On the mechanism of erythroid cell sensitivity to chloramphenicol: Studies on mitochondria isolated from erythroid and myeloid tumors

Erythroleukemia mitochondria (E. Mito) and chloroma mitochondria (C. Mito) were isolated from tumors grown in their hosts, DBA/2J mice and Long-Evans rats, respectively. Oxypolarographic tests showed respiratory control and ADP/O ratios typical for well-coupled mitochondria. Therapeutic concentration of chloramphenicol (CAP) had no effect on the energy transfer of those mitochondria. l-[¹⁴C]leucine incorporation into protein was comparable in both types of mitochondria. Although the incorporation at 15 min appeared higher in C. Mito, at 60 min it became similar to that in E. Mito. When CAP was used at the therapeutic concentration of 20 μg/ml about 80% inhibition was observed in both mitochondria. The exogenous amino acid mixture added to the medium was an important determinant in both the rate of leucine incorporation as well as the sensitivity to CAP. Thus, if no amino acids were added the incorporation was reduced to 18–25%. Under these conditions, however E. Mito were significantly more sensitive to the same concentration of CAP than C. Mito. The results suggest that mitochondrial amino acid pool may be involved in the greater sensitivity of erythroid precursors to CAP.     

The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

The mixed lymphocyte response of senescent mice: sensitivity to alloantigen and cell replication time.

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2^d, Mls^b) and DBA (H-2^d, Mls^a) spleen cells served as stimulating cells differing from C57B1/6J (H-2^b, Mls^b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G₂, G₁, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.     

Analysis of glial cell differentiation in peripheral nervous tissue: II. Neurons promote S100 synthesis by purified glial precursor cell populations

Isolated sensory neurons in vitro do not contain or synthesize S100, whereas glial cell precursor populations do. These precursor cells, when isolated from other cell types, produce low levels of S100 but never undergo the developmental transition to produce high levels of S100. When glial cell precursors are combined with isolated, live or paraformaldehyde-fixed sensory neurons, the precursor cells do undergo the second transition, and accumulate high levels of S100. Peroxidase-anti-peroxidase immunohistochemical staining for S100 confirms previous conclusions (B. Holton and J. A. Weston, 1982, Develop. Biol.89, 64–71) that only those glial cells which are closely apposed to neurons contain augmented levels of S100. This stimulation appears to be specific to neuronal/glial interactions since live or fixed fibroblasts, when cocultured with glial precursor cells, do not promote accumulation of S100 by the glial cells.     

Effect of procaine on development of ‘ridges’ and ‘domes’ in primary cell cultures from mammary glands of untreated virgin mice: The role of cell density in the occurrence of ‘domes’

Primary cell cultures from mammary glands of virgin mice that were not pretreated with hormones were subjected to: (1) procaine; (2) insulin+ prolactin +hydrocortisone; (3) a combination of (1) and (2). Procaine caused a ‘ridge’ effect similar to that of the hormones. The combination of procaine with the hormones caused a still stronger ‘ridge’ effect as well as the formation of ‘domes’. The formation of ‘domes’ is suggested to be dependent on cell density.     

The use of hormone-supplemented serum-free media in primary cultures.

Recent advances in tissue culture and endocrinology have made possible the growth of established cell lines in hormone-supplemented serum-free media. The hormone requirements differ for different cell types but are similar or identical for the same cell types. The hormone supplements derived for four different cell types, a melanoma, GH3 pituitary tumor, and testicular cell lines TM3 and TM4 are used in preparing primary cultures for organs to detect melanoma metastasis, and grow normal pituitary and normal Leydig and Sertoli cells, respectively. This hormone supplementation and the concomitant elimination or reduction of the serum requirement is shown to have several advantages in the preparation of primary cultures including prolonged viability and function, partial or total selection of the desired cell type and inhibition of fibroblast overgrowth.It is felt that such culture systems will significantly expand the range of problems which can be approached using primary culture systems.     

Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: separation from hemoglobin by subunit-exchange chromatography.

Myoglobin is extracted from muscle and separated from blood hemoglobin by subunit-exchange chromatography on a column of Sepharose 4B to which hemoglobin α-β subunits are linked covalently. Hemoglobin is retained on the column. Myoglobin in the effluent is determined spectrophotometrically as ferrous myoglobin or as carbon monoxide ferrous myoglobin. The method is applicable to cardiac, smooth, or skeletal muscle from mammals, reptiles, birds, and teleost fish, but failed with the one amphibian and the one shark tested.     

M-protein in chicken cardiac muscle.

