the equivalent Arabidopsis experiment. Homology searches using a canola expressed sequence tag database with approximately 6000 unique clones led to identification of canola defence genes. Pathogen-responsive transcripts included those associated to known defence genes, reactive oxygen species metabolism, disease resistance and regulatory genes, and cell maintenance/metabolism genes. Using specific primers for quantitative real-time reverse transcriptase PCR, gene expression profiles in canola were obtained that demonstrated coordinated defence responses, including systemic responses in distal tissue and salicylic acid- and methyl jasmonate-mediated signalling against A. brassicicola.

Inhibition of Human Metapneumovirus Replication by Small Interfering RNA

Human metapneumovirus (hMPV) is a major respiratory viral pathogen in young children, elderly individuals and immunocompromised patients. Despite its major effects related to bronchiolitis, pneumonia and its potential role in recurrent wheezing episodes, there is still no commercial treatment or vaccine available against this paramyxovirus. We tested a therapeutic strategy for hMPV that was based on RNA interference. An hMPV genome-wide search for small interfering RNAs (siRNAs) by computational analysis revealed 200 potentially effective 21-mer siRNAs. Initial screening with a luciferase assay identified 57 siRNAs of interest. Further evaluation of their inhibitory potential against the four hMPV subgroups by quantitative real-time reverse transcriptase PCR and plaque immunoassay identified two highly potent siRNAs with 50% inhibitory concentration (IC50) values in the subnanomolar range. siRNA45 targets the nucleoprotein messenger RNA (mRNA) and had IC50 values <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values between 0.090-<0.078 nM against the same panel of hMPV strains. Longer25/27-mer siRNAs known as Dicer substrates designed from the top two siRNA candidates were also evaluated and were at least as effective as their corresponding 21-mer siRNAs. Interestingly, the presence of one or two nucleotide mismatches in the target mRNA sequence of some hMPV subgroups did not always affect hMPV inhibition in vitro. We successfully identified two highly efficient siRNAs against hMPV targeting essential components of the hMPV replication complex.

Astragaloside IV improves high glucose-induced podocyte adhesion dysfunction via α 3β 1 integrin upregulation and integrin-linked kinase inhibition

Impaired podocyte adhesion to glomerular basement membrane (GBM) may contribute to podocyte detachment from GBM, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we examined the effects of Astragaloside IV (AS-IV), a saponin purified from Astragalus membranaceus (Fisch) Bge, on high glucose-induced cell adhesion dysfunction in cultured mouse podocytes. Cells were seeded into 96-well plates coated with basement membrane protein complex (BMC). The cells were incubated for 12 h in media containing 30 mM glucose (HG) with 10, 50 and 100 μg/ml of AS-IV. The cells were also exposed to HG media with 100 μg/ml of AS-IV for 3, 6, 12 and 24 h. Cell adhesion assays were performed by fluorescence and centrifugation methods, respectively. Levels of mRNA were determined by quantitative reverse transcriptase real-time PCR and protein expression was analyzed by immunoblotting. HG strongly inhibited adhesion of podocytes to BMC, accompanied by reduction in α 3β 1 integrin mRNA and protein expression, as well as increase in integrin-linked kinase (ILK) activity and expression. When podocytes under HG stimulation were treated with AS-IV, a dose- and time-dependent increase in cell–matrix adhesion was observed, which was significant from 10 μg/ml of AS-IV and from 6 h of incubation of AS-IV with 100 μg/ml. This was accompanied by significant increases in α 3β 1 integrin mRNA and protein expression, as well as inhibition of ILK activation and overexpression. These results suggest that AS-IV improve HG-induced podocyte adhesion dysfunction, which is partly attributed to α 3β 1 integrin upregulation and ILK inhibition.

MicroRNAs Regulate Ocular Neovascularization

In this study, we used ischemia-induced retinal neovascularization (NV) as a model to investigate the possible role of microRNAs in a clinically important disease process. Microarray analysis demonstrated seven microRNAs (miR-106a, -146, -181, -199a, -214, -424, and -451) that were substantially increased and three microRNAs (miR-31, -150, and -184) that were substantially decreased in ischemic retina. Potential targets for the upregulated microRNAs were not identified, but bioinformatic analysis suggested target genes for the downregulated microRNAs, and these were confirmed using a luciferase reporter assay. Real-time reverse transcriptase PCR confirmed that the substantial levels of miR-31, -150, and -184 present in normal retina were significantly reduced in ischemic retina. Interestingly, constitutive levels of miR-31 and -184 are high in the cornea and lens, two avascular tissues. Intraocular injection of pre-miR-31, -150, or -184 significantly reduced ischemia-induced retinal NV, and injection of pre-miR-31 or -150 also significantly reduced choroidal NV. These data suggest that alteration of microRNA levels contributes to two types of ocular NV, and that injection or enhanced expression of microRNAs is a potential therapeutic strategy.

