Reverse Transcriptase-polymerase Chain Reaction Procedure Research Articles

Detection of BCR–ABL using one step reverse transcriptase- polymerase chain reaction and microchip electrophoresis

One-step reverse transcriptase polymerase chain reaction (RT-PCR) coupled with microchip electrophoresis (MCE) was established to analyze BCR–ABL fusion gene. The use of one-step RT-PCR could simplify the RT-PCR procedure and thus reduced the risk of contamination and sample consumption. This method also enhanced the sensitivity for amplified target DNA and dramatically shorted the analysis time. Moreover, this assay can simultaneously identify b2a2 and b3a2. Orthogonal array design, which can investigated mutual effects of PCR parameters, was used to optimize the reaction system. This approach was highly effective, reproducible and sensitive, and would be suitable for the determination of BCR–ABL in clinic diagnosis.

Transmissible Viral Proventriculitis Identified in Broiler Breeder and Layer Hens

Transmissible viral proventriculitis (TVP) is a recognized cause of production losses in broiler chickens, but previously it has not been reported in broiler breeder and commercial layer hens. In this study, TVP was identified in broiler breeder and commercial layer hens, 9-20 wk of age, based on histopathologic detection of characteristic microscopic lesions. Microscopic lesions in proventriculi of affected hens consisted of glandular epithelial necrosis, ductal epithelial hyperplasia, replacement of glandular epithelium with ductal epithelium, and diffuse interstitial lymphoid infiltration. Additionally, chicken proventricular necrosis virus (CPNV), a virus previously identified as the etiology of TVP in broiler chickens, was detected in proventriculi of TVP-affected hens using a reverse transcriptase-polymerase chain reaction procedure. The findings identify TVP as a potential cause of production losses in broiler breeder and commercial layer hens and provide additional evidence for etiologic involvement in TVP by CPNV.

The mycotoxin, patulin, increases the expression of PXR and AhR and their target cytochrome P450s in primary cultured human hepatocytes

The mycotoxin, patulin (PAT), which is frequently found in apples, grapes, oranges, pear, peaches, and in apple juices, has previously been shown to be cytotoxic, genotoxic, and mutagenic. In this study, we have investigated the effect of PAT on mRNA level of pregnane X receptor (PXR), constitutive androstane receptor (CAR), aryl hydrocarbon receptor (AhR), and their corresponding target cytochrome P450s. Using primary cultures of adult human hepatocytes, we evaluated PAT cytotoxicity on hepatocytes after 24 hours of treatment. Real time reverse-transcriptase polymerase chain reaction procedure was employed to determine the effect of PAT on receptors (PXR, CAR, and AhR) and cytochrome (CYP3A4, 2B6, 3A5, 2C9, 1A1, and 1A2) genes. Our results showed that PAT reduced hepatocyte viability. At a noncytotoxic range of PAT concentrations, PAT induced an upregulation of the PXR gene in the three treated hepatocytes cultures, whereas CAR was overexpressed in only 1 treated liver. PXR gene induction was accompanied by the enhancement of CYP2B6, 3A5, 2C9, and 3A4 expression. PAT was also found to induce an overexpression of AhR and CYP1A1 and CYP1A2 mRNA expression. These findings suggested that PAT may activate PXR and/or CAR and AhR. However, further investigations are needed to confirm nuclear receptor activation by PAT and to elucidate the molecular mechanism of PAT action.

Detection of Chicken Proventricular Necrosis Virus (R11/3 Virus) in Experimental and Naturally Occurring Cases of Transmissible Viral Proventriculitis with the Use of a Reverse Transcriptase-PCR Procedure

