[Osteoporosis: multidisciplinary and radiologic aspects].

Osteoporosis in one of the most common diseases among the elderly individuals. Bone hardness is reduced to the critically low level. Even the smallest trauma makes previously healthy individual severely ill patient. In the USA, 1.6 million people sustain fractures due to osteoporosis each year, while approximately 14 billion dollars is spent on their treatment. The costs of osteoporosis treatment exceed the costs of prevention. The studies on the incidence, prevalence and socioeconomic aspects of osteoporosis, its prevention and treatment are expected to be carried out in our country in the future. Prevention of osteoporosis is not an individual problem, but it has wide social implications. Osteoporosis may be prevented and further loss of bone substance may be discontinued.

Search for compartments of glucose metabolism in the microinjected frog oocyte.

Microinjection of frog oocytes allows the modification of intracellular levels of substrates, intermediates, cofactors and enzymes. Use of labeled glucose at specific positions has led us to conclude that oocytes utilize glucose mainly for glycogen synthesis and to a lesser extent for the pentose-P pathway. Glycolysis, glycogenolysis and gluconeogenesis are not operative in these cells. The subject of compartmentation of glucose utilization has been addressed in this paper. First, we show that microinjection of glucose results in a 30-fold increase of carbon incorporation into glycogen when compared to oocytes incubated at saturating glucose concentrations. On the other hand, carbon incorporation into CO2, remains at about the same levels in both conditions Second, microinjection of NADP+ increases CO2 release and inhibits glycogen synthesis from glucose. Third, co-injection of unlabeled intermediates affects differentially glycogen synthesis and CO2 production from labeled glucose. Finally, microinjection of pure yeast hexokinase stimulates markedly 14CO2 release and inhibits glycogen synthesis. We conclude that two separate pools of glucose-6-P exists in oocytes: one pool is committed to the pathway of glycogen synthesis while a second pool serves as substrate for the operation of the pentose-P pathway.

Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.

The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.

A modified form of mitochondrial hexokinase produced by ATP-induced solubilization.

Mitochondrial hexokinases from several rat tissues were analyzed by DEAE-cellulose chromatography. Solubilization by glucose-6-P or Triton X-100 released hexokinases A and B. Solubilization by ATP resulted in a decrease of hexokinase A and the concomitant appearance of a new fraction of lower net charge (hexokinase Am) which readily reverts to hexokinase A by dialysis or dilution. Treatment of homogeneous or partially purified hexokinase A with ATP did not generate hexokinase Am. Hexokinases Am and A were equally inhibited by an anti-hexokinase immune serum and displayed the same Km values for glucose and ATP. Hexokinase Am may represent a conformer or an oligomer produced during ATP-induced solubilization of hexokinase A from mitochondria.

Glucose utilization in vertebrates as a molecular probe for the study of evolution.

Hexokinase isozymic profiles from the liver of 68 vertebrate species are presented. The comparison of the diverse patterns observed, as well as the kinetic and physicochemical properties of the isozymes, reveals that the hexokinases from mammals are very similar to those from turtles and amphibians. The hexokinases from birds, lizards and snakes on the other hand are similar within themselves and different from the enzymes from mammals and amphibians. Liver pyruvate kinases show about the same behavior. The hexokinase system from vertebrate muscle however is very uniform in all the species studied consisting mainly of hexokinase B.

An in vitro immuno-enzymatic assay of tumor antigens in the mouse with beta-galactosidase.

A method for detection of the primary binding of soluble tumor-associated antigens by antibodies has been developed by using an enzyme immunoassay (EIA). A heteroantiserum was produced by injecting tumor cells from a chemically induced murine sarcoma into rabbits, and antibodies reacting with most normal tissue components were removed by exhaustive in vivo absorption. A soluble preparation of tumor cells, obtained by 3 M KCl extraction, was conjugated to beta-galactosidase from Escherichia coli. The antibody binding was measured by determining the enzyme activity that could be separated by anti-antibody coprecepitation. The reaction follows saturation kinetics, and nonlabeled antigen can be readily quantitated by inhibition. The present method detects determinants common to several MC-induced tumors on the same mouse strain but absent in normal cells and nonrelated tumors in addition to individual tumor-specific transplantation antigens. The sensitivity and simplicity of the new method compare favorably with a binding assay that utilizes radioactive iodine as a label. Thus, EIA becomes a flexible tool for the further characterization and purification of these antigens.

Affinity and specificity of penicillin-antibody interaction determined by an enzyme (E. coli beta-galactosidase) immunoassay.

An enzyme immunoassay (EIA) for a penicillin derivative is described with a sensitivity at least at the nanogram level. The label, E. coli beta-galactosidase is a macromolecule of 540 000 daltons: the size of the enzyme and the ease of linking penicilloyl residues to it make it an interesting model to study the effect of the degree of haptenic substitution (DS) in the tracer on the parameters of EIA. Our results show that the affinity of the binding reaction between antibody and tracer is proportional to the DS but the sensitivity of inhibition is not affected, at least not between 1 and 10 penicilloyl residues per GZ molecule. The theoretical consequences and practical applications of multivalent tracers in EIA are discussed.