Chicken cardiac muscle myofibrils lack a visible M-line. Antibodies against chicken breast muscle M-protein, an M-line component with M_r = 165 000, were used to demonstrate the presence of a similar protein in chicken heart muscle. The immunoreplica technique showed the heart protein to have about the same molecular weight as the breast muscle M-protein on polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS). Positive staining within the H-zone was observed when the indirect immunofluorescence technique was used to localize the M-protein in isolated heart myofibrils. This result was confirmed by electron microscopic investigations on longitudinal sections of antibody-incubated heart muscle fiber bundles showing the antibody against M-protein to be bound within a region corresponding to the M-line region of breast muscle myofibrils.     

Size and sequence homology of masked maternal and embryonic histone messenger RNAs.

The messenger RNAs for five classes of histone proteins are shown by competitive RNA-DNA hybridization to be stored in the unfertilized egg of the sea urchin, Lytechinus pictus. The masked mRNAs for f2b, f2a2, f3 and f2al histones migrate in polyacrylamide slab gels with the same mobility as the histone mRNAs that are synthesized after fertilization and are found engaged in protein synthesis on polysomes. The masked maternal and embryonic mRNAs for histone f2a1 are identical in mobility when analyzed in a gel system capable of resolving differences estimated as small as 4–5 nucleotides in length. We conclude that these histone mRNAs synthesized during oogenesis and inactive prior to fertilization are not activated during embryogeny by alteration in their molecular size.     

5-Bromodeoxyuridine inhibition of differentiation. Kinetics of inhibition and reversal in myoblasts

This paper describes experiments on the kinetics of inhibition of muscle differentiation in vitro in the presence of 5-bromodeoxyuridine (BrdUrd) and the recovery phenomena that occur when such inhibited cells are permitted growth in normal medium. The studies consist of a quantitation of cell fusion in the presence of the analog and during recovery in its absence coupled with simultaneous studies on changes in buoyant density of cellular DNA. We find that if myoblasts are exposed to BrdUrd during the last doubling before cell fusion would normally occur, most cells do not differentiate, but as many as 18% of the cells can fuse in spite of the incorporation of BrdUrd into their nuclei. These nuclei contain approximately the amount of BrdUrd expected for a full round of DNA synthesis. Studies on the rate of recovery of inhibition of cell fusion following one generation in BrdUrd reveal that after one doubling of inhibited cells in the presence of normal medium. fusion reaches about 50% of the control value; after two doublings it reaches 75% of control value; and after 2.5 doublings of reversal, recovery is essentially complete. We find that both the degree of inhibition after approximately one round of BrdUrd incorporation and the rate of cell differentiation after two generations of reversal are consistent with a model which assumes that BrdUrd “sensitivity” resides on single pair of chromosomes and that inhibition occurs in a dominant fashion if approximately 30% or more of the thymidine is replaced by BrdUrd in the readout strand of either chromosome.     

Restriction endonuclease analysis and nucleotide composition of satellite III DNA from Drosophila virilis

Satellite III DNA from $\text{Drosophila virilis}$ is composed of a tandemly repeated heptanucleotide containing the sequence which is normally cleaved by EcoRI¹ restriction endonuclease activity. However, we observed only a very limited amount of cleavage of satellite III DNA by this activity. Although methyldeoxyadenosine is known to block EcoRI¹ activity, no modified nucleotides were detected in satellite III DNA subjected to nucleotide composition analysis. Since the proportion of each nucleotide present was consistent with the heptanucleotide sequence, we speculate that the resistance of satellite III DNA to EcoRI¹ cleavage may result from the highly repetitive nature of this DNA.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Commitment of hydra interstitial cells to nerve cell differentiation occurs by late S-phase

Nerve cells in hydra differentiate from the interstitial cell, a multipotent stem cell. Decapitation elicits a sharp increase in the fraction of the interstitial cells committed to nerve cell differentiation in the tissue which forms the new head. To investigate when during the cell cycle nerve cell commitment can be stimulated, hydra were pulse-labeled with [³H]thymidine at times from 18 hr before to 15 hr following decapitation; the resulting cohorts of labeled interstitial cells were in the various phases of the cell cycle at the time of decapitation. Increased commitment to nerve cell differentiation within a single cell cycle (≈24 hr) was observed in those cohorts which were at least 6 hr before the end of S-phase (12 hr) at the time of decapitation. The lag time required for decapitation to produce an effective stimulus for nerve cell differentiation was measured by transplanting the stem cells from the regenerating tissue to a neutral environment. Following decapitation, 3 to 6 hr were required for increased nerve cell commitment to be stable to such transplantation. These results suggest that interstitial cells must be stimulated by late S-phase to become committed to undergo nerve cell differentiation following the subsequent mitosis. However, when head regeneration was reversed by grafting a new head onto the regenerating surface, nerve cell differentiation by such committed stem cells was greatly reduced. This indicates that an appropriate tissue environment is required for committed interstitial cells to complete the nerve cell differentiation pathway.     