A reverse-transcriptase-polymerase-chain-reaction (RT-PCR) procedure was evaluated for detection of chicken proventricular necrosis virus (CPNV) in transmissible viral proventriculitis (TVP) -affected chickens. The RT-PCR procedure was compared with indirect immunofluorescence (IFA) and virus isolation for detection of CPNV in experimentally infected chickens. Microscopic lesions characteristic of TVP were detected on days 5-35 postexposure (PE) in CPNV-infected chickens; CPNV was detected by RT-PCR on days 3-14 PE in freshly collected proventriculi, and on days 1-14 PE in formalin-fixed paraffin-embedded (FFPE) proventriculi. CPNV was detected in proventriculi of experimentally infected chickens by IFA on days 3-10 PE, and by virus isolation on days 1-14 PE. With IFA used as a reference, sensitivity of the RT-PCR procedure with freshly collected and FFPE proventriculi was 88% and 100%, respectively; specificity was 83% and 86%, respectively. Proventriculi (FFPE) obtained from suspect TVP cases (n=19) were evaluated for presence of CPNV by RT-PCR and microscopic lesions consistentwith TVP. CPNV was detected by RT-PCR in proventriculi from 8/11 TVP (+) cases (24/36 tissue sections). TVP (+) cases were defined by microscopic lesions characteristic of TVP; CPNV was not detected in proventriculi (0/8 cases, 0/32 tissue sections) in the absence of these lesions. The association between presence of TVP-characteristic microscopic lesions and presence of CPNV was highly significant (P = 0.0014). These findings indicate the utility of the RT-PCR procedure for detection of CPNV and provide additional evidence for an etiologic role for this virus in TVP.

Reliability and Reproducibility of a RNA Preamplification Method for Low-density Array Analysis From Formalin-fixed Paraffin-embedded Breast Cancer Samples

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.

Effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium

To evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin receptor of murine endometrium. Experimental study using the RNA extraction to detect tissue prolactin receptor isoforms by reverse-transcriptase polymerase chain reaction (RT-PCR). University-based laboratory. Seventy-two female swiss albino mice (Mus musculus), approximately 100 days old, were divided into six 12-animal groups: (GI) nonoophorectomized mice given vehicle; (GII) nonoophorectomized mice treated with metoclopramide; (GIII) oophorectomized mice treated with metoclopramide; (GIV) oophorectomized mice treated with metoclopramide and 17beta-estradiol; (GV) oophorectomized mice treated with metoclopramide and micronized progesterone; (GVI) oophorectomized mice treated with metoclopramide and a solution of 17beta-estradiol and micronized progesterone. Drugs were administered for 50 days. Following euthanasia, the middle portions of the uterine horns were removed, sectioned, and immediately frozen for RT-PCR procedures. Blood was collected for the dosage of prolactin and serum estrogen and progesterone using radioimmune assay. Identification of uterine prolactin receptor isoforms. The PRL receptor and its isoform L were identified only in GI (control group) and GII (metoclopramide), the two groups with nonoophorectomized animals. The amount of PRL receptor mRNA and that of its isoform L from GII were the largest. No other isoforms of the prolactin receptor were identified in any of the groups. Our results suggest that replacement of estrogen and progestin may not increase the mRNA of endometrial PRL receptor in metoclopromide-induced hyperprolactinemia in rats after castration.

Rearrangement Analysis in Archival Thyroid Tissues: Punching Microdissection and Artificial RET/PTC 1–12 Transcripts

In few papillary thyroid carcinomas (PTC) and oxyphilic thyroid carcinoma, the clinical impact of the 15 known RET hybrid oncogene variants (RET/PTC 1 to 12, 1L, 3r2, 3r3) is subject to controversial discussions. Large patient cohorts and exploitation of pathological thyroid tissue archives are essential to study the prognostic significance of RET/PTC chimeras. Formalin-fixed and paraffin-embedded thyroid neoplasms were subjected to manual punching macrodissection and subsequent extraction of total RNA. Following reverse transcriptase polymerase chain reaction (RT-PCR)-based screening for RET rearrangements, hybrid-specific expression analyses were carried out for samples indicative of chimeric transcripts. Due to lack of tissue specimen harboring the rare RET chimeras, artificially constructed hybrid sequences of all known RET/PTC variants served as PCR controls. Manual punching dissection successfully diminished RET wild-type contamination originating from C-cells dispersed throughout normal thyroid tissues. The average amount of 27.4 mug RNA extracted allowed for repeated molecular analyses (>60 PCRs). Hybrid-specific expression analysis identified 10 of 15 RET rearrangements (8x RET/PTC 1, 2x RET/PTC 3, 5x RET/PTC x) to be found in 54 oxyphilic thyroid tumors examined. Successful amplification of each artificial hybrid sequence ensured the absence of rare chimeric transcripts. Therefore, RET/PTC x represent either common chimeras not amplifiable due to archival RNA degradation or truly novel hybrid oncoproducts. The fast and simple techniques described here were used to examine oxyphilic carcinomas and adenomas. These microdissection and RT-PCR procedures can easily be put into practice in any molecular biology research laboratory to enable screening of large numbers of archival thyroid tumors for known as well as yet unknown RET rearrangements.

