Reverse Transcription-polymerase Chain Reaction Results Research Articles

Microarray analysis reveals differential gene expression in hybrid sunflower species

This paper describes the creation of a cDNA microarray for annual sunflowers and its use to elucidate patterns of gene expression in Helianthus annuus, Helianthus petiolaris, and the homoploid hybrid species Helianthus deserticola. The array comprises 3743 ESTs (expressed sequence tags) representing approximately 2897 unique genes. It has an average clone/EST identity rate of 91%, is applicable across species boundaries within the annual sunflowers, and shows patterns of gene expression that are highly reproducible according to real-time RT-PCR (reverse transcription-polymerase chain reaction) results. Overall, 12.8% of genes on the array showed statistically significant differential expression across the three species. Helianthus deserticola displayed transgressive, or extreme, expression for 58 genes, with roughly equal numbers exhibiting up- or down-regulation relative to both parental species. Transport-related proteins were strongly over-represented among the transgressively expressed genes, which makes functional sense given the extreme desert floor habitat of H. deserticola. The potential adaptive value of differential gene expression was evaluated for five genes in two populations of early generation (BC2) hybrids between the parental species grown in the H. deserticola habitat. One gene (a G protein-coupled receptor) had a significant association with fitness and maps close to a QTL controlling traits that may be adaptive in the desert habitat.

Significance of Fas expression alteration during tumor progression of renal cell carcinoma

In order to characterize the alteration of apoptotic regulatory molecule expression during tumor progression of renal cell carcinoma (RCC), we compared the expression between tumor and normal tissues, and evaluated the relationship of the expression in tumors with pathological and clinical characteristics. Competitive reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) allowed the determination of Fas and bcl-2 mRNA and protein expression in surgically resected tumor and normal tissue of 50 RCC. The mRNA expression of Fas and bcl-2 in RCC was significantly reduced compared to that in normal tissues. An IHC analysis was supportive of the RT-PCR results. In terms of relationships with pathological and clinical characteristics, the mRNA and protein expression of Fas in high-stage or high-grade tumors was significantly higher than that in low-stage or low-grade tumors. Moreover, a statistically poor prognosis was observed in tumor cases expressing a high amount of mRNA. In bcl-2 analysis, the mRNA and protein expression was significantly reduced in clear cell tumors compared to chromophobe cell tumors. It is suggested that the reduced expression of Fas and bcl-2 in RCC compared with the expression in normal kidney is a prominent alteration of apoptotic regulatory molecules. The alteration of the up-regulated Fas expression might be characterized during the tumor progression stage. It is also suggested that the effect of alteration of bcl-2 expression might be minimal during the tumor progression stage because of the reduced expression in tumors of the clear cell type, which is the most dominant cell type in RCC.

Regulated Production of Mature Insulin in Rat Hepatoma Cells: Insulin Production is Up-regulated by Dexamethasone and Down-regulated by Insulin

We engineered an artificial beta cell line that produces an up-regulation of insulin in response to dexamethasone, and a down-regulation in response to insulin. A regulatory secretion system was devised by placing proinsulin cDNA containing genetically engineered furin endoprotease cleavage sites and a regulatory promoter for phosphoenolpyruvate carboxykinase (PEPCK), and an insulin expressing retrovirus vector (pN-PEPCK-mINS) was constructed and transfected into Hepa1-6 cells. The levels of insulin in culture medium and expression of insulin gene was estimated by radioimmunoassay and reverse transcription-polymerase chain reaction (RT-PCR), respectively. The clone (Hepa1-6/INS21), which secreted the highest level of insulin (10.79 microIU/106 cells per day), was selected for the regulation experiment. Compared with the non-treated Hepa1-6/INS21 cells, insulin production was augmented 3.6-fold by the addition of 10-7 M of dexamethasone. Addition of exogenous insulin to the culture medium decreased insulin mRNA expression remarkably on RT-PCR results, while dexamethasone increased insulin gene expression at the transcriptional level. The data indicated that genetically engineered Hepa1-6 cells could synthesize process and secrete insulin in a physiological manner.