Zinc accumulation and metabolism in primary cultures of adult rat liver cells. Regulation by glucocorticoids

Adult rat liver parenchymal cells were isolated by the collagenase perfusion technique and cultured as a monolayer for up to 20 h. The quantity of zinc accumulated from the extracellular environment was significantly increased by adding physiological concentrations of certain glucocorticoids to the medium. The degree of stimulation was directly related to glucocorticosteroids potency. Sex steroids, certain peptide hormones and prostaglandins E₂ and F_2α did not influence zinc accumulation.Control cells exhibited a decline of zinc accumulation after 4 h in culture although uptake processes were still operative. When dexamethasone, the most potent glucocorticoid used, was present in the medium the cells accumulated zinc at a linear rate greater than that seen in control cells, for at least 20 h. The dexamethasone-induced stimulation of zinc accumulation was relatively specific since ⁴⁵Ca, ¹⁴C-labelled amino acids and [³⁵S]cystine accumulation was not influenced by the hormone. A lag of 4 h was observed before an effect of dexamethasone on zinc accumulation could be detected. Moreover, the hormone-stimulated phase of accumulation was blocked when the cells were simultaneously incubated with either actinomycin D or cycloheximide. The additional complement of zinc accumulated by the dexamethasone-treated cells was localized in the cytosol fraction. Gel filtration and ion-exchange chromatorgraphy confirmed that this additional cytosol zinc was bound to metallothionein. [³⁵S]Cystine was incorporated into metallothionein in hormone-treated cells indicating that the protein was synthesized de novo during periods of enhanced zinc accumulation.     

Properties of rat 6-N-trimethyl-l-lysine hydroxylases: Similarities among the kidney,liver, heart,and skeletal muscle activities

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me₃)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me₃)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K_m values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me₃) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me₃) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me₃). The most effective inhibitors found, competitive with lys(Me₃), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd²⁺, Zn²⁺) but not by those of “hard metals” (e.g., Ca²⁺, Mg²⁺). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me₃) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.     

Antigen-inhibited B-lymphocyte differentiation. X. Sedimentation velocity characterization of antigen-binding cells and antibody-forming-cell progenitors in the in vitro microculture immune response of unprimed CBA mice to NIP--POL antigen.

The B lymphocytes serving as antibody-forming-cell (AFC) progenitors have been investigated using two different types of separation methods. Sedimentation velocity fractionation was used to separate subsets of B lymphocytes differing primarily in size. Fractionation on a 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP)-gelatin matrix was used to separate NIP-binding cells, a population highly enriched for cells with surface Ig receptors specific for the NIP hapten. Assessment of the functional capacity of the separated B cells was by culture at limiting dilution in the presence of thymus “filler” cells, using the T-independent antigen NIP-POL (polymerized bacterial flagellin) to induce antibody formation. Splenic AFC-progenitors from both adult conventional and neonatal germ-free mice were a physically heterogeneous population, with activity in small, medium, and large lymphocytes. The cells enriched by NIP-gelatin binding, whether isolated and counted directly or isolated and assayed as AFC-progenitors, were no less heterogeneous. These NIP-binding cells resembled in sedimentation characteristics the overall B-cell and overall NIP-specific AFC-progenitor populations, except for some relative enrichment of medium-sized cells (S value, 5.5 mm/hr). The small (S value, 3.4 mm/hr) B-cell region of adult mouse spleen contained both NIP-binding cells and cells responsive as AFC-progenitors in the microculture assay. This contrasts with the results of the in vivo adoptive immune assay, where the smaller B-cell region is unresponsive in unprimed adult animals.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Colorimetric determination of catechol siderophores in microbial cultures

A highly sensitive spectrophotometric method for the selective detection of catechol compounds such as catechol siderophores (e.g., enterobactin) is described. The basis of the method involves the ability of the vicinal aromatic hydroxyl groups under acidic conditions to bring about a reduction of Fe3+ (from ferric ammonium citrate) to Fe2+. Detection of Fe2+ in the presence of Fe3+ is made with 1,10-phenanthroline under previously established conditions. The assay mixture is heated at 60 degrees C for 1 h to accelerate the development of color which is subsequently measured at 510 nm. The Beer-Lambert law is obeyed over the range of 0.16 to 60 microM 2,3-dihydroxybenzoic acid. Compared to the Arnow nitration method, the assay is more responsive, is approximately seven times more sensitive, and is effective with catechols substituted at positions 3 and 4. The method gives positive results with catechols such as DL-DOPA, L-dopamine, (+/-)-epinephrine, and DL-norepinephrine. Very rapid color development is obtained with ascorbic acid and p-diols, while m-diols are poorly detected. Low degrees of reactivity are shown by hydroxylamino and hydroxamate compounds. Phenolic, sulfydryl, indolyl, and quinonyl derivatives do not interfere with the reaction. The method has been adapted to determine catechol compounds in the culture medium of bacterial cells grown at different iron concentrations.