Developmental and hormonal regulation of meltrin beta (ADAM19) expression in mouse testes during embryonic and postnatal life

More than half of ADAM (a disintegrin and metalloprotease) family members are expressed in mammalian male reproductive organs such as testis and epididymis. The ADAM19 gene identified in mouse is a member of the ADAM family and is highly enriched in testes of a newborn mouse. The present study was performed to determine its expression pattern in whole mouse testes in vivo as well as its in vitro action and regulation in testis cells from 2-day-old mice. Reverse transcriptase polymerase chain reaction (RT-PCR) detected ADAM19 mRNA from 15.5 days postcoitum (dpc) to 21 days postpartum (dpp), with high expression during the perinatal period. Immunohistochemistry demonstrated ADAM19 protein localization to the seminiferous cords at both embryonic and postnatal ages examined (from 15.5–19.5 dpc to 2 dpp). In particular, we obtained new evidence that a neutralizing antibody to ADAM19 had no influence on the proliferation of 2 dpp testis cells cultured in serum-free medium when compared to controls. Interestingly, it inhibited the 2 dpp testis cell proliferation elicited by stimulation with 10% FCS ( P < 0.01) or FSH ( P < 0.05). Lastly, using a model of 2 dpp testis cell cultures and RT-PCR procedures, we demonstrated that follicle stimulating-hormone (FSH) reduced the levels of ADAM19 mRNA in a time-dependent manner. Taken together, these results indicate that the expression of ADAM19 may be subject to regulation by FSH during mouse testis development. Furthermore, ADAM19 can act to regulate the proliferation of perinatal testis cells in the perinatal period.

Involvement of chemokines and type 1 cytokines in the pathogenesis of hepatitis C virus–associated mixed cryoglobulinemia vasculitis neuropathy

To examine the expression profiles of a large number of genes within typical vasculitic nerve lesions in patients with mixed cryoglobulinemia (MC) vasculitis in order to better characterize the molecules involved in cellular tissue activation and trafficking. The quantitative expression of 19 genes coding for cytokines, chemokines, and their receptors in the nerve lesions of 9 patients with hepatitis C virus (HCV)-associated MC vasculitis, 7 with idiopathic polyarteritis nodosa (PAN) (rheumatic disease controls), and 8 patients with noninflammatory idiopathic neuropathy (noninflammatory neuropathy controls) was assessed using a real-time reverse transcriptase-polymerase chain reaction procedure. Compared with the noninflammatory controls, HCV-MC vasculitis patients had a significantly higher expression of Th1 cytokines in vasculitic nerve lesions (mean +/- SEM fold increase 33.7 +/- 11.6 for interferon-gamma and 7.2 +/- 1.9 for tumor necrosis factor alpha), whereas Th2 cytokines were absent (interleukin-4 [IL-4], IL-5, and IL-13) or were not significantly different (IL-10). Chemokines involved in T cell and monocyte trafficking were also significantly up-regulated in the HCV-MC vasculitis patients (mean +/- SEM fold increase 27.4 +/- 8.3 for macrophage inflammatory protein 1alpha [MIP-1alpha], 19.9 +/- 5.7 for MIP-1beta, and 7.2 +/- 1.5 for CXCR3). Compared with patients with idiopathic PAN, there was a trend toward higher expression of MIP-1alpha and CXCR3 in HCV-MC vasculitis patients (mean +/- SEM fold increase 27.4 +/- 8.3 versus 5.3 +/- 3.4 for MIP-1alpha and 7.2 +/- 1.5 versus 2.5 +/- 0.9 for CXCR3). This study is the first to demonstrate a role of cellular immunity and Th1 lymphocytes in the pathogenesis of HCV-MC vasculitic nerve lesions.

