Establishment and characterization of new mammary adenocarcinoma cell lines derived from double transgenic mice expressing GFP and neu oncogene.

A new murine cell line, named GFPneu, was established from a mammary adenocarcinoma arising in double transgenic MMTVneu x CMV-GFP mice. Breast tumours develop in 100% of females after 2 months latency, as a result of the over-expression of the activated rat neu oncogene in the mammary glands. All tissues, and in particular the breast tumours, express the GFP protein. This cell line was tumorigenic when inoculated into nude mice and the derived tumours showed the same histological features as the primaries from which they were isolated. Their histopathology reproduces many characteristics of human breast adenocarcinomas, in particular their ability to metastasize. The GFP marker allows us to visualize the presence of lung metastases in fresh tissues immediately, to confirm the histopathology. From a lung metastatic fluorescent nodule, we derived a further cell line, named MTP-GFP, which we also characterized. These two cell lines could be useful to study the role played by the neu oncogene in the maintenance of the transformed phenotype, in the metastatic process, to test novel therapeutic strategies to inhibit primary tumour growth and to observe the generation of distant metastases.

Development of a one tube-one step RT-PCR protocol for the detection of seven viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid and Pospiviroid.

A one tube-one step RT-PCR was developed for the detection of seven viroids (Apple scar skin viroid, Apple dimple fruit viroid, Pear blister canker viroid, Hop stunt viroid, Chrysanthemum stunt viroid, Citrus exocortis viroid and Peach latent mosaic viroid) in four genera that infect eight plant species. The efficiency and specificity of this method were optimized by the use of Moloney-murine leukemia virus (M-MLV) reverse-transcriptase and HotStarTaq DNA polymerase which allowed increase sensitivity of viroid detection. The method was assessed with 56 viroid-infected field plants. The multiplex one tube-one step RT-PCR has the advantage of requiring less hands-on time to set up an assay than standard multiplex one, it also reduces the possibility of false positive tests because all steps are performed in the same tube thus, avoiding cross-contamination. The method may be used routinely for viroid detection in sanitary and certification programmes.

A novel multiplex RT-PCR probe capture hybridization (RT-PCR-ELISA) for simultaneous detection of six viroids in four genera: Apscaviroid, Hostuviroid, Pelamoviroid, and Pospiviroid.

A rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (RT-PCR)-probe capture hybridization (RT-PCR-ELISA). The assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: Potato spindle tuber viroid (Pospiviroid), Peach latent mosaic viroid (Pelamoviroid), Apple scar skin viroid (Apscaviroid), Apple dimple fruit viroid (Apscaviroid), Pear blister canker viroid (Apscaviroid), and Hop stunt viroid (Hostuviroid). Total RNA was obtained from infected tissue by the Qiagen RNeasy kit and, then viroid cDNA was synthesized using viroid specific complementary DNA primer. To identify and differentiate the amplicons of the six viroids, each amplicon was digoxigenin (DIG)-labelled during the amplification process, and then detected by a colorimetric system using a biotinylated cDNA capture probe specific for each viroid. The results revealed that each capture probe hybridized only to its complementary DIG-labelled amplicon. Thus the six viroids can be detected and differentiated in a multiplex RT-PCR-ELISA assay. In the multiplex assay, cDNAs of six viroids were synthesized simultaneously in one tube, DIG-labelled during amplification, then a portion of the DIG-labelled amplified products was hybridized with selected capture probe. All the six viroid capture probes hybridized to their respective complementary DIG-labelled RT-PCR-amplified product. These findings are important for viroid detection and identification for studying host-viroid interactions and for management and control viroid diseases.

[Ventricular pre-excitation in Wolff Parkinson White syndrome in the aged]. / La pre-eccitazione ventricolare da sindrome di Wolff Parkinson white nell'anziano.

AIM: To evaluate any differences in ventricular pre-excitation secondary to Wolff Parkinson White syndrome in the aged compared to young and adult patients. EXPERIMENTAL DESIGN: a clinical study was performed using a comparative prospective criterion with retrospective analysis. The duration of follow-up ranged between one and ten years. SETTING: the series was collected from the Cardiology Clinic of the Health District and the Cardiology Division of Gorizia, both forming part of no. 2 Isontina Health Service. PATIENTS OR PARTICIPANTS: the series included 17 patients suffering from Wolff Parkinson White syndrome who were divided into two study groups: 9 elderly patients and 8 young patients. The latter were subdivided into a first subgroup of 4 cases with Wolff Parkinson White syndrome with ECG positive for the presence of delta waves, and a second subgroup also with Wolff Parkinson White syndrome secondary to bundle. INTERVENTIONS: some young patients with Wolff Parkinson White syndrome who were symptomatic for tachycardia underwent ablative surgery with radiofrequency of the bundle. PARAMETERS: all patients underwent cardiological screening focused in particular on surface electrocardiogram. Those cases with Wolff-Parkinson White syndrome with occult bundle underwent transesophageal electrostimulation to find the conduction threshold of the anomalous bundle. RESULTS: Adult-elderly patients: six subjects were diagnosed with antero-septal and left ventricular Kent's bundle (type B common) and 3 cases with Mahaim-Wiston bundle (type A rare). Surface ECG revealed the presence of left ventricular hypertrophy in 6 cases, left anterior hemiblock and total block of the left branch in 3 cases, as well as myocardial pseudonecrosis correlated to Wolff Parkinson White syndrome. Young patients: four out of this group were affected by Kent's bundle with type B Wolff Parkinson White syndrome and the same number suffered from the same syndrome caused by occult bundle. Patients in the first subgroup showed an antero-septal, transitional and left ventricular orientation of Kent's bundle, with the onset of 2 cases of orthodromic and antidromic reciprocal rhythm respectively and 1 case of atrial fibrillation. The refractory nature of the anomalous pathway was not very high in 2 cases, equal to 60 milliseconds and 240 milliseconds with the proposed ablation of the anomalous bundle.

