Assay of nucleotides and other phosphate-containing compounds in isolated chloroplasts by ion exchange chromatography

This report describes a method for a sensitive assay of phosphate-containing metabolites in chloroplasts. The first step was to preincubate the chloroplasts with inorganic [³²P]phosphate in order to label the stromal phosphate pool. On illumination, the intermediates of the reductive CO₂ fixation cycle, and also ADP and ATP, were radioactively labeled. After a metabolic stop by filtering centrifugation into perchloric acid, the resulting extract was chromatographed on a small ion exchange column with a formic acid-formate gradient. The separated substances which appeared in the eluate were detected by continuous monitoring of ³²P radioactivity. The identity of the peaks in the chromatogram and the reproducibility of the assay procedure were investigated.     

Augmentation of interleukin 2-activated cytotoxicity after treatment of cells with inhibitors of interferon production.

Low-density human lymphocytes cultured with recombinant interleukin 2 (rIL-2) generated a high level of interferon(s) (IFN). Consistently more IFN including IFN-tau was produced during the first 3 days of culture with rIL-2 than during the subsequent 4 days. That ability was mainly associated with mepacrine+ cells and was decreased by low concentrations of leucine methyl ester (Leu-O-Me) or ammonium chloride. Leu-O-Me was employed either for the pretreatment of cells before the culture or as the additive to culture medium. The decrease in IFN production after pretreatment was associated with enhanced rIL-2-activated cytotoxicity. Similarly, 1 mM of ammonium chloride or 1 mM of Leu-O-Me added to rIL-2 supplemented cultures for 3 days showed an association between inhibition of IFN-tau generation and increased activation of cytotoxic activity. Thus NK cells appear to regulate their own response to rIL-2 activation and the control mechanism seems to be associated with the ability of the cells to produce IFN(s) and possibly other cytokines.     

The influence of substrate peptide length on human beta-tryptase specificity.

Combinatorial chemistry approach was applied to design chromogenic substrates of human beta-tryptase. The most active substrate, Ala-Ala-Pro-Ile-Arg-Asn-Lys-ANB-NH(2), was selected from among over 9 million heptapeptides. The amide of 5-amino-2-nitrobenzoic acid (ANB-NH(2)) attached at the C-terminus served as a chromophore. In order to determine the optimal length of the tryptase substrate, a series of N-terminally truncated fragments of this substrate was synthesized. Pro-Ile-Arg-Asn-Lys-ANB-NH(2), with the determined value of the specificity constant (k(cat)/K(M)) above 9 x 10(6) M(-1) s(-1), appeared to be the most specific substrate of tryptase. This substrate was twice as active as the parent heptapeptide substrate. We postulate that the optimal size of the pentapeptide substrate for the interaction with human beta-tryptase is associated with the unique structure of this proteinase, comprising four almost identical monomer subunits arranged in a square flat ring with its substrate pockets faced inside, forming a tetramer with a central pore that can be penetrated by this short peptide. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

The lower molecular weight acid phosphatase from the frog liver: isolation of homogeneous AcPase III and IV representing glycoforms with different bioactivity

1. AcPase III and AcPase IV, the major enzyme forms of the LMW AcPase of the frog (Rana esculenta) liver were resolved and purified to homogeneity. 2. AcPase III and IV showed a single protein band on SDS-PAGE corresponding to a mol. wt (Mr) of about 35,000. The Mr of the native enzyme forms were 33,200 (gel electrophoresis) and 38,200 +/- 5000 (gel filtration). This indicates that they are monomeric proteins sharing the same protein molecule. 3. AcPase III and IV differ essentially in thermostability and the activating effect of ConA binding. 4. AcPase III and IV are considerably activated with DTT but they differed markedly by the extent of this activation and the accompanying changes of their pH-activity curves. 5. It is suggested that the frog liver LMW AcPase represents a set of glycoforms whose different bioactivity is determined by the redox states of their essential cysteine residues.     

Role of chemotherapy in the treatment of anaplastic thyroid cancer --personal observations]

Results of chemotherapy applied in 23 patients with anaplastic thyroid cancer are presented. Chemotherapy combined with local treatment is a suggested therapy for treatment the locally advanced anaplastic thyroid cancer.     

Triglycerides, fatty acids and cortisol in simple obesity in children

In 50 children with obesitas simplex, 6-14 years of age, the triglycerides (TG), free fatty acids (FFA) and cortisol (F) levels in venous blood serum were estimated. In agreement with the development stages, studied patients were divided into the group of younger children in prematurity stage and the group of older children approaching the maturity. Obtained mean values of TG, FFA and F concentrations were analysed in the particular groups of obese children and compared with the healthy children of the same age. Mean concentrations of TG, FFA and cortisol in obese children were within the normal values and statistically did not differ from those of control healthy children. Also there was no difference in parameters studied if compared the younger and older groups of obese children. There was no interrelationship between the high birth weight and the degree of overweight as well as between the duration of obesity and the blood serum TG levels. In the course of obesitas simplex in children no detectable disturbances in the levels of TG, FFA and cortisol were found. It may depends on the more efficient adaptational mechanism connected with metabolism which are acting in the course of overfeed in the period of growth and development.     

Effect of air ions on L 1210 cells: changes in fluorescence of membrane-bound 1,8-aniline-naphthalene-sulfonate (ANS) after in vitro exposure of cells to air ions

The ability of air ions to produce changes in the electrical properties of L 1210 mouse leukemia cells was tested. The fluorescence of ANS incorporated into a membrane lipid bilayer (measured microphotometrically) was used as a probe. It was shown that the action of air ions of both signs could change (negative ions by increasing and the positive ones by decreasing) the fluorescence intensity of ANS in the cell surface structures or to an imbalance of ions inside and outside the cell. Both possibilities are discussed in the light of the results of experiments using ouabain or biguanide as factors diminishing the intensity of ANS fluorescence.