Gene-specific Reverse Transcription-polymerase Chain Reaction Research Articles

Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India

In late 2009/early 2010, approximately 2000 people were affected by a mysterious viral outbreak in a southern district of Tamil Nadu; this particularly affected those living in coastal areas. Blood samples from affected patients were sent for clinical analysis to determine the actual cause of the illness, but reports were inconclusive. The present study describes the clinical observations and laboratory investigations involving molecular methods performed on 170 of the 2000 clinically suspected cases. These were patients who were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assays were used to detect West Nile virus (WNV) infection. Further investigation of the genetic diversity of the WNV implicated in ocular complications was undertaken by sequence phylogeny. Out of 170 samples, 25 (15%) were positive for chikungunya IgM antibody, 10 (6%) for chikungunya antigen, and 30 (18%) were positive for dengue IgM antibody. The remaining 105 seronegative samples were further processed for WNV detection by IgM capture ELISA and molecular methods. Out of the 105 samples, 35 (33%) were positive for WNV IgM antibody, 15 (14%) were positive for WNV by RT-PCR, and 27 (26%) were found to be positive for WNV by both real-time RT-PCR and RT-LAMP assays. Comparative evaluation with acute-phase patient serum samples revealed 100% concordance between the real-time RT-PCR and RT-LAMP assays. These assays had an overall higher sensitivity than the conventional RT-PCR as they picked up 12 additional samples with a low copy number of template. Further genotyping through sequence phylogeny revealed that all the WNV isolates were grouped in lineage I. The association of West Nile virus with ocular infection in South India during an epidemic of mysterious fever in the first half of 2010 was clearly established through molecular approaches employing envelope gene-specific real-time RT-PCR and RT-LAMP assays followed by nucleotide sequencing.

Expression Analysis of the LFY and TFL1 Homologs in Floral Buds of Japanese Pear (Pyrus pyrifolia Nakai) and Quince (Cydonia oblonga Mill.)

We investigated the expression of Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga) homologs of Arabidopsis thaliana flowering-related genes LEAFY (LFY) and TERMINAL FLOWER 1 (TFL1) in dissected floral tissues. Japanese pear and quince belong to the subfamily Maloideae (Rosaceae), which is an ancient polyploid; thus, the genome of each plant contains at least two different homologs of LFY and TFL1. We examined the expression of each homolog separately using gene-specific reverse transcription-polymerase chain reaction (RT-PCR). Under the experimental conditions used in this study, there was no difference in the expression pattern of LFY homologs, regardless of the species and homolog type. In contrast, TFL1 homologs were differentially expressed in Japanese pear and quince. In Japanese pear, PpTFL1-1 and PpTFL1-2 were expressed in the apical meristem prior to floral differentiation; however, during floral differentiation, PpTFL1-2 expression disappeared and PpTFL1-1 alone continued to be expressed in the developing inflorescence. In quince, CoTFL1-1 was the only TFL1 homolog detected prior to floral differentiation. Upon floral differentiation, both CoTFL1-1 and CoTFL1-2 were expressed mainly in the domed apical meristem. These expressions decreased to an undetectable level once the apical meristem had transformed into a single flower meristem. Thus, Japanese pear and quince TFL1 homologs are supposed to be involved in the formation of domed apical meristems during early reproductive development. Our results imply the importance of PpTFL1-1 in morphological development of Japanese pear inflorescence.

Antioxidant and Antibacterial Genes Are Upregulated in Early Involution of the Mouse Mammary Gland: Sharp Increase of Ceruloplasmin and Lactoferrin in Accumulating Breast Milk

The mammary gland develops mainly after birth, and shows a repeated cycle of pregnancy-triggered proliferation, differentiation for lactation, and a regressive phase after weaning known as involution. Compared to the proliferation and differentiation phases, the molecular mechanisms of involution are largely unknown. In the present study we screened genes that could play a potential role in early involution of the mouse mammary gland using fluorescent differential display followed by gene-specific reverse transcription-polymerase chain reaction. We found that five genes were upregulated more than twofold 48 h after weaning: ceruloplasmin, chemokine (CXC motif) ligand 4, epoxide hydrolase 1, lactoferrin, and properdin P factor. The products of these genes can be linked to defense against oxidative stress and/or infectious bacteria. Electrophoretic analysis and mass spectrometry of milk proteins showed that the concentrations of ceruloplasmin and lactoferrin in milk were increased fivefold and more than 38-fold, respectively, within 48 h after weaning. These increases were in contrast to the constant presence of other major proteins including albumin, caseins, transferrin, and whey acidic protein. Ceruloplasmin and lactoferrin may cooperate in the defense of the mammary gland in the postlactation period.