Cloning the prophenoloxidase cDNA and monitoring the expression of proPO mRNA in prawns ( Macrobrachium rosenbergii) stimulated in vivo by CpG oligodeoxynucleotides

The prophenoloxidase (proPO) system forms the basis of the non-specific defence system in crustaceans. The aim of this study was to develop an RT-PCR procedure to determine proPO gene expression. We used several degenerate primers designed from the conserved regions in amino acid sequences of proPOs from other species to clone the possible cDNA(s) from the haemocytes of the prawn Macrobrachium rosenbergii. One DNA fragment, 2016 bp long, was cloned by a combination of reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (3′/5′-RACE). This fragment could encode a putative polypeptide with 671 amino acids. Further analysis showed that this putative polypeptide contained six histidine residues and a thiol ester-like motif (GCGWPRHM) just like the structural features of proPOs from the shrimp Penaeus monodon. A partial fragment of the sequence with 934 bp containing three histidine residues and the thiol ester-like motif was used as a target to monitor the activation of the proPO gene by the semi-quantitative RT-PCR analysis. The result showed that the highest level of proPO mRNA was detected at 1 h after in vivo injection with 5 μg of CpG oligodeoxynucleotide 2006 per prawn; in the same experiment, the highest PO activity was detected at 6 h after injection. In the control, a continuous and slow elevation of PO activity was observed during the experimental period, but such elevation of proPO mRNA was not observed. From our previous study and the time course of gene expression of proPO and enzymatic activity of PO in this study, it can be concluded that the enhancement effect was through its transcriptional level, its translational level and then its post-translational level sequentially. These results suggest that, both for reliability, sensitivity and for the timing of sampling, the change in proPO mRNA is more useful than the PO activity in monitoring the activation of the proPO non-specific defence system of prawns after treatment with stimulants.

Detection of West Nile virus-infected mosquitoes and seropositive juvenile birds in the vicinity of virus-positive dead birds.

Mosquitoes and wild birds were collected from three sites near locations in the New York City metropolitan area where single, West Nile (WN) virus-positive dead birds were found early in the 2000 transmission season. The mosquitoes were tested for the presence of infectious virus with a Vero cell culture assay and for WN viral RNA by using reverse transcriptase-polymerase chain reaction (RT-PCR) protocols. Serum samples from wild birds were tested for the presence of neutralizing antibodies against WN virus. Infectious WN virus and WN viral RNA were found in Culex species adult mosquitoes from each of the three sites, and a seropositive hatch-year house sparrow (Passer domesticus) was found in one of the three sites. Molecular techniques used to identify the species in the positive mosquito pools found that most of the pools contained a combination of Culex pipiens and Cx. restuans. The minimum infection rate in Culex species mosquitoes from the sites ranged from 0.2 to 6.0 per 1,000 specimens tested. The results demonstrated that, at least early in the transmission season, detection of a WN virus-positive dead bird indicates a local WN virus transmission cycle. This information is valuable in focusing subsequent surveillance and vector management programs. In addition, the RT-PCR procedure for detecting WN viral RNA in mosquito pools detected more positive pools than did the Vero cell plaque assay.

Expression of CCR9 β-chemokine receptor is modulated in thymocyte differentiation and is selectively maintained in CD8+ T cells from secondary lymphoid organs