Comparison of different methods of RNA isolation for plum pox virus detection by reverse transcription-polymerase chain reaction.

The diagnosis of plum pox virus (PPV) is still considered one of the most important aspects of the "sharka" problem. In fact, different studies demonstrated an uneven distribution of the virus in infected trees due to a high variability in virus concentration. These aspects complicate the PPV diagnosis. To date, biological, serological and molecular assays have been successively developed in order to obtain sensitive and efficient PPV detection techniques. In particular, the polymerase chain reaction (PCR) technique seems to be promising and can be considered the most sensitive and reliable one. Preparation of viral RNA is still a fundamental step in reverse transcription-PCR (RT-PCR) technique, especially when applied to large scale testing, i.e., for certification purposes. In order to find the most rapid and efficient procedure, we have compared three different procedures of extraction of viral RNA to be processed RT-PCR. Their common characteristics is their capacity to extract the RNA from a small amount of plant tissue without organic solvents in the extraction fluid. The procedures were as follows: an immuno-capture (IC) method using a specific antiserum, a silica-capture (SC) method using a non-specific matrix, and a simple and rapid RNA extraction (RE) method. They all were followed by one-tube RT-PCR. The obtained results show that all the three techniques allowed a successful amplification and detection of PPV in tested samples except the SC-PCR method which proved less effective. In fact, the IC-PCR and RE-PCR methods amplified and detected PPV in all isolates tested, while the SC-PCR method was able to reveal the presence of the virus in apricot and infected control samples only.

Occurrence of Peach Latent Mosaic Viroid (PLMVd) on Plum in Italy.

Peach latent mosaic disease was originally described in France in 1976 (1). The disease is often latent in peach trees but may induce mosaic symptoms on leaves, irregularly shaped colorless fruit with cracked sutures and enlarged pits, bud necrosis, and delay in foliation, flowering, and fruit maturity. Recently, the disease has been shown to be caused by peach latent mosaic viroid (PLMVd) (2). PLMVd is distributed worldwide on peach. A recent survey in Italian plum orchards has showed the occurrence of PLMVd in two plum cultivars (Black Diamond and Angeleno) at different geographical locations in central Italy. Plum samples showed bark necrosis and bark split on branches and trunk; moreover, 1-or 2-year-old shoots and stem appeared dwarfed because of shortening of internodes and reduced growth. Budwood from plum samples were grafted, in greenhouse, onto peach GF305 indicator plants. Total nucleic acids (TNA) were then extracted from peach GF 305, or plum leaves, after 6 months of postinoculation. The presence or absence of PLMVd was ascertained by bidirectional polyacrylamide gel electrophoretic (dPAGE) analysis and by spot hybridization assay with PLMVd cRNA probe provided by A. Hadidi. A dPAGE analysis of TNA extracted from grafted peach GF305 showed a band with the same electrophoretic mobility as that of D-168 strain of PLMVd positive control. No bands were observed in healthy peach GF305 or from healthy or diseased plum extracts. The lack of detection of a viroid band from diseased plum samples by dPAGE analysis may be due to low titer of the viroid in infected plum. Molecular hybridization analysis of total nucleic acids with PLMVd cRNA probe, however, showed positive hybridization signals with nucleic acids from grafted peach GF305 or infected plum. Thus, PLMVd or a very closely related viroid infects plum trees. Moreover, on the basis of biological and serological assays, apple chlorotic leaf spot trichovirus, in a sample, and prunus necrotic ring spot ilarvirus, in the second one, were found in association with PLMVd. The same symptoms observed in the two PLMVd-infected plums occurred in other plants infected only by the two viruses (alone or in association). These data suggest that, very probably, PLMVd does not play a specific role in the etiology of the field symptomatology described above. In any case, this is the first report for the occurrence of PLMVd on plum in Italy. References: (1) J. C. Desvignes. Acta Hortic. 67: 315, 1976. (2) R. Flores and G. Llacer. Acta Hortic. 235:325, 1988.