Regulation of Glycan Structures in Animal Tissues: TRANSCRIPT PROFILING OF GLYCAN-RELATED GENES

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.

Human High Molecular Weight–Melanoma-Associated Antigen: Utility for Detection of Metastatic Melanoma in Sentinel Lymph Nodes

Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection. Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection. Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases. HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Edysone receptor isoforms play distinct roles in controlling molting and metamorphosis in the red flour beetle, Tribolium castaneum

Ecdysteroids regulate insect growth and development through a heterodimeric complex of nuclear receptors consisting of ecdysone receptor (EcR) and ultraspiracle (USP). In the red flour beetle, Tribolium castaneum, two isoforms each of EcR and USP have been identified. Quantitative real-time reverse-transcriptase PCR (qRT-PCR) analysis showed isoform-specific developmental expression of both EcR and USP in the epidermis and the midgut dissected from the final instar larvae and pupae. Injection of double-stranded RNA (dsRNA) prepared using the common or isoform-specific regions of EcR or USP as templates caused derailment of development. EcR common region (EcRC) or EcRA dsRNA caused more severe effects, and most of the treated larvae died prior to pupation. EcRB dsRNA caused less severe effects and most of the treated larvae became pupae but showed developmental defects. Only dsRNA prepared against USP common region but not against USPA or USPB isoform-specific region caused developmental defects during larval-pupal metamorphosis. Determination of mRNA levels of EcR isoforms and 20-hydroxyecdysone-response (20E) genes (broad, E75, E74, HR3 and FTZ-F1) by qRT-PCR in the larvae injected with EcRA, EcRB or EcRC dsRNA showed that EcRA initiates ecdysteroid action by regulation the expression of EcRB and 20E-response genes. These data suggest that the EcR but not USP isoforms play distinct roles during the larval-pupal metamorphosis and EcRA plays a dominant role in transduction of ecdysteroid response in T. castaneum.

Localized Gene Expression in Pseudomonas aeruginosa Biofilms

Gene expression in biofilms is dependent on bacterial responses to the local environmental conditions. Most techniques for studying bacterial gene expression in biofilms characterize average values across the entire population. Here, we describe the use of laser capture microdissection microscopy (LCMM) combined with multiplex quantitative real-time reverse transcriptase PCR (qRT-PCR) to isolate and quantify RNA transcripts from small groups of cells at spatially resolved sites within biofilms. The approach was first tested and analytical parameters were determined for Pseudomonas aeruginosa containing an isopropyl-beta-D-thiogalactopyranoside-inducible gene for the green fluorescent protein (gfp). The results show that the amounts of gfp mRNA were greatest in the top zones of the biofilms, and that gfp mRNA levels correlated with the zone of active green fluorescent protein fluorescence. The method then was used to quantify transcripts from wild-type P. aeruginosa biofilms for a housekeeping gene, acpP; the 16S rRNA; and two genes regulated by quorum sensing, phzA1 and aprA. The results demonstrated that the amount of acpP mRNA was greatest in the top 30 microm of the biofilm, with little or no mRNA for this gene at the base of the biofilms. In contrast, 16S rRNA amounts were relatively uniform throughout biofilm strata. Using this strategy, the RNA amounts of individual genes were determined, and therefore the results are dependent on both gene expression and the half-life of the transcripts. Therefore, the uniform amount of rRNA throughout the biofilms likely is due to the stability of the rRNA within ribosomes. The levels of aprA mRNA showed stratification, with the largest amounts in the upper 30-microm zone of these biofilms. The results demonstrate that mRNA levels for individual genes are not uniformly distributed throughout biofilms but may vary by orders of magnitude over small distances. The LCMM/qRT-PCR technique can be used to resolve and quantify this RNA variability at high spatial resolution.

