Evaluation of experimental combined toxicity by use of dose-frequency curves: comparison with theoretical additivity as well as independence

Dose-frequency curves of toxic effects of a substance A were evaluated in the absence and in the presence of a fixed dose of a second substance B. Data were fitted by the curve-fitting program ALLFIT. Observed combined frequencies of A + B were compared statistically with the expected frequencies of additivity and (or) independence by the phi 2-square goodness-of-fit test. The theoretical dose-frequency curves expected for an additive response were obtained by a solely graphical procedure and the theoretical curves for independent effects were calculated from the effects of B and A at certain doses. In rotarod tests with trained mice, the combined deteriorating effect of ethanol and benzodiazepines were significantly over-additive. However, their lethal interaction appeared underadditive in mice. The lethal underadditive interaction of ethanol and phencyclidine (PCP) can be ascribed largely to independent actions of these compounds. Loss of righting reflex was additively enhanced by PCP, whereas PCP overadditively enhanced the effect of ethanol. The insecticidal action of the cholinesterase inhibitors malathion and parathion appeared additive and significantly different from independent interaction. A comparison of results from dose-response curves with isoboles showed good agreement. The method appears as an attractive alternative or as a complementary procedure to the isobolographic analysis. Combination experiments as described can be carried out and evaluated rather simply, with a minimum of expenditure and a maximum of information.     

An ultrafiltration assay for nucleotide binding to ribonucleotide reductase

Direct partition through ultrafiltration was applied to develop a method for the study of nucleotide binding to ribonucleotide reductase from Escherichia coli. The assay involved a 0.5- to 1-min centrifugation step where bound and unbound nucleotides are separated over an ultrafiltration membrane. No effects were seen due to hyperconcentration of protein at the membrane surface. The method was verified by measuring binding of dATP, ATP, dTTP, dGTP, and GDP at 25 and 4 degrees C with dissociation constants ranging from 0.1 to 80 microM. The results were in good agreement with earlier data obtained by other techniques and extend our knowledge in the case of ATP and dGTP binding at 25 degrees C.     

Structural characterization of glycoprotein carbohydrate chains by using diagoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.     

Phase diagrams of pseudo-binary phospholipid systems. II. Selected calorimetric studies on the influence of branching on the mixing properties of phosphatidylcholines

The miscibility properties of branched phosphatidylcholines in mixtures of aqueous dispersions were studied by means of differential scanning calorimetry. The phase diagrams of four pseudo-binary systems from mixing type unbranched phosphatidylcholine/branched phosphatidylcholine/water (50 wt. % water) were investigated and discussed. The unbranched dipalmitoylphosphatidylcholine acts as a reference component of the mixtures. The phase diagrams of these four pseudo-binary phosphatidylcholine systems showed some connections between chain structure of the branched phosphatidylcholines and miscibility of the components. A change of the phase diagram type has been observed according to the branching and/or chain length differences of the phosphatidylcholines: complete miscibility and peritectic mixing behaviour. Generally we observed complete miscibility in the high-temperature phase (La-phase) and demixing in the low-temperature phases (gel phase). This is dependent on the branching and chain length differences of the mixing components.     

In vivo and in vitro effects of temperature on monoamine oxidase activity in brain and other tissues of the goldfish. Carassius auratus L

1. The maximum velocity (Vmax) and apparent Michaelis constant (Km) of brain and liver monoamine oxidase (MAO) in goldfish were different in fish acclimated to 22 degrees C and to 7 degrees C ambient temperature. 2. In brain, Vmax and Km were dependent upon incubation temperature, but both parameters were lower in 7 degrees C, adapted fish over most of the incubation temperature range. 3. The values obtained for Km showed a plateau at incubation temperatures at and below 25 degrees C for warm water fish, and at and below 20 degrees C for cold water fish. The activation energy of brain MAO was lower in fish adapted to the colder water. 4. These results show that goldfish MAO displays changes in functional activity in response to a change in environmental temperature. Apparently the purpose of this adaptation is to compensate for a reduction in enzyme concentration.     

Uncommon pneumonia associated with acute poststreptococcal glomerulonephritis

184 cases of acute poststreptococcal glomerulonephritis were investigated and six of these were associated with a peculiar, uncommon pneumonia, and another one had a lethal course. The clinicoradiological and especially pathological data summarized in this study attempt to demonstrate the individuality of this type of pneumonia. Pneumonia associated with acute poststreptococcal glomerulonephritis is similar or identical to rheumatic pneumonia. In both entities, pneumonia and pulmonary edema may and do coexist, and the differentiation of pneumonia from congestive heart failure is difficult and often impossible without pathological evidence. The most attractive pathogenic interpretation is the hypothesis of an immune mechanism in the induction of pneumonia. Authors attributed the lung changes, consecutive to a vascular damage, to a hypersensitivity phenomenon, with accumulation of fibrinogen in alveoli where it is converted to fibrin. Then, the hyaline membrane lining the alveoli, an important feature of pneumonia associated with glomerulonephritis is built up. It is, however, plausible that the effect of the immune reaction is associated with those of hydrosaline retention, arterial hypertension and congestive heart failure. Analogous to rheumatic pneumonia, the "peculiar pneumonia" associated with acute poststreptococcal glomerulonephritis should be named nephritic pneumonia.     

