Effect of proteases on the beta-thromboglobulin radioimmunoassay

Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of beta-thromboglobulin and platelet factor 4. The initial assays indicated that a beta-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the beta-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of alpha 1-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases.     

Somatic hybrids between Solanum brevidens and Solanum tuberosum: Expression of a late blight resistance gene and potato leaf roll resistance

Hexaploid somatic hybrids resulting from mesophyll protoplast fusions between Solanum brevidens Phil., PI 218228, and Solanum tuberosum L., PI 203900 were tested for late blight resistance using two races of Phytophthora infestans Monte., de Bary. The S. tuberosum parent was a late blight differential possessing the R4 gene which confers resistance to race 0. The S. brevidens parent is resistant to potato leaf roll virus. Inoculations with both compatible (race 1.3.4.5) and incompatible (race 0) races of P. infestans clearly demonstrated the expression of the late blight resistance gene in all of the hybrid progeny tested. Most of the hybrids tested were also resistant to potato leaf roll virus (PLRV), indicating that the S. brevidens genes for PLRV resistance were present and expressed.     

Control of logarithmic growth rates of tobacco callus tissue by cytokinins

The growth rates of tobacco callus tissues on media containing 10⁻⁶ to 10 μm 6-(γ,γ-dimethylallylamino)purine (2iP) were measured. At concentrations of 10⁻⁴ μm and above growth rates were exponential and dependent on cytokinin concentration. At 2iP concentrations of 10⁻⁴ to 0.33 μm, the exponential rate was maintained for 4 to 5 doublings of fresh and dry weight. After this period a linear phase, resulting in approximately 1 doubling of weight, occurred. The growth of tissues on media containing higher than 0.33 μm 2iP was exponential for only about 15 days. At the end of this time, and well before they achieved half their final weight, they exhibited growth which was less rapid than logarithmic but more rapid than linear. Comparisons with zeatin, 6-benzylaminopurine and kinetin indicated that, although the maximum growth rates obtained with relatively high concentrations (0.1-1 μm) were similar, the naturally occurring cytokinins, 2iP and zeatin, promoted faster rates at lower concentrations (10⁻³-10⁻² μm) than did 6-benzylaminopurine and kinetin.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Interaction of a hydroxyproline-rich glycoprotein from tobacco callus with potential pathogens

A hydroxyproline-rich glycoprotein was isolated from tobacco (Nicotiana tabacum L.) callus tissue cultures by an acidic-ethanol extraction procedure and purified to about 95% homogeneity by ion exchange chromatography on carboxymethyl cellulose. This glycoprotein agglutinated cells of an avirulent strain (B-1) of the bacterial pathogen Pseudomonas solanacearum but not its parental, virulent isolate (K-60). Bacterial lipopolysaccharide (from K-60 strain) inhibited this agglutination. The tobacco glycoprotein also agglutinated zoospores of both compatible and incompatible races of Phytophthora parasitica var. nicotianae. Although 34 potential haptens were tested, no low-molecular-weight carbohydrate that inhibited bacterial or fungal agglutination was found. The agglutination activity of the tobacco glycoprotein was sensitive to pronase and sodium periodate. The apparent molecular weight of the glycoprotein was 120,000. The protein moiety was basic (12% lysine and 5% histidine) and contained 38% hydroxyproline. The carbohydrate moiety comprised 26% (by weight) of the glycoprotein, and contained 87% arabinose, 8% galactose, and 5% glucose. The glycoprotein labeled with fluorescein isothiocyanate bound significantly better to the avirulent isolate (B-1) of P. solanacearum than to the virulent strain (K-60). Binding to the avirulent cells was inhibited by incubation in a higher ionic strength medium (e.g. 0.2 m NaCl). The labeled glycoprotein also bound to cystospores and mycelia of both races of P. parasitica var. nicotianae. This fungal-glycoprotein interaction was inhibited by the lipopolysaccharide from strain K-60 and by higher ionic strength conditions.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.