Molecular cloning and mRNA expression of cyclophilin A gene in black tiger shrimp ( Penaeus monodon)

The techniques of homology cloning and anchored PCR were used to clone the cyclophilin A (CypA) gene from black tiger shrimp ( Penaeus monodon). The full-length cDNA of black tiger shrimp CypA (btsCypA) contained a 5′ untranslated region (UTR) of 81 bp, an ORF (open reading frame) of 495 bp encoding a polypeptide of 164 amino acids with an estimated molecular mass of 17.68 kDa and a 3' UTR of 308 bp. The predicted amino acid sequence of btsCypA shared high identity with CypA in other organisms. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of btsCypA in different tissues and the temporal expression of btsCypA in the hepatopancreas challenged by lipopolyssacharide (LPS). Higher-level mRNA expression of btsCypA was detected in the tissues of hepatopancreas and blood. The expression of btsCypA in the hepatopancreas was up regulated after stimulated by LPS. The results indicated that btsCypA was a constitutive and inducible expressed protein and could be induced by LPS.

Collateral Effects of Antibiotics: Carbadox and Metronidazole Induce VSH-1 and Facilitate Gene Transfer among Brachyspira hyodysenteriae Strains

Brachyspira hyodysenteriae is an anaerobic spirochete and the etiologic agent of swine dysentery. The genome of this spirochete contains a mitomycin C-inducible, prophage-like gene transfer agent designated VSH-1. VSH-1 particles package random 7.5-kb fragments of the B. hyodysenteriae genome and transfer genes between B. hyodysenteriae cells. The chemicals and conditions inducing VSH-1 production are largely unknown. Antibiotics used in swine management and stressors inducing traditional prophages might induce VSH-1 and thereby stimulate lateral gene transfer between B. hyodysenteriae cells. In these studies, VSH-1 induction was initially detected by a quantitative real-time reverse transcriptase PCR assay evaluating increased transcription of hvp38 (VSH-1 head protein gene). VSH-1 induction was confirmed by detecting VSH-1-associated 7.5-kb DNA and VSH-1 particles in B. hyodysenteriae cultures. Nine antibiotics (chlortetracycline, lincomycin, tylosin, tiamulin, virginiamycin, ampicillin, ceftriaxone, vancomycin, and florfenicol) at concentrations affecting B. hyodysenteriae growth did not induce VSH-1 production. By contrast, VSH-1 was detected in B. hyodysenteriae cultures treated with mitomycin C (10 microg/ml), carbadox (0.5 microg/ml), metronidazole (0.5 microg/ml), and H(2)O(2) (300 microM). Carbadox- and metronidazole-induced VSH-1 particles transmitted tylosin and chloramphenicol resistance determinants between B. hyodysenteriae strains. The results of these studies suggest that certain antibiotics may induce the production of prophage or prophage-like elements by intestinal bacteria and thereby impact intestinal microbial ecology.

Renin-Angiotensin System Blockade Is Renoprotective in Immune Complex–Mediated Glomerulonephritis

Blockade of the renin-angiotensin system is renoprotective in a variety of chronic nephropathies, but the direct effect of such treatment in active, immune complex-mediated glomerulonephritis is unknown. This study investigated the short- and long-term effects of an angiotensin-converting enzyme inhibitor (enalapril) and an angiotensin II type 1 receptor blocker (losartan) in thymic stromal lymphopoietin transgenic (TSLPtg) mice, which develop mixed cryoglobulinemia and severe cryoglobulinemia-associated membranoproliferative glomerulonephritis. Enalapril and losartan each reduced hypertension, proteinuria, glomerular extracellular matrix deposition, and mesangial cell activation in TSLPtg mice. These renoprotective effects were not observed with hydralazine treatment, despite a similar antihypertensive effect. Treatment with enalapril or losartan also decreased renal plasminogen activator inhibitor-1 in TSLPtg mice, assessed by immunohistochemistry and quantitative real-time reverse transcriptase-PCR. None of the treatments affected immune complex deposition or macrophage infiltration. Overall, enalapril- and losartan-treated TSLPtg mice survived significantly longer than untreated TSLPtg mice. These studies demonstrate that angiotensin blockade may provide renoprotective benefits, independent of its BP-lowering effect, in the treatment of active immune complex-mediated glomerulonephritis.