AbstractChemokines appear to have an important role in the seeding of lymphoid progenitors in the thymus, the regulation of the coordinated movements of the maturing T cells within this organ, and the egress of the resulting naive T cells to secondary lymphoid organs. CCR9, the specific receptor for the β-chemokine TECK/CCL25, is selectively expressed in thymus, lymph node, and spleen. Using a specific anti-CCR9 polyclonal antibody, K629, and a semiquantitative reverse transcriptase–polymerase chain reaction procedure, a detailed study of CCR9 expression in the thymus and secondary lymphoid organs was performed. The results show that CD4+CD8+ double-positive thymocytes have the highest CCR9 expression in thymus. Single-positive CD8+ thymocytes continue to express this receptor after abandoning the thymus as mature naive T cells, as suggested by the existence of a CD8+CD69lowCD62LhighCCR9+ cell subset. Consistent with this, CD8+lymphocytes from lymph nodes, spleen, and Peyer patches express a functional CCR9, as its expression correlates with migration in response to CCL25. Conversely, CD4+ thymocytes lose CCR9 before abandoning the thymus, and CD4+ T cells from secondary lymphoid organs also lack CCR9 expression. Analysis of CCR9 expression in thymocytes from mice of different ages showed that CCR9 levels are affected by age, as this receptor is more abundant, and its response to CCL25 is more potent in newborn animals. Collectively, these results suggest that CCR9 has a role in thymocyte development throughout murine life, with clear differences between the CD4+ and CD8+ lineages.

Regulation of TNF-α by 1α,25-dihydroxyvitamin D3 in human macrophages from CAPD patients

We have previously reported that 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] accumulates in the dialysis fluid of uremic patients treated by continuous ambulatory peritoneal dialysis (CAPD). It has been reported that this metabolite regulates the production of cytokines by monocytes/macrophages. Since tumor necrosis factor-alpha (TNF-alpha) initiates an inflammatory cascade during peritonitis, the aim of the present study was to investigate the effect of 1alpha, 25(OH)2D3 on the production of TNF-alpha by human peritoneal macrophages (HPMs). HPMs were obtained from patients on CAPD. Cells were incubated with various concentrations of 1alpha, 25(OH)2D3, 1alpha,24(S) dihydroxyvitamin D2 [1alpha,24(S)(OH)2D2] or 25-hydroxyvitamin D3 (25-OH-D3) for 16 hours. This was followed by lipopolysaccharide (LPS; 1 microg/mL) incubation for 2.5 to 6 hours. TNF-alpha protein production was determined by enzyme-linked immunosorbent assay. TNF-alpha mRNA was assayed by the reverse transcriptase-polymerase chain reaction procedure, using internal synthetic mRNA standards for quantitative results. Incubation of HPMs with 1alpha,25(OH)2D3 prior to stimulation with LPS dose dependently inhibited the expression of TNF-alpha on both mRNA and protein levels. Similar results were obtained with the less calcemic vitamin D2 analogue 1alpha,24(S)(OH)2D2. Incubation of HPMs with 25-OH-D3 also revealed a down-regulation of TNF-alpha expression. Since this down-regulatory effect was blocked by ketoconazole, it is likely that this effect was caused by the conversion of 25-OH-D3 into 1alpha,25(OH)2D3 by HPMs. 1alpha,25(OH)2D3 has a potent inhibitory effect on the production of TNF-alpha by LPS-activated HPMs. We hypothesize that 1alpha, 25(OH)2D3 may constitute a regulatory mechanism that, by controlling the intensity of the inflammatory response of the peritoneum, will moderate tissue damage during peritonitis.

Morphine Induces Gene Expression of CCR5 in Human CEM x174 Lymphocytes

All HIV-1 strains studied to date use CCR5, CXCR4, or both receptors to enter cells. Simian immunodeficiency virus (SIV) infection of non-human primates has served as a useful model for understanding AIDS pathogenesis in humans. Research on several genetically divergent SIV isolates has revealed that SIV uses CCR5, and not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been routinely used to cultivate and maintain various SIV strains. However, questions have arisen about how CEM x174, which reportedly was unable to express detectable amounts of CCR5 transcripts, efficiently supports the growth of SIV. In searching for an answer, we resorted to a sensitive competitive reverse transcriptase-polymerase chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of CCR5 is increased in cells pretreated with morphine. These results correlate well with our previous observations that morphine treatment causes CEM x174 cells to be more susceptible to SIV infection. Similar morphine effect was not observed on CEM x174 cells infected with simian retroviruses, which do not depend on CCR5 for entry. These findings suggest a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations.

