Incorporation of acetate-1-14C into lipids by the perfused liver of normal, X-irradiated, or partially hepatectomized rats

In order to study lipid metabolism in the liver without interference due to transport from and to the liver the isolated livers of normal, X-irradiated, and partially hepatectomized rats were perfused with acetate-1-(14)C and the distribution of radioactivity in total lipids, total fatty acids, individual lipids, and fatty acids of individual lipids was determined. In X-irradiated animals, an increased incorporation of acetate into many lipids, particularly into cholesterol, was observed. Lipids in the liver of the partially hepatectomized rats exhibited a marked increase in triglyceride content together with a decreased rate of incorporation into all but the phospholipid fractions. It is concluded that the increase usually observed in lipid pontent of the regenerating liver is due to the changes in transcort rather than to changes in synthesis. The changes observed in irradiated liver could be the result of alterations in the metabolism of precursors common to most lipids.     

Cholinergic fiber growth in co-cultures of CNS tissue.

In co-cultures prepared from the septum and the hippocampus, cholinergic fibers originating in the septal slices grew into the neighboring hippocampal tissue and established functional cholinergic connections with pyramidal cells. To get further insight into the mechanisms governing cholinergic fiber growth, we have added TTX to the growth medium (2 x 10(-7) M) to block propagated electrical activity. Under these conditions, considerably fewer cholinergic cells appeared to survive. A few cholinergic fibers still invaded hippocampal target tissue, but their number was markedly reduced compared with control cultures. Simultaneous application of NGF together with TTX, however, not only increased enzyme levels and enhanced survival of cholinergic neurons, but also led to hippocampal ingrowth in virtually all septo-hippocampal co-cultures. These data, therefore, suggest, that in the absence of spiking activity, cholinergic fibers are capable of growing into a co-cultured target tissue. To test the specificity of growth of septal cholinergic fibers, we have co-cultured septal slices with slices of various brain areas which in situ lack a major cholinergic innervation, in particular the cerebellum. In the vast majority of such co-cultures, cholinergic fibers remained restricted within the septal slices, without innervating cerebellar tissue. This failure might in part be related to the lack of trophic factors released by the target tissue. We have, therefore, grown septo-cerebellar cultures in the presence and absence of NGF. Following application of 100 ng/ml NGF during the entire growth of the cultures, numerous AChE-positive fibers originating in the septal slices invaded the co-cultured cerebellar slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.     

Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in the Xenopus oocyte

The Xenopus oocyte is widely used to study the various aspects of eukaryotic cell structure and function. It is also being used increasingly in expression cloning of cDNAs encoding proteins for which there are no structural data. One of the drawbacks of the Xenopus oocyte system is that individual oocytes taken at the same time from the same frog vary considerably in the amount of protein synthesized from the same amount of injected mRNA. In this report we describe the preparation and use of the mRNA for a secreted mutant form of human placental alkaline phosphatase as an internal, coinjected standard to monitor translation in oocytes. Secreted alkaline phosphatase can be readily determined in the medium of cultured oocytes by using a standard colorimetric assay. The amounts of alkaline phosphatase secreted into the medium were shown to parallel the level of expression of two membrane proteins. This permits rapid identification and selection of those oocytes that efficiently express injected mRNAs. The procedure yields more precise data and results in an enormous saving of time and expense, especially in investigations that involve complex measurements on individual oocytes.     

Colloidal aggregates of insoluble inclusions in human goiters.

To shed some light on the physicochemical properties of the thyroid follicular colloid, we have screened retrospectively the autoradiographs of 60 human nodular goiters labeled 17 h preoperatively with 100 microCi 125I for evidence of colloid compartmentalization. In 87% (52/60) of all goiters examined, sporadic or multiple colloidal inclusions ('colloid stones') not mixing with newly labeled Tg were detected. The detailed analysis of 17 goiters revealed a mean incidence of 0.09+/-0.11 'colloid stones' of variable size per follicle ranging from 0.02+/-0.01 (10) to 0.43+/-0.09 (5) (mean values +/- S.D., number of sections examined in brackets). In this study we did not find a clear-cut association of incidence of 'colloid stones' with sex, age or nosologic group (hyperthyroid, preclinically hyperthyroid, euthyroid). The existence of different colloidal compartments as demonstrated in this and other studies is of considerable importance for thyroid function, interpretation of iodine kinetics, and studies on the role of iodine on growth and function of the thyrocytes. Different thyroidal iodine compartments could well be of functional relevance, for example in the adaptation of thyroid hormone secretion to antithyroid drugs or in severe and prolonged iodine deficiency, when very slow compartments become an important source of minimal quantities of iodine and thyroid hormone. 'Colloid stones', for example, may well explain the repeatedly observed, surprisingly large total iodine store in human endemic goiters, even in the presence of severe iodine deficiency. It is evident that the existence of multiple iodine compartments and, in particular, of particulate slow-turnover pools complicates the interpretation of total glandular iodine measurements with modern techniques such as X-ray fluorescence and positron emission tomography.     

Measurement of arachidonic acid in the plasma by gas-liquid chromatography-flame ionization using dihomo-gamma-linolenic acid as an internal standard.

A gas-liquid chromatography-flame ionization method is described for measuring arachidonic acid in plasma using dihomo-gamma-linolenic acid as an internal standard. We found this technique to -e reproducible, and quicker and superior to previously described techniques because of the similar physico-chemical properties of the unsaturated fatty acid internal standard and arachidonic acid. In addition, we observed that the use of the saturated fatty acid, n-tricosanoic acid, was unsatisfactory as an internal standard because of its poor extractability from plasma as compared to arachidonic acid.     

Changes in the nucleotide metabolism of Ehrlich ascites tumour cells during their growth in vivo

During the transition of Ehrlich mouse ascites tumour cells from the proliferating into the resting phase of growth a tremendous loss of purine and pyrimidine compounds was quantitated by ion-pair reversed-phase high performance liquid chromatography. This change is accompanied by a distinct decline in the incorporation rates of adenine, hypoxanthine, and adenosine. Inorganic phosphate stimulates the low rate of hypoxanthine incorporation of cells in the plateau phase, but lacks any effect on the high rate during proliferation. The mitochondria suffer structural deteriorations and decrease in their cellular content in the course of the plateau phase; however, other changes were not seen by morphometric analysis. The interrelations between nucleotide metabolism, mitochondrial content and the rates of formation and consumption of ATP are discussed.