Effect of diuretics on the distribution and renal excretion of lithium in rats of different ages

The postnatal development of the renal lithium elimination is maturated earlier than that of renal sodium excretion. The filtered lithium is reabsorbed to a great amount in the kidney (70-80%). 90% of the administered lithium is eliminated by the kidney. Acetazolamide stimulates the renal lithium excretion in young and adult rats. Other diuretics with different sites of attack are not able to influence the elimination of lithium. Also, a forced diuresis does not change the elimination rate of lithium. The well-known interactions between sodium and ithium must have their cause in extrarenal processes.     

The need of protein synthesis for the effect of LH--RH on the cAMP level in the anterior pituitary in vitro

The effect of LH--RH on cAMP accumulation in the anterior pituitary of male rats was investigated. The effect consisted of two phases. In the first phase of incubation (about 2 hours), there was no change in the cAMP level, while in the second phase a significant increase was observed. Cycloheximide (10(-5)M) given simultaneously with the LH--RH totally blocked the observed effect of LH--RH. If cycloheximide was given in the second phase of incubation, when the cAMP level was already elevated, its further rise was prevented and the elevated cAMP level remained unchanged for 2 or more hours. Actinomycin D (10(-5)M) added together with LH--RH totally abolished the action of the latter, but if actinomycin D was given 1 hour after LH--RH a significant increase of the cAMP level was found at the end of the 4-hour incubation.     

The increase of cerebellar cAMP level after decapitation: the effect of propranolol

The increase of cAMP level of the rat cerebellum induced by decapitation was studied. Administration of 5 mg/100 g propranolol 1 hour before decapitation completely prevented this increase. Neither the depletion of catecholamine pools, inhibition of their synthesis, nor barbiturate treatment influenced the increase of cAMP level evoked by decapitation. It has been concluded that noradrenergic neurotransmission is involved in the cerebellar cAMP level increase after decapitation.     

Disinfecting properties of performic acid against bacteriophage phi X 174 as a model of small envelope--free viruses

Performic acid HCOOH (PFA) is a wide-spectrum disinfectant. It inactivates viruses, bacteria and bacterial spores, mycobacteria as well as microscopic fungi. Its main drawback is its instability, which makes it a logical necessity that it is to be prepared prior to use from its components HCOOH and H2O2. The mixing of 8 ml HCOOH of the concentration 850 ml/l and 17 ml H2O2 of the concentration 300 ml/l in a 100 ml-volume reagent bottle with a ground-in glass stopper gives, after an 1-hour rest at room temperature and after another 1 hour in a refrigerator, a stock solution that contains about 50 ml/l of PFA the actual concentration of which is determined iodometrically. Bacteriophage phi X 174 (host E. coli C) is characterized by cubic ikosahedral-type symmetry of particles free of envelope, has 27 mm in diameter and contains single-strand cyclic DNA; formerly was classed among Parvoviridae. The possibility of plaque assay-based quantitative determination of the number of infectious particles makes if it a feasible model for assessing disinfectant action on small hydrophilic viruses under conditions close to those of practical disinfection procedures. PFA stock solution diluted to 1 X 10(-3) (0.05 ml/l of effective component) inactivates the model virus of a concentration 10(8) pfu/ml aqueous suspension within 5 min so that no virus is detectable; the drop in the number of pfu amounts to 7 log orders of magnitude. In the presence of 400 ml/l of serum, the identical effect is achieved within 5 min by PFA stock solution diluted to 5 X 10(-3). The lowest PFA concentration that reliably inactivates bacteriophage phi X 174 in aqueous suspension is identical with the lowest concentration inactivating Coxsackie B 1 virus in tissue cultures. On textile, glass, plastic, rubber and metal carriers contaminated by swabbing or by a dried drop of bacteriophage suspension containing about 1 X 10(9) pfu/ml, the lowest reliably effective concentrations of PFA range within 0.25-0.025 ml/l, i.e. PFA stock solutions diluted to 5 X 10(-3)-5 X 10(-4), depending on the type of carrier and the type of contamination.