Field Trials of Plum Clones Transformed with the Plum pox virus Coat Protein (PPV-CP) Gene.

Transgenic clones C2, C3, C4, C5, C6, and PT-6, of plum (Prunus domestica L.) transformed with the coat protein (CP) gene of Plum pox virus (PPV), PT-23 transformed with marker genes only, and nontransgenic B70146 were evaluated for sharka resistance under high infection pressure in field trials in Poland and Spain. These sites differed in climatic conditions and virus isolates. Transgenic clone C5 showed high resistance to PPV at both sites. None of the C5 trees became naturally infected by aphids during seven (Spain) or eight (Poland) years of the test, although up to 100% of other plum trees (transgenic clones and nontransgenic control plants) grown in the same conditions showed disease symptoms and tested positively for PPV. Although highly resistant, C5 trees could be infected artificially by chip budding or via susceptible rootstock. Infected C5 trees showed only a few mild symptoms on single, isolated shoots, even up to 8 years post inoculation. These results clearly indicate the long-term nature and high level of resistance to PPV obtained through genetically engineered resistance.

Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV).

The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription--polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequence of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D.

Expression of the plum pox coat protein gene in transgenic Nicotiana tabacum plants.

Plant expression vector pBI 121 containing the gene encoding coat protein of Plum Pox Virus of the Skierniewice isolate (CP PPV-S) was prepared (clone pCM1). The construct was used for transformation of Nicotiana tabacum plants using an Agrobacterium tumefaciens based system. About 82% of kanamycin resistant plant lines contained a transgene (the sequence of CP PPV-S) but only 81% of them actively expressed the PPV-S coat protein gene as measured by RT-PCR.

Preliminary report on the apparent breaking of resistance of a transgenic plum by chip bud inoculation of plum pox virus PPV-S.

Five transgenic clones of Prunus domestica L. containing plum pox virus (PPV) coat protein (CP) gene and one non-transformed control clone were challenged with PPV-S in the field. Symptoms developed on C2, C3, C4, C6 and B70146 but not C5 trees inoculated by chip budding (CBI) (2/2, 2/2, 1/1, 2/2 and 2/2, positive/inoculated) in the first summer after inoculation. However, in the second year, symptoms appeared on CBI C5 trees. The presence of the virus in the plants was confirmed by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) amplification of a fragment of viral polymerase gene. During two years, symptoms of infection developed on 3 to 4 of 8 non-inoculated trees of clones C2, C3, C4, C6 and B70146. Eight non-inoculated C5 trees remained symptomless and ELISA-negative as of spring 1998, in spite of the presence of aphid vectors and inoculum sources.

Serum creatine kinase kinetics after endomyocardial biopsy in cardiac transplant patients.

The objective of this study was to define the changes in serum creatine kinase (CK) and creatine kinase-MB isoenzyme (CK-MB) activities following endomyocardial biopsy. Ten cardiac transplant recipients underwent routine surveillance endomyocardial biopsy via the right internal jugular vein with single-use disposable bioptomes. Serum CK and CK-MB levels were measured at baseline and post-biopsy at 15, 30, and 45 minutes, 1, 1.5, 2, 4, 6, 8, 12, 16, 20, and 24 hours. There was no significant increase in serum total CK after biopsy. No measurable change in CK-MB was observed in 40% of patients. CK-MB increased significantly in the remaining patients (N = 6) at 30 minutes (p < 0.05), and continued to rise to a peak at 4 hours post-biopsy, after which CK-MB gradually decreased to baseline. However, the increase in CK-MB did not meet criteria for the diagnosis of myocardial necrosis, hence should not be confused with the change in CK-MB occurring with myocardial infarction. These findings suggest that serum CK-MB activity is useful in the differential diagnosis of chest pain in cardiac transplant patients in the setting of recent biopsy.

[Occupational allergy in miners]. / Alergia zawodowa wsród zalóg górniczych.

The authors allergologically analysed 141 coal miners afflicted with upper respiratory tract inflammations. In 7% of cases the disease was found to be induced by coal dust. All the subjects exhibited positive dermal tests with common allergens (plants pollens, household dust, saprophytes, moulds). The authors suggest that atopic status may contraindicate the work in coal mining. 57% of cases exhibited changes in the nose inhibiting its patency (hypertrophy of nasal concha, nasal mucosa polyps, lateral deviation of the nasal septum).

[Histamine inhalation test in patients with specific bronchial hyperreactivity]. / Wziewny test z histamina u chorych ze swoista nadreaktywnoscia oskrzeli.

In 46 patients with a clinical suspicion of bronchial hyperreactivity tests of non-and specific bronchial hyperreactivity with the sensitizing allergen were carried out. The results were compared with the results of other allergological tests (skin prick tests, IgE and allergen specific IgE serum levels). The results of this study demonstrate the importance of duration, character and concentration of allergen exposition on formation of specific bronchial hyperreactivity. Non-specific and specific bronchial hyperreactivity demonstrates tissue dependent variations, this being seen especially in seasonal allergens. The authors have also shown a relation between both types of hyperreactivity and the results of other allergological tests.

Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus.

Detection of Apple stem pitting virus (ASPV) using RT-PCR based methods was studied in infected apple and pear trees. Three virus-specific primers (ASPF1CP, ASPF2CP, ASPR3CP) were designed to target the most conservative regions of the coat protein gene of 10 virus isolates in Poland and 7 other ASPV sequences available in GenBank. The suitability of the primer pairs ASPF1CP-ASPR3CP and ASPF2CP-ASPR3CP for detection of 19 virus isolates was checked. Both new primer pairs initiated amplification of a specific product from all sources tested. From 1 to 11 isolates were not detected with the primer sets published previously. Detection of the virus in the samples collected in March, using ASPF1CP-ASPR3CP primer pair, was possible up to 512 times dilution. For the samples collected in July, virus was detected in the extracts from infected plants diluted eight times. More than 100-fold increase of sensitivity could be obtained by semi-nested PCR with primers ASPF2CP-ASPR3CP following the first round with ASPF1CP-ASPR3CP. Identification of virus isolates with different number of deletions in the coat protein gene was possible using RT-PCR with newly designed reverse primer ASPDEL in combination with the published primer ASPV7956. Besides, the comparative analysis of silicacapture-RT-PCR (SC-RT-PCR) versus immunocapture-RT-PCR (IC-RT-PCR) assays was carried out. Few ASPV isolates escaped detection by IC-RT-PCR, while all isolates tested were detected using the SC-RT-PCR with the new primers.