Evaluation of the polarographic technique for assay of the viability of freeze-dried BCG vaccine: I. The polarographic technique.

In this paper, a new assay based on oxygen uptake assessed by polarography was evaluated with the aim of establishing the viability of freeze-dried BCG vaccine. An oxygen electrode possessing a temperature sensor was designed for this purpose. The polarographic method used had several advantages, particularly its rapidity and use of small amounts of biological material. These advantages are ideal for quality control of BCG vaccine.

Evaluation of the polarographic technique for assay of the viability of freeze-dried BCG vaccine: II. Viability of the vaccine assessed by polarography, Warburg respirometry and colony counting.

A comparative study of the viability of 34 lots of freeze-dried BCG vaccine has been carried out using an oxygen electrode polarographic technique, Warburg respirometry and colony counting. There were no statistical differences between the results obtained with the polarographic and Warburg techniques. Both methods gave reliable and similar results and showed a positive correlation (r = 0.8615). Comparison between the polarographic and colony-counting methods showed a positive correlation (r = 0.6530); for comparison between the Warburg and colony-counting methods, the correlation value was r = 0.6868. All the tests were significant at the level of alpha = 0.05. The advantages of the polarographic technique are that it is much less time- and material-consuming than other methods. It is a reliable, inexpensive and convenient method for BCG vaccine quality control.

Blood glucose partition and levels of glycolytic enzymes in erythrocytes and somatic tissues of penguins.

1. A comparative study was carried out on blood glucose partition and glucose metabolism of penguin erythrocytes and somatic tissues. Pygoscelidae penguins (Pygoscelis antarctica and P. papua) were used in these experiments. 2. Blood glucose partition was established by assaying whole blood and plasma glucose in several individuals of the gentoo and chinstrap penguins. 3. It was found that almost all the whole blood sugar is compartmentalized at the plasma site, the red blood cells being ineffective in regard to glucose metabolism. 4. Levels of hexokinase, phosphoglucose isomerase, phosphofructokinase, fructose bisphosphate aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, phosphopyruvate hydratase (enolase), pyruvate kinase, alpha-glycerolphosphate dehydrogenase and fructose bisphosphate phosphatase were estimated in the erythrocytes of both gentoo and chinstrap penguins, the same determinations being carried out also on the somatic tissues (leg muscle, breast muscle, heart muscle, liver and brain) of the gentoo.

Metabolic activity of the Sticker's lymphosarcoma.

Some parameters (glycolysis, respiration, levels of glycolytic enzymes) of the lymphoid cells from the Sticker's lymphosarcoma were established in order to better define the biochemical behavior of the venereal tumor of the dog. For comparative purposes lymphocytes from peripheral blood of normal and tumor-bearing dogs were also studied. Lactic acid produced by the tumor cells during aerobic glycolysis is liberated in the reaction medium. Oxygen uptake is enhanced in the presence of succinate, but not with pyruvate, alpha-ketoglutarate, or malate as substrates. Higher levels of some of the enzymes from the glycolytic pathways as well as differences on the physicochemical and kinetic properties of the glycolytic regulatory enzymes are found in Sticker's tumor cells, when compared with the lymphocytes from peripheral blood of normal and tumor-bearing dogs. A fructose-bisphosphate positively modulated pyruvatekinase is found in the tumor cells.

A technique for the determination of the available oxygen in living carp (Cyprinus carpio) muscle.

A polarographic method for the measurement of the available oxygen in the muscle of living carp by the use of a platinum microelectrode is proposed. The oxygen and the reference electrodes were assembled in a single insertion piece which was implanted in the muscle of a living carp maintained in a special experimental chamber. Curves for normal oxygen levels corresponding to air-saturated water, as well as to a carbogene-saturated water, were obtained. The method can be considered adequate for the measurement of tissue oxygen in living fishes.

Comparative levels between enzymes of the glycolytic pathway from erythrocytes and somatic tissues of the chicken Gallus gallus domesticus.

A comparative study on the glycolytic enzymes from chicken erythrocytes and somatic tissues has been carried out, the results being shown as active units per mg protein in supernatants of 1085, 12,100 and 106,000 g fractionated centrifugation. The profiles of the glycolytic enzymes have been analyzed in terms of their activity relative to hexokinase and as the ratios between pairs of enzymes bearing a product-substrate relationship. Chicken erythrocyte displays a very peculiar profile of glycolytic enzymes. It possesses a FruP2-activated pyruvate kinase of the L isoenzyme type, which does not seem to be the predominant isoenzyme together with the M type, the content in glycolytic enzymes being much lower than in the somatic tissues.

Studies on the structure of aldolase A from chicken muscle.

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I-V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB3H4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB3H4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.

General effects of beryllium sulfate on isolated rat heart muscle mitochondrial fraction.

The effects caused by Be2+ on the properties of isolated rat heart muscle mitochondria were investigated. It has been shown that Be2+ enhances oxygen uptake by the mitochondrial fraction in the presence of oxidizable substrates. This is due to the uncoupling effect caused by Be2+ on the mitochondria, which also undergoes a swelling effect by this divalent ion that can be accounted by the retention of Be2+ by the mitochondria. The saturation of the organellae binding sites occurs when the concentration of the ion reaches a maximum of 6.2 mumoles/mg protein.

Comparative levels of muscle glycolytic enzymes in mammals, fish, echinoderm and molluscs.

1. Levels of glycolytic enzymes were determined in terms of units of enzyme/mg protein in rat striated muscle, carp lateral muscle, holothuria longitudinal muscle of the body wall, and a snail foot muscle. 2. An attempt has been made to correlate levels of glycolytic enzymes as a parameter to establish a "biochemical distance" at molecular level and correlate this with the phylogenetic position in animals sufficiently separated in the animal tree of evolution. 3. The possibility of a peculiar kinetic behaviour of the glycolytic pathway in each muscle tissue studied, has been analyzed as the profiles of the ratios of pairs of enzymes bearing a substrate-product dependence. 4. A possible "futile synthesis" of some glycolytic enzymes, such as FDP-aldolase in the case of fish muscle, is proposed.

General effects of beryllium sulfate on isolated rat heart muscle mitochondrial fraction.

The effects caused by Be2+ on the properties of isolated rat heart muscle mitochondria were investigated. It has been shown that Be2+ enhances oxygen uptake by the mitochondrial fraction in the presence of oxidizable substrates. This is due to the uncoupling effect caused by Be2+ on the mitochondria, which also undergoes a swelling effect by this divalent ion that can be accounted by the retention of Be2+ by the mitochondria. The saturation of the organellae binding sites occurs when the concentration of the ion reaches a maximum of 6.2 mumoles/mg protein.

General effects of beryllium sulfate on isolated rat heart muscle mitochondrial fraction

The effects caused by Be2+ on the properties of isolated rat heart muscle mitochondria were investigated. It has been shown that Be2+ enhances oxygen uptake by the mitochondrial fraction in the presence of oxidizable substrates. This is due to the uncoupling effect caused by Be2+ on the mitochondria, which also undergoes a swelling effect by this divalent ion that can be accounted by the retention of Be2+ by the mitochondria. The saturation of the organellae binding sites occurs when the concentration of the ion reaches a maximum of 6.2 mumoles/mg protein.