Dysregulation of TAp63 mRNA and Protein Levels in Psoriasis

Psoriasis is a chronic and excessive inflammation of the skin and is currently incurable. The cause of psoriasis remains poorly understood and a central and cooperative role for keratinocytes and T-cells in triggering the disease is highlighted. The p63 gene encodes six different proteins with homology to the tumor suppressor protein p53 that are crucial for normal development of ectodermally derived structures such as skin and oral mucosa. In this study, we have analyzed levels of the different p63 isoforms using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry in 15 patients diagnosed with psoriasis. Quantitative RT-PCR results showed downregulation of the full-length TAp63 in psoriatic lesions compared to both clinically normal skin from patients (P<0.001) and matched healthy controls (P<0.001); however, p63 protein levels detected by immunohistochemistry were similar. All psoriasis lesions also had detectable levels of activated Stat3, a protein indicated in development of the disease, whereas control tissue lacked this protein. The present data show a different regulation of TAp63 in psoriasis, where the discrepancy between mRNA levels and protein expression indicates a post-transcriptional regulation analogous to that seen in p53.

Association Between Recurrence of Hepatocellular Carcinoma and α-Fetoprotein Messenger RNA Levels in Peripheral Blood

Intra- and extrahepatic recurrence is common, even after curative resection for hepatocellular carcinoma (HCC), suggesting preoperative or intraoperative tumor cell dissemination. Reverse transcription - polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP) is used to detect circulating liver cancer cells. We previously developed a quantitative method that allows estimation of the AFP mRNA level by real-time PCR. In the present study, we used this method to measure the AFP mRNA level before and after resection of HCC, then correlated the findings with various clinicopathological characteristics and prognosis. We prospectively examined peripheral blood samples from 38 patients with HCC, and bone marrow aspirate from 25 of these patients. As a control, we examined bone marrow from 20 patients with benign diseases. The follow-up period ranged from 32 to 66 months. Real-time RT-PCR was used to detect AFP mRNA levels in the samples. AFP was expressed in 9 (23.7%) of the 38 peripheral blood samples. The detection of AFP mRNA was significantly correlated with extrahepatic metastasis after primary surgery, and a shorter disease-free survival time (P = 0.0245 each). Bone marrow samples were defined as positive if they expressed AFP mRNA at levels higher than the maximum expressed level in the controls, because only 1 (5%) of the 20 control bone marrow samples had low AFP mRNA expression. Using this cutoff level, 12 (48%) of the 25 patients with HCC had positivity for AFP mRNA. The results of bone marrow RT-PCR did not correlate with the clinocopathological characteristics of prognosis. Using real-time PCR to measure the AFP mRNA level in blood, but not bone marrow, could be useful for predicting postoperative tumor recurrence.

The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces

When fungi grow on plant or insect surfaces coated with wax polyesters that protect against pathogens, the fungi generally form aerial hyphae to contact the surfaces. Aerial structures such as hyphae and conidiophores are coated with hydrophobins, which are surface-active proteins involved in adhesion to hydrophobic surfaces. When the industrial fungus Aspergillus oryzae was cultivated in a liquid medium containing the biodegradable polyester polybutylene succinate-coadipate (PBSA), the rolA gene encoding hydrophobin RolA was highly transcribed. High levels of RolA and its localization on the cell surface in the presence of PBSA were confirmed by immunostaining. Under these conditions, A. oryzae simultaneously produced the cutinase CutL1, which hydrolyses PBSA. Pre-incubation of PBSA with RolA stimulated PBSA degradation by CutL1, suggesting that RolA bound to the PBSA surface was required for the stimulation. Immunostaining revealed that PBSA films coated with RolA specifically adsorbed CutL1. Quartz crystal microbalance analyses further demonstrated that RolA attached to a hydrophobic sensor chip specifically adsorbed CutL1. Circular dichroism spectra of soluble-state RolA and bound RolA suggested that RolA underwent a conformational change after its adsorption to hydrophobic surfaces. These results suggest that RolA adsorbed to the hydrophobic surface of PBSA recruits CutL1, resulting in condensation of CutL1 on the PBSA surface and consequent stimulation of PBSA hydrolysis. A fluorescence recovery after photobleaching experiment on PBSA films coated with FITC-labelled RolA suggested that RolA moves laterally on the film. We discuss the novel molecular functions of RolA with regard to plastic degradation.