Up‐regulated expression of the α7 nicotinic acetylcholine receptor subunit on inflammatory infiltrates during Dictyocaulus viviparus infection

Cholinergic signalling is known to affect immune cell function, but few studies have addressed its relevance during nematode infection. We therefore analysed the anatomical distribution and expression pattern of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in lungs obtained from Dictyocaulus viviparus-infected and uninfected control cattle. The analysis was performed on trachea and lung parenchyma from uninfected animals and animals necropsied at 15, 22 and 43 days post-infection (DPI). Localization of the alpha7 nAChR was evaluated by immunohistology and mRNA expression analysed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR). In uninfected animals, tracheal, bronchial and bronchiolar epithelium and smooth muscle cells constitutively expressed the alpha7 nAChR, as did type I and II alveolar epithelial cells and alveolar macrophages and a few infiltrating leucocytes. By 15 DPI, immunohistology revealed a massive influx of alpha7 nAChR+ inflammatory cells into the lung parenchyma and tracheal wall. This was reflected in the RT-PCR results. At later time points, both parenchyma and tracheal wall contained large numbers of alpha7 nAChR+ leucocytes, but detection of transcript was restricted to the trachea. Recruitment of nAChR-containing leucocytes to the lungs of D. viviparus-infected cattle suggests that these cells may represent possible downstream targets for parasite-secreted acetylcholinesterases.

INCREASING EXPRESSION OF GST-pi MIF, AND ID1 GENES IN CHEMORESISTANT PROSTATE CANCER CELLS

The differential expression of genes and related proteins of multidrug resistance in chemoresistant prostate cancer cell lines were elucidated in this study. RNA extracted from doxorubicin-resistant rat prostate cancer (PCa) cells (AT3/ADR1000) and native PCa cells was hybridized to expression arrays containing cDNAs from 588 known genes. Differential expression of selected genes was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Protein contents were measured by fluorescent flow cytometry and immunoblotting. Localization of selected proteins in cells was observed by immunocytochemical staining. Up-regulation of eleven genes and down-regulation of one single gene were displayed in the chemoresistant prostate cancer cells. Overexpression of mRNAs in macrophage migration inhibitory factor (MIF), DNA binding protein inhibitor 1 (ID1), and glutathione S-transferase-pi (GST-pi) were confirmed by gene-specific RT-PCR. Protein over-expression of GST-pi, MIF, and ID1 in resistant cells were 3.3-, 1.5-, and 1.5-fold to native cells, respectively. Immunocytochemistry revealed that GST-pi, MIF, and ID1 were present primarily in the cytoplasm of tumor cells, but ID1 also could be found in the nucleus. AT3/ADR1000 drug-resistant PCa cells displayed significantly increased expression of GST-pi, MIF, and ID1 proteins when compared with native PCa cells. It indicates these genes may play a role in drug resistance of prostate cancer.

The differentiation of sepal and petal morphologies in Commelinaceae

The morphological transition of the first whorl of tepals into sepals occurs frequently during the diversification of angiosperms. Such transitions may play important roles in pollination modes. The B class genes, APETALA3 ( AP3) and PISTILLATA ( PI) in Arabidopsis thaliana and GLOBOSA ( GLO) and DEFICIENS ( DEF) in Antirrhinum majus, are required for the development of petals in the second whorl, and its homologs have been isolated and characterized from various plants. A recent study on tulip, a monocotyledonous plant, indicates that the morphology of petaloid tepals in the first and second whorls is consistent with the expansion of B class gene expression. Here, we report five B class genes, TRGLOA, TRGLOB, CCGLO, TRDEF and CCDEF, isolated and characterized from two commelinaceous plants, Tradescantia reflexa and Commelina communis, with distinct sepal and petal morphologies in monocots. Northern blot analysis and gene-specific reverse transcription–polymerase chain reaction (RT–PCR) studies using dissected floral organs reveal a lack or low level of DEF-like gene expression in these commelinaceous species in the first whorl, in contrast to previous results. The expression data suggest that DEF-like gene expression in Commelinaceae correlates with the production of petaloid organs in the first whorl.

Can the reverse transcriptase-polymerase chain reaction for prostate specific antigen and prostate specific membrane antigen improve staging and predict biochemical recurrence?