Comparison of Virus Isolation, Immunohistochemistry, and Reverse Transcriptase-Polymerase Chain Reaction Procedures for Detection of Turkey Coronavirus

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure and two monoclonal antibody (MAb)-based immunohistochemical procedures were developed for detection of turkey coronavirus (TCV) in tissues and intestinal contents/dropping samples. The RT-PCR, MAb-based fluorescent antibody (FA), and MAb-based immunoperoxidase (IP) procedures were compared with virus isolation (VI) for detection of TCV in experimentally infected turkeys. TCV was detected in experimentally infected turkeys as early as day 1 postexposure (PE) by each of the four detection procedures. TCV was detected as late as day 35 PE by FA or IP and days 42 and 49 PE by VI and RT-PCR, respectively. With VI as a reference, sensitivity and specificity of RT-PCR were 93% and 92%, respectively; specificity of both FA and IP was 96%, and sensitivities were 69% and 61%, respectively. Each of the examined procedures was highly specific, but the RT-PCR procedure was also highly sensitive. These findings demonstrate the utility of both immunohistochemistry and RT-PCR for detection of TCV. In addition, the findings indicate that RT-PCR is a highly sensitive and specific alternative to conventional diagnostic procedures.

DETECTION OF CIRCULATING PROSTATE DERIVED CELLS IN PATIENTS WITH PROSTATE ADENOCARCINOMA IS AN INDEPENDENT RISK FACTOR FOR TUMOR RECURRENCE

To determine whether the presence of prostate-derived cells in the peripheral blood circulation is a marker of prostate cancer and to define the clinical impact of the test. We tested the peripheral blood of 99 patients with prostate adenocarcinoma (PAC), 79 of them undergoing radical prostatectomy, and 92 controls (31 healthy volunteers, 50 patients with adenoma and 11 with prostatitis) using a highly controlled procedure including reverse-transcriptase polymerase chain reaction (RT-PCR) targeted to prostate-specific antigen (PSA) mRNA. Patients were followed for 26 +/- 12 (range: 4 to 49) months. Forty tumor tissues were analyzed by immunohistochemistry for expression of p53 and E-cadherin antigens. Thirty three (33%) patients with PAC and 2 (2%) controls scored positive (p <0.0001) for the test. Detection of circulating prostatic cells was associated with development of metastases (p <0. 001), with relapse (p <0.001) and with a serum PSA level at diagnosis higher than 15 ng./ml. (p = 0.009). The rate of development of metastases according to time was significantly higher in patients who scored positive for the test (p <0.04). In a multivariate analysis, only the RT-PCR test was an independent risk factor associated with relapse (RR: 6.7). Finally, E-cadherin expression was significantly lower in the tumor tissues of positive patients as compared with those who scored negative for the test (p <0.01). This RT-PCR procedure, performed at diagnosis and with appropriate controls, is a clinically useful assay in evaluating the risk of tumor recurrence after radical prostatectomy in patients with PAC.

Evidence for expression of EcR and USP components of the 20-hydroxyecdysone receptor by a mosquito cell line.

The reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine whether the C7-10 cell line from the mosquito, Aedes albopictus, expresses transcripts encoding 20-hydroxyecdysone receptor (EcR) and ultraspiracle (USP) isoforms known to constitute a functional 20-hydroxyecdysone receptor. Here we describe recovery and analysis of products with high similarity to the EcR and to the USP isoform "a" that have been reported from the related mosquito, Aedes aegypti. The C7-10 EcR was 97% identical to Aedes aegypti EcR in amino acid sequence. Key features of the nuclear/steroid hormone receptor superfamily, including the zinc fingers, proximal (P)-box, and distal (D)-box were well conserved. However, the C7-10 EcR contained 5 additional amino acids in the C-terminal domain F, which required introduction of gaps to maximize alignment. The 5'-untranslated regions of the two mosquito EcRs were 98% identical, but the function of this region remains unknown. The C7-10 USP was 95% identical in amino acid sequence to the longer Aedes aegypti isoform "a." Although only the C7-10 EcR was detected on Northern blots using total RNA from the cell line, transcripts for both EcR and USP were detected using the RT-PCR procedure. These transcripts appeared to be expressed constitutively and expression levels were not affected by treatment of cells with 20-hydroxyecdysone. Arch.