Induction of vitellogenin mRNA in juvenile chinese sturgeon (acipenser sinensis gray) treated with 17β‐estradiol and 4‐nonylphenol

It has been demonstrated that 4-nonylphenol (4-NP) exerts estrogenic effects in diverse fishes. The present study investigated the effects of 4-NP on Chinese sturgeon (Acipenser sinensis Gray) vitellogenin (VTG) gene expression. By reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers, a 462-base pair fragment of Chinese sturgeon VTG, corresponding to a 154-amino acid sequence, was amplified and sequenced. This sequence exhibited 152/154 identity to the amino acid sequence of white sturgeon (A. transmontano) VTG. Conventional RT-PCR and quantitative real-time RT-PCR were established and used to study the VTG and beta-actin gene expression in the liver of juvenile Chinese sturgeon injected three times with 17beta-estradiol (5 mg/kg body wt/week) or 4-NP (10 or 100 mg/kg/week) in the course of three weeks. Significant induction of VTG gene expression was detected in all treated groups, and no VTG mRNA was detected in the control group. The ratio of VTG to beta-actin analyzed from the results of quantitative real-time RT-PCR reached 0.041 +/- 0.024 (mean +/- SD) in the group receiving 10 mg/kg/week of 4-NP and 4.51 +/- 1.68 in the group receiving 100 mg/kg/week of 4-NP. Chemical analysis of 4-NP showed that the concentrations of 4-NP in the 10 mg/kg/week group and the 100 mg/kg/week group were 2.78 +/- 2.41 and 31.38 +/- 0.26 mcirog/ g wet weight, respectively. Compared with the 4-NP concentrations (0.8-1.92 microg/g wet wt) in fish from the Yangtze River, China, a potential hazard exists regarding 4-NP in Chinese sturgeon. These results represent the first indication of the risk of endocrine-disrupting chemicals for Chinese sturgeon.

A Novel Immunocompetent Rat Model of HCV Infection and Hepatitis

Hepatitis C virus (HCV) infects millions of people worldwide. Therapy is limited, and treatment does not produce a sustained response in the majority of patients. Development of new agents has been hampered by the lack of a convenient animal model. The aim of this study was to determine whether an immunocompetent rat, tolerized and transplanted with a human hepatoma cell line (Huh 7 cells), could be used to sustain an HCV infection. Fetal rats were tolerized in utero with 10(5) Huh 7 cells. One day after birth, rats were transplanted with 5 x 10(6) Huh 7 cells and, a week later, inoculated with HCV, genotype 1. In tolerized, transplanted, and HCV-infected rats, Huh 7 cells were found in the liver, and HCV viral replication was detected by the presence of negative strand HCV RNA. HCV levels in serum were measured at 11,000 copies/mL at week 4, peaked at 22,500 copies/mL by week 12. In tolerized, transplanted, inoculated rats, but not controls, serum alanine aminotransferase (ALT) values increased to 60 IU/L by week 4 and reached a peak of approximately 120 IU/L by week 13. Histology showed foci of mononuclear infiltrates in portal and central regions. HCV-inoculated immunocompetent rats tolerized and transplanted with Huh 7 cells support HCV gene expression, viral replication, and develop biochemical and histologic evidence of hepatitis.

A Chlorotic Spot Disease on Calla Lilies (Zantedeschia spp.) Is Caused by a Tospovirus Serologically but Distantly Related to Watermelon silver mottle virus.

A new tospovirus, Calla lily chlorotic spot virus (CCSV), was isolated from calla lilies (Zantedeschia spp.) in Taiwan. Chlorotic spots, ranging from light green to yellow, appear on the middle leaves of the affected plants. Virions measuring 75 to 105 nm, similar in size to tospovirus particles, were present in crude extracts and ultrathin sections of diseased leaves. Of 35 plant species inoculated mechanically, 24, including wax gourd (Benincasa hispida) and zucchini squash (Cucurbita pepo), were susceptible to the virus. CCSV was transmitted from infected wax gourd by Thrips palmi to healthy wax gourd and zucchini squash. The virus was weakly related to Watermelon silver mottle virus (WSMoV) in enzyme-linked immunosorbent assay (ELISA) and western blot tests. WSMoV-specific N gene primers, however, failed to produce DNA fragments from total RNA extracts of CCSV-infected plants in reverse transcription-polymerase chain reaction (RT-PCR). Results of RT-PCR show that the conserved regions of the L genes of tospoviruses are present in CCSV.

First Incidence of Plum Pox Virus on Apricot Trees in China.

