Protein reagent modification of cholera toxin: characterization of effects on antigenic, receptor-binding and toxic properties.

The effects of protein modification procedures on the biologically most important properties of cholera toxin, i.e. the toxic activity, the GM1 receptor-binding capacity and the antigenic (antibody-fixing) properties, have been studied quantitatively using microgram amounts or less of toxin protein. Most of the 24 group-specific reagents used had either no inhibitory effect on the toxic or the combination of GM1-binding and antibody-fixing properties of cholera toxin, or they had a concomitant inhibitory effect on these activities. Separate testing of GM1- and antibody-binding revealed a close, but not absolute, structural association between these properties, Amino group reactive substances were particularly effective in decreasing the GM1-binding activity, while leucine aminopeptidase had no effect. This suggests that lysine residues may be involved in binding toxin to the acidic GM1 receptor. Sodium dodecylsulphate and mercaptoethanol, which caused dissociation of the subunits of cholera toxin as indicated by polyacrylamide gel electrophoresis, abolished toxicity without inhibiting the concomitant GM1- and antibody-binding properties of the toxin. Similar differential effects were also obtained with three reagents which did not seem to change the aggregation state of the toxin. These substances all had specificity for arginine, suggesting that arginyl residues of the toxin molecule may be involved in a 'toxic site' distinct from the receptor-binding site(s). A selective effect on the toxic site was also found by treating the toxin with carboxypeptidase or trypsin in the presence of urea; in the absence of urea no enzymic effect on any toxin property was noted.     

Diabetogenic effect of triamcinolone in man with impaired thyroid function: serum free fatty acids, inorganic phosphorus, calcium and total protein pattern after an oral glucose load.

In a group of 35 subjects (12 hypothyroid, 11 euthyroid and 12 hyperthyroid) the changes of FFA, inorganic phosphorus, calcium and total proteins were investigated during glucose tolerance test with (TGTT) or without (OGTT) triamcinolone pretreatment. In hypothyroidism the decrease of FFA during TGTT was blunted, whereas the pattern of phosphatemia was unaltered and total proteins were increased. In euthyroidism triamcinolone exerts the opposite effect on the behaviour of FFA (decline was smaller and shorter) and phosphatemia (decline was pronounced and persists for longer period). In hyperthyroidism the fasting level of FFA decreased in TGTT but during the test no significant differences in FFA and phosphatemia patterns were observed. The calcemia was not influenced by triamcinolone in any subgroup. Our results suggest the importance of thyroid function as modifying factor in some metabolic effects of triamcinolone in man. Changes in hypothyroidism are in good agreement with changes of blood glucose and insulinemia, as described previously. Opposite effect of triamcinolone on FFA and phosphatemia pattern in euthyroidism may suggest the changes of sensitivity of some peripheral tissues to insulin action. The decline of fasting level of FFA after triamcinolone in hyperthyroidism is surprising and might be of importance in maintaining unaltered glucose tolerance after triamcinolone in this subgroup.     

Effect of ovarian sex hormones on the pool of free amino acids in maternal tissues of pregnant rats fed a protein-free diet.

The effects of s.c. injections of 5 mug oestrone/kg and 50 mg progesterone/kg on Days 5-7 of gestation were investigated in rats fed a protein-free diet. The decrease in essential and non-essential amino acid concentrations in maternal liver and the implantation site caused by dietary protein deficiency was restored to control values, whereas the alterations of the plasma free amino acid concentrations, except for arginine and glycine, were not reversed by hormone treatment.     

Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3'':5''-cyclic monophosphate in foetal rat liver maintained in organ culture.

Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2'-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.     

Comparative study of the repeated nucleotide sequences in DNA from differentiated tissues and tumors]

DNA renaturation kinetics was measured for the genomes of normal (spleen) and malignant (plasmacytoma) mouse tissues and for DNA from liver, sperm and developing embryos of the loach. It has been shown that the measuring of DNA renaturation kinetics makes it possible to reveal differences in the content of certain fractions of the repetitions in the genomes of different species. Moreover, differences in the distribution of the repetitions between hetero- and euchromatine can be identified. Loach embryo DNA (blastula stage) was shown to contain larger amount of the fraction renaturing at Cot less than 10(-2) as compared to liver and sperm DNAs (by 5%). An enrichment with respect to the intermediate repetitions (10(-2) less than Cot less than 10(2)) was found in the mouse plasmacytoma DNA as compared to the spleen DNA. The nature of these distinctions is discussed.     

Experience in the study of the peculiarities of the distribution of the tick Ixodes ricinus in a large territory]

Attempts were undertaken to study the character of the distribution of I. ricinus over a large territory. Methodic principles of the land survey of arthropods distribution and laboratory processing of its results, that were used before for I. persulcatus, were found to be quite suitable for I. ricinus. It was established that hungry adults of I. ricinus have at least three types of the distribution throughout the forests of Lithuania. Types of the distribution can be ascertained by the results of the record of I. ricinus nymphs with a flag.     

Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.     

Specifically cytotoxic human and mouse lymphoid cells induced with antibody or antigen-antibody complexes.

Human or mouse lymphoid cells could be "armed" with anti-NIP antibodies to become cytotoxic to NNP-conjugated fowl erythrocytes (NNP and NIP are closely related haptens). The arming factor was neutralized by a sufficient concentration of NIP-BSA (twice the concentration causing maximal precipitation) but low concentrations (e.g., 7% of the maximal precipitation concentration) increased the arming capacity.     

Induction of histamine release and desensitization in human leukocytes. Effect of anaphylatoxin.

Hog anaphylatoxin (AT) in concentrations from 0.5 to 5 mug/ml gives a dose-dependent histamine release from human leukocytes. Concentration of 100 mug/ml AT give the same high histamine release as 5 mug/ml. This is in contrast to the histamine release obtained with anti-IgE or allergen, which give low histamine release with high doses. The histamine release obtained with AT is completed in 20 sec and the reaction is temperature- and calcium-dependent. Treatment of cells with AT in the presence or absence of calcium makes them insensitive to another challenge with AT. Such treated cells are fully responsive, however, to challenge with anti-IgE if the pretreatment has been performed in the absence of calcium. This, together with the calcium- and temperature-dependence indicates that the AT-induced histamine release is nontoxic. Treatment of cells with AT in the presence of calcium induces, besides histamine release, decrease in sensitivity to anti-IgE, indicating that both AT and anti-IgE release histamine from the same cells. We discuss to what extent AT and cell-bound Ig share intracellular mechanisms for induction of histamine release.