Tetracycline alters drug susceptibility in Candida albicans and other pathogenic fungi

The tetracycline (TET) promoter has been used in several systems as an inducible regulator of gene expression. In control analyses, the standard Candida albicans laboratory strain SC5314 was found to have altered susceptibility to a variety of antifungal drugs in the presence of relatively high concentrations (50-200 microg ml(-1)) of TET. Altered susceptibility was most notable with exposure to amphotericin B (AMB), with a 32-fold increase in susceptibility, and terbinafine (TRB), with a 32-fold decrease in susceptibility. The TET/AMB synergy was observed in several clinical isolates of C. albicans and in the distantly related species Aspergillus fumigatus and Cryptococcus neoformans. The TET/AMB synergy is not related to efflux pump activity, as determined by FACS analyses and by analysis of a strain containing efflux pump deletions. Gene expression analyses by luciferase and by quantitative real-time reverse transcriptase PCR failed to identify significant alterations in expression of any genes associated with resistance. C. albicans grown with TET for 48 h does show a reduction in total cellular ergosterol. Analysis of growth curves suggests that the TET effect is associated with lack of a diauxic shift, which is related to a loss of mitochondrial function. MitoTracker fluorescent dye was used to demonstrate that TET has a direct effect on mitochondrial function. These results demonstrate the need for careful analysis of TET effects when using a TET-inducible promoter, especially in studies that involve antifungal drugs. This study defines some limits to the use of the TET-inducible promoter, and identifies effects on cells that are the result of TET exposure alone, not the result of expression of a targeted gene.

Development of a real-time quantitative reverse transcriptase PCR assay for detection of the Friend leukemia virus load in murine plasma

Friend leukemia virus (FLV), a murine retrovirus, has been used as a model for elucidation of human immunodeficiency virus (HIV) immunopathogenesis and evaluation of anti-HIV drug effects for several decades. However, no method for direct detection of the plasma viral load has yet been reported. In this study, a TaqMan real-time quantitative reverse transcriptase PCR (qRT-PCR) assay was established for the rapid detection and quantitation of FLV. Measurement of the absolute FLV load was achieved through synthesis of a standard RNA from within the FLV envelope gene for generation of a standard curve. The assay allows quantitation over a range from 20 to 2×108 RNA copies per reaction in a two-step real-time quantitative reverse transcriptase PCR protocol. The relationships between the initially injected FLV dose and the plasma FLV load and spleen index were explored. Following this, the in vivo effects of zidovudine, adefovir dipivoxil, and entecavir on mice infected with FLV were evaluated. The results showed that the plasma FLV load was not proportional to the spleen index over the same FLV injection dosage series, although a trend was observed. When evaluated using plasma viral load, high dose (15mg/(kgd)) adefovir dipivoxil was capable of significant inhibition of FLV replication in mice. The qRT-PCR assay described here allows specific, sensitive and direct detection of FLV and may also provide more precise measurement of FLV load.

Anti-Inflammatory Effects of Thyme Essential Oil in Mice

Plant essential oils are plant secondary metabolites possessing various pharmacological properties, primarily anti-oxidative, antimicrobial or immunomodulatory ones. The aim of this work was to study the effects of thyme essential oil dietary administration in murine DTH/ CHS reaction, carrageenan paw oedema and TNBS colitis. Thyme essential oil was added to the murine diet at three concentrations (5000, 2500 and 1250 ppm) and fed to Balb/c mice. The extent of ear swelling in DTH/CHS reaction and paw oedema induced by carrageenan application was measured using the Mitutoyo thickness gauge. In the model of TNBS colitis we evaluated the changes in body weight, the colon weight : body weight ratio, bacterial translocation to mesenteric lymph nodes, and macroscopical and histological scores. IL-1β and IL-6 messenger RNA expression in colonic samples of one experimental group were assessed using quantitative real-time reverse transcriptase PCR. Dietary supplementation with 5000 ppm of thyme essential oil significantly decreased paw oedema and ear swelling. This thyme essential oil concentration caused a significant inhibition of total mRNA IL-1β expression in the mouse colon, and markedly decreased the macroscopic and microscopic scores of colitis. On the other hand, the 1250 ppm of thyme essential oil in diet increased ear oedema induced by oxazolone application in mice. Our study indicates that thyme essential oil is able to affect murine experimental inflammatory models depending on the concentration used. It is concluded that the anti-inflammatory effects of thyme essential oil should be interpreted with a caution due to its contradictory, dose-related effects.

Dicarbonyl/L-xylulose reductase: a potential biomarker identified by laser-capture microdissection-micro serial analysis of gene expression of human prostate adenocarcinoma.