Plum pox disease, caused by Plum pox virus (PPV), is the most severe virus disease of plums, apricots, and peaches. The disease causes heavy losses for fruit growers and the international trade of propagation materials and fresh fruits. PPV was first reported in Bulgaria in 1917 (1). It is now widespread in Europe and has been reported from Cyprus, Syria, Egypt, India, Kazakhstan, Chile, the United States, and Canada. Leaves on symptomatic apricot trees (Prunus armeniaca cvs. Hong Mei and Bai Mei and a selected genotype) in the Hunan Province of China showed typical yellow rings and diffused chlorotic spots. Samples from three suspected trees were repeatedly analyzed using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) in the summers of 2001-2003. PPV was detected in leaves, bark, and leaf buds of all three trees using ELISA with polyclonal and monoclonal antibodies provided by M. Navratil, Palacky University, Olomouc, Czech Republic (3). The results were confirmed using RT-PCR amplification of a 243-bp of the coat protein gene with a PPV-specific primer pair (2). BLAST analysis of two RT-PCR product sequences (GenBank Accession Nos. AY750961 and AY795603) showed 100% homology to multiple sequences of the PPV-D strain (GenBank Accession Nos. X81080, AF440743, and AF401295). The third sequence (GenBank Accession No. AY795602) had a C at position 112 rather than the T found in the other sequences. The ELISA, RT-PCR, and sequence results indicate that PPV-D was present in the apricot trees. To our knowledge, this is the first indication of PPV occurrence in China. This sporadic incidence of PPV on apricot trees requires addressing problems with the occurrence and spread of plum pox diseases in China and starting an eradication program.

Methotrexate induction of human sulfotransferases in Hep G2 and Caco-2 cells

Methotrexate (MTX) was the first antifolate drug developed for the treatment of cancer. It is also effective in treating inflammatory and autoimmune diseases. Sulfotransferases are phase II drug-metabolizing enzymes and their induction by hormones and endogenous molecules is relatively well known, although xenobiotic drug induction of sulfotransferases has not been well studied. In the present investigation, MTX is shown to be a xenobiotic inducer of human sulfotransferases in transformed human liver (Hep G2) and intestinal (Caco-2) cells. Following MTX treatment, various sulfotransferases were induced in both cell lines. Enzyme assay, Western blot and reverse-transcription polymerase chain reaction (RT-PCR) results demonstrated that protein and mRNA expressions of human simple phenol sulfotransferase (P-PST), human monoamine sulfotransferase (M-PST), human dehydroepiandrosterone sulfotransferase (DHEA-ST) and human estrogen sulfotransferase (EST) were induced in Hep G2 cells; M-PST and DHEA-ST were induced in Caco-2 cells. Inductions in both cell lines were dose dependent. Enzyme activity and Western blot results were in good agreement with RT-PCR results, suggesting that the induction is at the gene transcription level. Folic acid had a significantly lesser effect on sulfotransferases compared with MTX. Interestingly, the induction of different sulfotransferases by MTX was inhibited by high doses of folic acid at both protein and mRNA levels in Hep G2 cells. Methotrexate is the first antifolate and apoptosis-inducing drug to show induction of sulfotransferases in Hep G2 cells and Caco-2 cells. The inhibition by folic acid suggests a possible mechanism for MTX induction.

First Report of Barley yellow mosaic virus in Barley in Spain.

Recently, the presence of Barley mild mosaic virus (BaMMV) and the weakly serological detection of Barley yellow mosaic virus (BaYMV) were reported in Spain (1); both viruses are members of the genus Bymovirus (family Potyviridae). Random and symptomatic surveys were conducted during February and March of 2003 in barley fields in northeastern Spain to determine the occurrence of BaMMV and BaYMV. Leaves from 316 samples collected in 15 fields were analyzed using enzyme-linked immunosorbent assay (ELISA) with commercial antisera specific for BaYMV and BaMMV (Loewe Biochemica, Munich) as well as antisera against both viruses (provided by T. Klumen). Positive ELISA samples were further analyzed using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers that amplify 445 bp of BaMMV and 433 bp of BaYMV (2). Complete agreement was observed between the ELISA and RT-PCR results. Mixed infections of BaYMV and BaMMV were detected in 10 samples, BaYMV in 5 samples and BaMMV in 3 samples. Samples positive for both viruses that exhibited clear mosaic symptoms were collected in two fields. RT-PCR products from five BaYMV-infected samples were cloned and sequenced and showed 96 to 98% identity to BaYMV isolates previously reported from Europe (Genbank Accession Nos. AJ1515479-85 and X95695-7) and 92 to 95% identity with isolates reported from Asia (GenBank Accession Nos. AB023585-96, AJ132268, AJ224619-22, AJ224624-28, AF536944-46, AF536948-58, D01091, D00544, and Z24677). Sequence identity of Spanish isolates were 96 to 99%. To our knowledge, this is the first report of BaYMV infecting barley in Spain and illustrates the association of both Bymoviruses infecting barley.

