In vitro labeling of peroxisomal cholesterol with radioactive precursors

Peroxisomes isolated from rat liver were incubated with [³H]squalene and [³H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern ofin vitro incorporation of [³H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterolin vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.     

Inactivation of bacteria by decay of incorporated radioactive phosphorus

Cultures of Escherichia coli will not grow in media containing very high specific activities of radiophosphorus P³², the inhibition of growth being due to the decay of assimilated P³² atoms. Experiments with a differentially labeled thymineless strain of E. coli show that the P³² disintegrations which occur in the bacterial deoxyribonucleic acid, i.e. in the nucleus, are mainly responsible for the inactivation of the cell. The kinetics with which radioactive bacterial populations are inactivated indicate that the function of several nuclei per bacterial cell must be eliminated by P³² decay before the ability to generate a colony is lost. The efficiency with which each P³² disintegration inactivates the nucleus in which it has occurred is calculated to be 0.02 (at –196°), i.e., similar in magnitude to the killing efficiency of P³² decay in bacteriophages. P³² decay and thymine starvation cooperate in bringing about the death of individuals of the thymineless strain, from which observation it is inferred that "thymineless death" is likewise a nuclear inactivation. The descendants of a non-radioactive bacterial culture grown for several generations in the presence of P³² and the descendants of a radioactive culture grown in the absence of P³² are inactivated by P³² decay in a manner which indicates that the phosphorus atoms of bacterial nuclei are dispersed among the progeny nuclei in their line of descendance.     

Inactivation of bacteriophages by decay of incorporated radioactive phosphorus

The inactivation of the phages T1, T2, T3, T5, T7, and lambda by decay of incorporated P(32) has been studied. It was found that these phages fall into two classes of sensitivity to P(32) decay: at the same specific activity of P(32) in their deoxyribonucleic acid (DNA), T2 and T5 are inactivated three times as rapidly as T1, T3, T7, and lambda. Since the strains of the first class were found to contain about three times as much total phosphorus per phage particle as those of the second) it appears that the fraction of all P(32) disintegrations which are lethal is very nearly the same in all the strains. This fraction alpha depends on the temperature at which decay is allowed to proceed, being 0.05 at -196 degrees C., 0.1 at +4 degrees C., and 0.3 at 65 degrees C. Decay of P(32) taking place only after the penetration of the DNA of a radioactive phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P(32) can be cross-reactivated; i.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P(32) disintegrations are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule.     

In vivo and in vitro turnover in dental microwear

Given the potential usefulness of dental microwear analyses in interpretations of archaeological and paleontological material, it is surprising how little we know about changes in individual microwear features through time. The purpose of this study was to document the turnover in primate dental microwear through in vivo dental studies of monkeys raised on different diets, and through in vitro studies of the abrasive effects of monkey chow biscuits on isolated monkey teeth. As in previous studies, epoxy replicas were prepared from dental impressions and examined under a scanning electron microscope. Results indicate that, under certain conditions, the turnover in primate dental microwear can be on the order of days, hours, or even minutes. Individual microscopic wear features can be obliterated within 24 hours on the molars of laboratory monkeys, and monkey chow biscuits can easily scratch the enamel of isolated monkey teeth. Monkeys raised on a hard diet showed more rapid turnover in dental microwear than monkeys raised on a soft diet. However, paired-sample tests revealed that, for all animals, the molar shearing facets were being abraded at a significantly slower rate than molar crushing/grinding facets. In light of these results, investigators should make every effort to use large samples in interspecific comparisons of dental microwear involving species with variable diets. Another implication of these results is that changes in dental microwear might be useful indicators of changes in oral behavior over relatively short periods of time.     

In vitro biosynthesis of radioactive estradiol-17 beta, 17-glucosiduronate by rhesus monkey liver.

A method for the preparation of radioactive estradiol-17 beta, 17-glucosiduronate by incubating 3H-estradiol with rhesus monkey liver microsomal preparation in the presence of uridine diphosphoglucuronic acid is described. Small but significant amounts of the conjugate were also obtained from the 150,00 pellet and cytosol fractions. The addition of NADPH to the incubation media increased the yield of radioactive-estradiol-17 beta, 17-glucosiduronate perhaps by preserving the integrity of the C-17-hydroxyl group. As expected, the effect of the reduced nucleotide was more pronounced in the fractions other than the microsome. The biosynthesized conjugate was isolated and purified by multiple column chromatography and the structure was confirmed by derivative formation, enzyme hydrolysis and crystallization of the aglycone.     

In vitro formation of thyroid hormones from 3,5-diiodothyronine by supernatant of submaxillary gland

Cell-free extracts of submaxillary glands from rat and goat iodinate exogenously added 3,5-diiodothyronine to form 3,5,3’-triidothyronine and thyroxine. This capacity was elevated after either thyroidectomy or exposure of rats to cold (3 ±1 ‡C) for 5 h. However, there was no further increase of iodination of 3,5-diiodothyronine when the thyroidectomized rats were exposed to cold stress. The submaxillary extracts have the ability to incorporate radioactive iodide into 3,5-diiodothyronine, 3,5,3’-triiodothyronine and thyroxine. The presence of 3,5-diiodothyronine was also detected in the soluble supernatant of submaxillary extract.