To identify genes involved in prostate carcinogenesis, we used laser-capture microdissection-micro serial analysis of gene expression to construct libraries of paired cancer and normal cells from human tissue samples. After computational comparison of the two libraries, we identified dicarbonyl/l-xylulose reductase (DCXR), an enzyme that catalyzes alpha-dicarbonyl and l-xylulose, as being significantly up-regulated in prostate cancer cells. The specificity of DCXR up-regulation for prostate cancer tissues was confirmed by quantitative real-time reverse transcriptase-PCR, virtual Northern blot, and Western blot analyses. Furthermore, DCXR expression at the protein level was assessed using fresh-frozen tissues and a tissue microarray consisting of 46 cases of organ-confined early-stage prostate cancer and 29 cases of chemohormonally treated prostate cancer. In most normal prostate epithelial cells, DCXR was expressed at low levels and was localized predominantly in the cytoplasmic membrane. In contrast, in virtually all grades of early-stage prostate cancer and in all chemohormonally treated cases, DCXR was strikingly overexpressed and was localized predominantly in the cytoplasm and nucleus. In all samples, the stromal cells were completely devoid of DCXR expression. Based on these findings, we suggest that DCXR overexpression has the potential to be an additional useful biomarker for prostate cancer.

MicroRNA-Mediated Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B-Cell Lymphomas.

PR (PRDI-BF1-RIZ-homology) domain zinc finger protein 1 (PRDM1) is a master regulator in plasma cell differentiation recently identified as a tumor suppressor target for inactivation in diffuse large B-cell lymphomas (DLBCL) of the activated B-cell (ABC) type, implying interference of B-cell terminal differentiation as a pathogenetic mechanism in DLBCL. Besides deleterious gene mutations, it has been suggested that PRDM1 may also be inactivated in DLBCL by an epigenetic mechanism. In this study, we examined the hypothesis of microRNA (miRNA)-mediated down-regulation of PRDM1 in DLBCL. 10 DLBCL cell lines and 25 clinical DLBCL samples were analyzed for PRDM1α (PRDM1 functional isoform) RNA and protein expression by quantitative real-time reverse transcriptase-PCR, Western blotting and immunohistochemistry. The clinical samples included 5 ABC-DLBCLs with PRDM1 gene deletions and inactivating mutations (Group I), 12 ABC-DLBCLs without PRDM1 mutations (Group II) and 8 germinal center B-cell (GCB) type (Group III). The myeloma cell line U266, which expresses relatively abundant PRDM1α mRNA and protein, was used as a reference standard (arbitrarily set as 1). These expression studies identify desynchrony in PRDM1α mRNA and protein expression. PRDM1α is weakly expressed or undetectable in DLBCL cell lines regardless of levels of PRDM1α transcripts. For the primary DLBCL cases, the mean levels of PRDM1α mRNA in groups I, II and III were: 2.21+0.53 (p<0.05 vs. II & III), 0.84+0.19, and 0.43+0.24, respectively. However, immunohistochemistry demonstrated that in all three DLBCL groups, including Group II which has relatively high PRDM1α mRNA and harbors no PRDM1 mutations, an average of only <5% (range: 0 to 10%) of the neoplastic B cells weakly expressed PRDM1. These results suggest epigenetic down-regulation of PRDM1 protein expression in some DLBCLs. Several lines of evidence support a role for miRNA let-7 in mediating translation repression of PRDM1 in DLBCLs: (1) let-7a levels in DLBCL cell lines and primary cases, as determined by quantitative modified Invader assays, are higher (∼2 to 30 fold) than in U266; (2) The lowest (let-7a)/(PRDM1α mRNA) ratio is found in those ABC-DLBCLs harboring PRDM1 mutations; (3) Enforced expression of let-7a caused binding site-dependent reduction in reporter gene activities of at least 50%. This reduction is due to translation repression; (4) Enforced let-7a expression reduces PRDM1α levels by ∼ 50% in U266 cell lines, suggesting functional in vivo interaction of let-7a with PRDM1 mRNA. In conclusion, PRDM1 protein levels correlate poorly with PRDM1 mRNA expression in DLBCLs. Our studies suggest miRNA-mediated down-regulation as a mechanism of lowering PRDM1 activity in DLBCL, apart from genetic mutations and transcription repression. In ABC-DLBCLs without PRDM1 gene mutations, PRDM1 inactivation is likely mediated at least in part via translation repression of PRDM1 transcripts by high levels of let-7. Those ABC-DLBCLs with PRDM1 gene mutations might have “escaped” let-7-mediated down-regulation, for example, via higher levels of induction of PRDM1 transcripts or some other mechanisms. let-7 may be considered a potential target for therapeutic inhibition to restore terminal differentiation in DLBCL cells.

Comparison between Quantitative Nucleic Acid Sequence-Based Amplification, Real-Time Reverse Transcriptase PCR, and Real-Time PCR for Quantification ofLeishmaniaParasites

DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.