EUROPEAN BROWN HARE SYNDROME IN FREE-LIVING MOUNTAIN HARES (LEPUS TIMIDUS) AND EUROPEAN BROWN HARES (LEPUS EUROPAEUS) IN FINLAND 1990–2002

We investigated the epidemiology of the European brown hare syndrome (EBHS) in Finland among free-living mountain hares (Lepus timidus) and European brown hares (Lepus europaeus). Carcasses of 297 European brown hares and 843 mountain hares submitted for postmortem examination were analyzed between 1990 and 2002. One hundred thirty-six cases of EBHS were detected during the examination, 20 cases in European brown hares and 116 in mountain hares. Liver samples from 56 histopathologically positive and 135 negative cases were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The results of RT-PCR correlated well with histopathology. Cases of EBHS appeared only sporadically in a restricted area in eastern Finland during 1990-97. In 1998, the number of cases increased, and the disease spread to the north. In 1998-99, EBHS was the most common disease of mountain hares in necropsy material from northern and eastern Finland. Special features were the occurrence of the disease in areas in which European brown hares are rare and the seasonal distribution of cases, most of which appeared in spring or summer. This is the first report of an outbreak of EBHS occurring mainly among free-living mountain hares from Finland.

Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells. The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with beta-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR. The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418. The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected. Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

Clinical Significance of AML/ETO Fusion Gene mRNA in Acute Myelogenous Leukemia.

Acute myelogenous leukemia (AML) patients with normal-karyotype may have undetected chromosome aberrant that could affect prognosis. Screening for known AML-specific genetic abnormalities using the reverse transcription polymerase chain reaction (RT-PCR) may help in arriving at a more definitive prognosis. And RT-PCR is the most sensitive method to detect Minimal residual disease (MRD). Nested PCR was a frequently used method to amplify AML1-ETO fusion gene. To further comprehend the relationship between AML1-ETO mRNA expression in AML and its clinical significance in Eastern China. We investigated the prevalence of t(8;21) (q22;q22) and AML1/ETO fusion gene in 461 unselected de novo patients with AML by single RT-PCR and compared the results of cytogenetic analysis with these of RT-PCR. The patients' median age was 29(1–76).The results were used in diagnosis of 461 de novo leukemic patients, and 70 AML1-ETO-postive patients were followed up. 107 in 461 patients (23.2%) AML1/ETO was detected by RT-PCR. 98 in 461 patients (21.3%) showed t(8;21) (q22;q22) in karyotype analysis. All patients who had t(8;21) (q22;q22) in conventional karyotyping also showed the gene rearrangement in molecular analysis. The results showed that AML1/ETO mRNA could be expressed in cells from AML-M1, AML-M2 and AML-M4 patients. The complete remission rate in AML1/ETO-positive patients was significantly higher than that in AML1-ETO-negative patients (without M3 subtype patients)[80.4% (86/107) vs 70.6% (191/269)]. The AML1-ETO-postive patients who became negative after chemotherapy had an optimistic outcome, but the patients who demonstrated persistence of AML1-ETO mRNA had a poor prognosis. In chemotherapeutic group, patients whose AML1/ETO expression turning from negative (3 cases) or faint positive (1 case) to positive relapsed later. So PCR becoming positive again indicated relapsing. Only 7 was detected in 48 M2 t(8:21) patients who had been maintaining remission for more than 18 months.RT-PCR detected the overall AML1-ETO-positive rate in AML was from 6% to 13% in Western country. But our results showed the rate is 23.2%. One reason might be that the patients involved in our observation were younger than those patients involved in other observation group reported.. The other reason, which might be more important, was that the differences between China and Western country in race and region.Our results showed single RT-PCR method was sensitive enough to detect AML1-ETO mRNA. These observations suggest that AML1/ETO mRNA could disappear after chemotherapy or bone marrow transplantation. The patients had a great probability to relapse if the results of RT-PCR are continuously positive or change from negative to positive. So Regular detection is necessary for leukemia patients. We can monitor the minimal residual disease by detecting AML1-ETO mRNA regularly to direct clinical therapy

First Report of Cherry virus A and Little cherry virus-1 in Poland.

