Simultaneous determination of leu-enkephalin localization and [3H]γ-aminobutyric acid uptake in rat striatal cell cultures

The purpose of this study was to investigate the coexistence of gamma-aminobutyric acid (GABA) and leu-enkephalin in single neurons from the corpus striatum. Monolayer cell cultures, started from newborn rat corpus striatum, were grown in serum-free medium and examined using GABA autoradiography and leu-enkephalin immunocytochemistry in a double-label protocol. Examples of cells were found which were positive for one or the other neurotransmitter or for neither transmitter, but not for both. Furthermore, cells which appear similar by morphological criteria alone differed in transmitter specificity. We conclude that the two transmitters tested are not localized within single cells and that morphology alone is inadequate to identify functional cell classes in this area.     

THE LYMPHOCYTES OF CHRONIC LYMPHOCYTIC LEUKEMIA: THEIR PROLIFERATION AND CELL CYCLE KINETICS

Lymphocytes obtained from CLL patients exhibited a delayed and reduced response to PHA when cultured in diffusion chambers. DNA synthesis (8–10 hr) and general time (15–19 hr) of the late-developing CLL blasts were consistent with normal values ( T _s: 8–10 hr; T _c: 14–17 hr). However, the G₂ period of CLL blasts seemed more variable, and their mitotic index during the response at 5–6 days was 30–50% of the values determined for normal blasts during their peak response at 2–3 days.     

The number of Na:K pump sites on red blood cells from HK and LK lambs

Lambs of known genotype with respect to the locus determining cation composition of red cells were obtained by selective matings. Numbers of K⁺ pump sites per cell were determined on HK and LK lambs 10–20 days postnatal by simultaneously determining [³H]ouabain binding and inhibition of active K⁺ transport. Red cells from HK lambs were indistinguishable from adult HK cells with regard to the K⁺ pump flux and number of pump sites. Cells from genetically LK lambs had pump fluxes and numbers of pump sites intermediate between those from adult HK and LK sheep. The results suggest that the change in cation composition and in the K⁺ pump during the first 60 days in genetically LK lambs can be correlated with a reduced number of K⁺ pump sites.     

THE EFFECTS OF IRON DEFICIENCY ON THE HEPATOCYTE: A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY

Effects of iron deficiency on the hepatocyte were studied quantitatively in the rat by combining ultrastructural and biochemical techniques. After 3–8 wk of an iron-deficient diet, the percentage of cytoplasm occupied by mitochondria increased progressively compared with complete diet values. The increment resulted primarily from an enlargement of individual mitochondria rather than from an increased mitochondrial number. Many mitochondria were completely divided by a double membrane, often at a point of constriction. After 2 days of iron administration, mitochondria were of heterogeneous size, shape, and electron opacity. After 5 days, essentially all mitochondria had become normal in configuration. The rate of reversal of the morphological abnormality was more rapid than would be anticipated if it coincided with known rates of renewal of mitochondrial DNA or protein. The concentrations of mitochondrial cytochromes were more rapidly depressed as a result of iron deprivation than those of microsomal cytochromes. Cytochromes c and a were decreased after 3 and 8 wk of exposure to the deficient regimen. Cytochrome P 450 was not decreased after a 3 wk exposure to the deficient diet and responded normally to phenobarbital treatment with a fourfold increase in total hepatic content; its concentration was depressed only after 8 wk of exposure to the deficient diet. There was no reduction in cytochrome b₅ concentration.     

Localization of Parental Deoxyribonucleic Acid from Superinfecting T4 Bacteriophage in Escherichia coli

High-resolution autoradiography has been employed to localize the nonsolubilized but genetically excluded deoxyribonucleic acid (DNA) of T4 bacteriophage superinfecting endonuclease I-deficient Escherichia coli. This DNA was found to be associated with the cell envelope (this term is used here to include all cellular components peripheral to and including the cytoplasmic membrane); in contrast, T4 DNA in primary infected cells, like host DNA in uninfected E. coli, was found to be near the cell center. The envelope-associated DNA from super-infecting phage was not located on the outermost surface of the cell since it was insensitive to deoxyribonuclease added to the medium. These results suggest that DNA from superinfecting T-even phage is trapped within the cell envelope.     

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrB1), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip(+) episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB(+) is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

Origin of the genetic code: A physical-chemical model of primitive codon assignments

Selective compartmentalization of amino acids and nucleotides according to their polarities is proposed as a physical-chemical model for the origin of the genetic code. Assumptions made in this hypothesis are: (1) an oil-slick covered the surface of the primitive ocean, constituents of which formed association colloids or micelles at the water-oil-air interfaces; (2) depending on the polarity of the media, these aggregates possessed hydrophilic and hydrophobic interiors where selective uptake of amino acids and nucleic acid constituents could take place; and 93) condensation and polymerization in the micellar phase were enhanced. According to the chromatographically observed polarities, for example, lysine and uridylate fall into the hydrophilic compartment, and phenylalanine and adenylate are enriched in the hydrophobic environment. These components could eventually be condensed to form a charged adaptor loop with an anticodon which is complementary to the presently valid codon. Only two groups of amino acids, hydrophilic and hydrophobic, were recognized by the primitive translation mechanism. Implications of this hypothesis for the further development of the genetic code is discussed. The catalytic power of micelles have been substantiated by successful synthesis of nucleotides under relatively mild conditions using thiophosphates as high energy phosphates.