Failure to detect the extracellular calcium-sensing receptor (CasR) in human osteoblast cell lines.

Whether the known calcium-sensing receptor (CasR) is present in osteoblasts is a source of considerable controversy. Prior studies failed to detect CasR in osteoblasts, but more recent investigations purport the detection of CasR in several osteoblast cell lines by immunoblot analysis with polyclonal anti-CasR antisera (4637) and low stringency reverse transcriptase-polymerase chain reaction (RT-PCR). To explain these disparate findings, we performed immunoblot analysis with the 4637 anti-CasR antisera and a highly specific monoclonal antibody to CasR (ADD), and we compared the ability of low and high stringency RT-PCR to amplify CasR transcripts. We found that the ADD antibody detected the anticipated CasR immunoreactive bands, including a approximately 165 kDa and approximately 140 kDa glycosylated doublet and a >250 kDa dimerized receptor, in positive control mouse kidney, human parathyroid, and human embryonic kidney (HEK) 293 cells transfected with rat CasR, but we did not detect these bands in either wild-type HEK 293 cells or Saos2, MG-63, or U-2 OS osteoblast-like cell lines. Standard two-step RT-PCR using CasR-specific primers confirmed these results by detecting CasR transcripts in positive controls but not in negative control HEK 293 cells or osteoblast cell lines. In contrast, the 4637 antisera did not recognize CasR by immunoblot analysis under the conditions studied and our low stringency RT-PCR procedure amplified nonspecific products in wild-type HEK 293 cells and osteoblasts. Since we failed to detect CasR in human osteoblast cell lines using either the highly specific ADD antibody or RT-PCR under standard conditions, it is possible that the cation response in osteoblasts is mediated by a functionally similar but molecularly distinct calcium sensing receptor.

The detection of bovine viral diarrhoea virus in bulk milk samples by the use of a single-tube RT-PCR

A single step, single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) test was developed to detect the presence of bovine viral diarrhoea virus (BVDV) in somatic cells from bulk milk samples. The test was configured using commercial kit-form RNA extraction and RT-PCR procedures. The test was validated by examining bulk milk samples from ∼80 herds with a history of BVDV and comparing results with those obtained from samples from a similar-sized control group. The test proved highly specific, giving a positive result in 20.5% of herds with a history of BVDV, with no control herds positive. Its sensitivity was likewise high, detecting, at its maximum, one persistently infected (PI) cow in a herd of 162 lactating animals. In 19 herds where follow-up blood tests were performed, the RT-PCR gave a positive result in all ten herds where at least one lactating PI animal was present. In control schemes involving the detection of PI cattle, the test provides a rapid and inexpensive alternative to individual animal testing for those cows in milk at the time of sampling.

Neuroendocrine differentiation in Ewing's sarcomas and primitive neuroectodermal tumors revealed by reverse transcriptase-polymerase chain reaction of chromogranin mRNA.

Ewing's sarcomas (ESs), primitive neuroectodermal tumors (PNETs), and neuroblastomas (NBs) are closely related neoplasms supposedly derived from the neural crest and belonging to the family of the small blue round cell tumors of infancy and childhood. We investigated the expression of the neuroendocrine and neuroectodermal markers chromogranin A (CgA) and secretogranin II (SgII) in ESs, PNETs, and NBs, both in primitive tumors (five, nine, and four cases, respectively) and in established cell lines (three ES and two PNET cell lines). Different technical approaches, namely immunohistochemistry, Northern blot analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR) were used in parallel. Chromogranin A and secretogranin II production was constantly detectable in NBs by all procedures. CgA mRNA was detectable in most ESs and PNETs only by RT-PCR, whereas SgII mRNA was detectable in some ESs and PNETs by Northern blot analysis and in all tumors by RT-PCR. CgA and SgII proteins were never detectable by immunohistochemistry in ESs and PNETs. We conclude that neuroendocrine differentiation is shared by all three tumor entities, being more overt in NBs and rudimentary in ESs and PNETs; traces of chromogranin mRNA are detectable only by a highly sensitive RT-PCR procedure.