Cherry virus A (CVA), a member of the genus Capillovirus, has been reported in sweet cherry in Germany, Canada, and Great Britain. No data are available on the effects of CVA on fruit quality and yield of infected trees. Little cherry disease (LChD) occurs in most cherry growing areas of the world. Symptoms on sensitive cultivars include discolored fruit that remain small, pointed in shape, and tasteless. Three Closterovirus spp. associated with LChD have been described (Little cherry virus-1 [LChV-1], LChV-2, and LChV-3). Diseased local and commercial cultivars of sour cherry trees were found in a Prunus sp. germplasm collection and orchards in Poland during the 2003 growing season. The foliar symptoms included irregular, chlorotic mottling, distortion, and premature falling of leaves. Some of the diseased trees developed rosette as a result of decreased growth and shortened internodes. Severely infected branches exhibited dieback symptoms. Because the symptoms were suggestive of a possible virus infection, leaf samples were collected from 38 trees and assayed for Prune dwarf virus and Prunus necrotic ringspot virus using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). RNA extracted from leaves was used in a reverse transcription-polymerase chain reaction (RT-PCR) with the One-Step RT-PCR with Platinum Taq (Invitrogen Life Technologies) and primer sets specific for CVA (1), LChV-1 (3), and LChV-2 (3). The RNA samples were also tested using RT-PCR for detection of Cherry mottle leaf virus (CMLV), Cherry necrotic rusty mottle virus (CNRMV), and Cherry green ring mottle virus (CGRMV) with specific primer sets (2). Amplification of a 397-bp coat protein gene product confirmed infection of 15 trees with CVA. A 419-bp fragment corresponding to the coat protein gene of LChV-1 was amplified from cv. Gisela rootstock and local cv. WVIII/1. To confirm RT-PCR results, CVA amplification products from local cv. WX/5 and LChV-1 from cvs. Gisela and WVIII/1 were cloned in bacterial vector pCR 2.1-TOPO and then sequenced. The sequences were analyzed with the Lasergene (DNASTAR, Madison, WI) computer program. The alignment indicated that the nucleotide sequence of cv. WX/5 was closely related to the published sequences of CVA (Genbank Accession No. NC_003689) and had an 89% homology to the corresponding region. The nucleotide sequence similarity between the 419-bp fragment obtained from cvs. Gisela and WVIII/1 was 87% and 91%, respectively, compared with the reference isolate of LChV-1 (Genbank Accession No. NC_001836). The sampled trees tested negative for LChV-2, CGRMV, CMLV, and CNRMV using RT-PCR. Some trees tested positive for PNRSV and PDV. To our knowledge, this is the first report of CVA and LChV-1 in Poland.

Expression profiles of fibrinolytic components in nasal mucosa.

Components of the fibrinolytic pathway contribute to diverse pathways in many tissues, in addition to their well-recognized role in degradation of fibrin clots. In this study of nasal mucosa, we investigated the presence of mRNA of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), and plasminogen activator inhibitor-2 (PAI-2) using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization, and compared these results with their localization in immunostained tissues. According to real-time RT-PCR results, t-PA, u-PA, PAI-1, and PAI-2 mRNA were noted in human nasal mucosa. Particularly, expression of u-PA and PAI-1 mRNA was significantly high in allergic nasal mucosa in comparison with normal mucosa. t-PA mRNA was detected in endothelial cells and epithelium in normal nasal mucosa. t-PA mRNA was detected in mucous cells of allergic submucosal glands, but not in normal glands. In allergic rhinitis, u-PA and PAI-2 mRNA were detected in mucinous cells and epithelium, and PAI-1 mRNA was detected in serous cells and epithelium. Expression of u-PA and PAI-1 mRNA in normal nasal tissues was decreased in contrast to that in allergic nasal tissues. u-PA staining was observed in mucous cells of allergic submucosal glands and the staining pattern of PAI-2 was similar to that of u-PA. PAI-1 was present in serous cells of submucosal glands from allergy samples, while epithelial cells were almost devoid of stain. In contrast, with allergy, immunohistochemical staining of t-PA was negative in submucosal glands, though positive in endothelial cells and epithelium. However, the expression of t-PA mRNA in allergic nasal mucosa was noted in mucous cells. In fibrin autography of nasal discharge, u-PA was markedly activated in the allergic patient. These results suggest that t-PA synthesized in mucous cells is promptly secreted and modifies watery nasal discharge in allergic rhinitis, and that u-PA activity may help the passage of large amounts of rhinorrhea by also reducing its viscosity. A lot of cellular infiltration (eosinophils in particular) was recognized in allergic nasal mucosa. It is most likely that the modifications in expression of PAs and PAIs are due to the local release of cytokines or growth factors from these inflammatory and immune cells.