To evaluate the perioperative gene-specific primed nested reverse transcription-polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) for staging patients undergoing radical prostatectomy and predicting biochemical recurrence. In 80 consecutive patients undergoing radical prostatectomy for prostate cancer, blood samples were drawn before, during and 1 and 7 days after removing the prostate. After buffy coat and mRNA extraction, gene-specific primed nested RT-PCR was performed for PSA, PSMA and glyceraldehyde-3-phosphate dehydrogenase mRNA, and Southern blot analysis of the PCR reaction. The sensitivity of gene-specific RT-PCR to detect tumour cells was comparable with random primed RT-PCR. In the 80 patients the stage distribution was pT1 in two (2.5%), pT2 in 30 (37.5%) and > or = pT3 in 48 (60%); the nodal status was pN0 in 57 (71%), pN1 in 11 (14%) and pN2 in 12 (15%). The gene-specific RT-PCR reaction for PSA and PSMA was positive in no patients with pT1, 11 (37%) with pT2 and 23 (48%) with stage > or = pT3 disease. The result for PSA was positive in 12 (52%) and for PSMA in 11 (48%) of those with positive nodal status. Neither gene-specific RT-PCR for PSA or PSMA was able to predict organ-confined disease (P > 0.5). After a median (range) follow-up of 37 (11-67) months a biochemical recurrence was predicted in 65% of patients by preoperative RT-PCR for both PSA and PSMA, with a sensitivity, specificity, positive and negative predictive value of 58%, 80%, 87% and 47%, respectively; the assay after surgery predicted a recurrence in 73%, with respective values of 68%, 84%, 84% and 57%. Gene-specific primed nested RT-PCR for PSA and PSMA is a sensitive and simple assay; it might add substantial information for tumour staging in individual patients. RT-PCR before surgery allows the prediction of recurrence in 65% of cases and after surgery in 73%.

Platelet Ca(2+)ATPases : a plural, species-specific, and multiple hypertension-regulated expression system.

Gaining insight into nonmuscle Ca(2+) signaling requires basic knowledge of the major structures involved. We investigated the expression of platelet Ca(2+)ATPases in normal and hypertension-associated abnormal Ca(2+) signaling. First, overall identification of normotensive Wistar-Kyoto rat Ca(2+)ATPases was attempted by looking for newly described human platelet 3'-end alternatively spliced sarco/endoplasmic reticulum Ca(2+)ATPases (SERCA) 3b mRNA and plasma membrane Ca(2+)ATPase (PMCA) 1b and 4b proteins, in addition to SERCA2b and SERCA3a isoforms. For SERCAs, comparative analyses of human and Wistar-Kyoto rat SERCA3 platelet mRNA by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing established that human platelets coexpressed SERCA3b and a third SERCA3c, while rat cells were devoid of them but expressed a still unknown splice variant that we termed rSERCA3b/3c. Its identification using 3'-end SERCA3 gene and rapid amplification of cDNA ends (RACE)-PCR studies showed that it results from an additional SERCA3 alternative splicing process, which uses a second alternative polyadenylation site located in the last intron. For PMCAs, with the use of gene-specific RT-PCR followed by sequencing and Western blotting using 5F10 monoclonal antibody, expression of human and rat platelet PMCA1b and PMCA4b was similar. Second, comparative analysis of these newly identified Ca(2+)ATPases and SERCA3a in age-matched spontaneously hypertensive rat platelets demonstrated (1) a marked downregulation of rSERCA3b/3c, which became null, and a 1.71-fold increase in SERCA3a and (2) an opposite regulation of the 2 PMCAs, namely, a 3.3-fold decrease in PMCA1b mRNA and a 3.7-fold increase in PMCA4b mRNA. Hence, platelets coexpress multiple, diverse, and species-specific Ca(2+)ATPases, including a novel fourth SERCA3. Moreover, expression of PMCA (1b and 4b), SERCA3a, and rSERCA3b/3c was modulated in rat hypertension. Hence, Ca(2+)ATPases should be regarded as constituting a new rational basis for the understanding of nonmuscle cell Ca(2+) signaling.

Upregulation of the RAS‐GTPase activating protein (GAP)‐binding protein (G3BP) in proliferating RPE cells

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)-based differential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3′-end of the coding sequence of the human RAS-GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA-PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation- and proliferation-dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy. J. Cell. Biochem. 74:194–201, 1999. © 1999 Wiley-Liss, Inc.

Upregulation of the RAS-GTPase activating protein (GAP)-binding protein (G3BP) in proliferating RPE cells

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)-based differential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3'-end of the coding sequence of the human RAS-GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA-PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation- and proliferation-dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy.