Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65–70 μg LTB g⁻¹ tissue whilst hairy roots of tomato contained ~10 μg LTB g⁻¹. Antigen at ~600 ng ml⁻¹ was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.     

Comparison by Controlled Clinical Trial of Streptokinase and Heparin in Treatment of Life-threatening Pulmonary Embolism

Treatment with heparin or streptokinase was allocated randomly to 30 patients with life-threatening pulmonary embolism verified by angiography. Treatment was given for 72 hours and pulmonary angiography was repeated. There was significantly greater (P < 0·001) evidence of thrombolysis in those patients treated with streptokinase compared with those treated with heparin. The reduction of systolic and mean pulmonary arterial pressures was also significantly greater (P < 0·05 and P < 0·02 respectively) in the streptokinase group.Seven patients failed to complete 72 hours of the trial treatment: five successfully underwent pulmonary embolectomy. Six of these “failures” had initial pulmonary angiographic scores of 24 or more and systemic systolic blood pressure recordings of 100 mm Hg or less. Patients with these features should probably be considered for pulmonary embolectomy as the initial treatment.A febrile reaction commonly occurred in the streptokinase group; otherwise side effects were no more common than in the heparin group.     

The use of the polymerase chain reaction in plant transformation studies

Summary Transformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.For tobacco species the PCR reaction worked efficiently with up to 2 g of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.Abbreviations c⁷dCTP 7-deaza-2-deoxyguanosine - GUS--Glucuronidase, NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - TBE Tris Borate - EtBr ethidium bromide These results were presented in part at the 7th International Congress on Plant Tissue and Cell Culture, Amsterdam, Netherlands 24–29th June 1990.     

Potential for use of nicotinic acid as a selective agent for isolation of high nicotine-producing lines of Nicotiana rustica hairy root cultures

The addition of exogenous nicotinic acid, nicotinamide or nicotine was studied with reference to their effects on growth and alkaloid production by hairy root cultures of Nicotiana rustica. Nicotinic acid and nicotinamide were toxic (50% phytostatic dose being 2.4 and 9 mM respectively) while nicotine was not toxic below 10 mM. Nicotinic acid (up to 5 mM) was found to be phytostatic rather than phytotoxic. Roots exposed to increasing nicotinic acid or nicotinamide levels had altered alkaloid accumulation patterns relative to the controls. The principal effects were to increase the intracellular and extracellular levels of anatabine and nicotine, with a markedly greater proportion of anatabine being produced. The use of nicotinic acid as a selection agent for the recovery of higher alkaloid-producing lines is identified and discussed.     

Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes

Hairy root cultures, obtained after transformation of seven plant species by A. rhizogenes, were examined cytologically to assess their chromosome number. All species had the correct 2n diploid number of chromosomes in root tip cells. Free cell suspensions of two of the species were also examined and were found to be variable with polyploids or aneuploids predominating. The DNA from the hairy root cultures was isolated and the presence of the Agrobacterium T-DNA confirmed by Southern blotting. The relevance of these observations to the use of hairy roots in the study of plant secondary metabolite production is discussed.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.