Gene expression profiling reveals novel targets of estramustine phosphate in prostate cancer cells

Estramustine phosphate (EMP) is a compound widely used for the treatment of hormone-refractory prostate cancer. In order to better understand the precise molecular mechanism(s) by which EMP exerts its effects on hormone-resistant PC3 prostate cancer cells, we have utilized microarray to interrogate 22,215 known genes to determine the gene expression profiles altered by EMP treatment. The purpose of this investigation was to identify gene expression profile first and then in future studies determine the specific role of these genes in EMP-induced apoptosis in prostate cancer cells. We found a total of 726 genes which showed >2 fold change after EMP treatment. Clustering analysis showed 12 different types of expression alteration. These genes were also subjected to cluster analysis according to their biological functions. We found that EMP regulated the expression of genes, which are critically involved in the regulation of cell growth, cell cycle, apoptosis, iron homeostasis, cytoskeleton and cell signaling transduction. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm the results of microarray, and the results of real-time quantitative RT-PCR were consistent with the microarray data. From these results, we conclude that EMP caused changes in the expression of a large number of genes that are related to the control of cell survival and physiological behaviors. The gene expression profiles may provide comprehensive molecular mechanism(s) by which EMP exerts its pleiotropic effects on prostate cancer cells. EMP-induced regulation of these genes may be further exploited for devising therapeutic strategies for prostate cancer.

Development of an optimized protocol for the detection of classical swine fever virus in formalin-fixed, paraffin-embedded tissues by seminested reverse transcription-polymerase chain reaction and comparison with in situ hybridization

An optimized protocol was developed for the detection of classical swine fever virus (CSFV) in formalin-fixed, paraffin-embedded tissues obtained from experimentally and naturally infected pigs by seminested reverse transcription-polymerase chain reaction (RT-PCR). The results for seminested RT-PCR were compared with those determined by in situ hybridization. The results obtained show that the use of deparaffinization with xylene, digestion with proteinase K, extraction with Trizol LS, followed by seminested RT-PCR is a reliable detection method. An increase in sensitivity was observed as amplicon size decreased. The highest sensitivity for RT-PCR on formalin-fixed, paraffin-embedded tissues RNA was obtained with amplicon sizes less than approximately 200 base pairs. An hybridization signal for CSFV was detected in lymph nodes from 12 experimentally and 12 naturally infected pigs. The sensitivity of seminested RT-PCR compared with in situ hybridization was 100% for CSFV. When only formalin-fixed tissues are available, seminested RT-PCR and in situ hybridization would be useful diagnostic methods for the detection of CSFV nucleic acid.

Gene expression profiling reveals novel targets of estramustine phosphate in prostate cancer cells

Estramustine phosphate (EMP) is a compound widely used for the treatment of hormone-refractory prostate cancer. In order to better understand the precise molecular mechanism(s) by which EMP exerts its effects on hormone-resistant PC3 prostate cancer cells, we have utilized microarray to interrogate 22,215 known genes to determine the gene expression profiles altered by EMP treatment. The purpose of this investigation was to identify gene expression profile first and then in future studies determine the specific role of these genes in EMP-induced apoptosis in prostate cancer cells. We found a total of 726 genes which showed >2 fold change after EMP treatment. Clustering analysis showed 12 different types of expression alteration. These genes were also subjected to cluster analysis according to their biological functions. We found that EMP regulated the expression of genes, which are critically involved in the regulation of cell growth, cell cycle, apoptosis, iron homeostasis, cytoskeleton and cell signaling transduction. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm the results of microarray, and the results of real-time quantitative RT-PCR were consistent with the microarray data. From these results, we conclude that EMP caused changes in the expression of a large number of genes that are related to the control of cell survival and physiological behaviors. The gene expression profiles may provide comprehensive molecular mechanism(s) by which EMP exerts its pleiotropic effects on prostate cancer cells. EMP-induced regulation of these genes may be further exploited for devising therapeutic strategies for prostate cancer.