Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42°C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42°C in semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42°C.     

Muramic Acid Assay in Sediments

An improved chromatographic assay for muramic acid which is sufficiently sensitive for marine sandy sediments is described; it involves acid hydrolysis, thin-layer chromatography, and gas-liquid chromatography.     

Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Chromium oxalate: A new spin label broadening agent for use with thylakoids

Potassium tris(oxalato)chromate(III) trihydrate (chromium oxalate) has been shown to be a more useful broadening agent than potassium ferricyanide for the spin label 2,2,6,6-tetramethylpiperidine-N-oxy-4-amine (Tempamine) in thylakoid suspensions. Our data show that chromium oxalate is less permeable than ferricyanide, does not inhibit thylakoid electron transport or photophosphorylation, and is not photoreduced by thylakoids.     

The preparation of a sarcolemmal fraction from evacuated muscle slices

A novel procedure is described for preparing a plasma membrane fraction from skeletal muscle (i.e., sarcolemma). The procedure entails evacuating the myoplasm from muscle slices as a preliminary step to homogenization and fractionation. The evacuated muscle slices are composed of a stroma-containing sarcolemma, which is then homogenized and fractionated, utilizing a sequence of differential and discontinuous sucrose density gradient centrifugations. On the basis of electron microscopy, selective enzyme markers and α-bungarotoxin binding in innervated and denervated muscles, the fraction most enriched with sarcolemma is recovered from the 0.5/0.7 M interface of a discontinuous sucrose gradient.     

Further investigation of the antiovulatory effects of the antiprogestational steroid RMI 12,936 in the rat.

Inhibition of ovulation by RMI 12,936 was associated with suppression of the pro-oestrous peak of hypothalamic dopamine. The antiovulatory effect was not reversed by administration of oestrogen, was partly reversed by progesterone and was fully reversed by oestrogen and progesterone. Hypophysial sensitivity to LH-RH, known to be reduced by RMI 12,936, remained low when ovulation was restored by steroid treatment. Administration of oestrogen did not restore the pro-oestrous peak of hypothalamic dopamine and ovulation was not induced following administration of L-DOPA in RMI 12,936-treated animals. It was concluded that RMI 12,936 is antioestrogenic as well as antiprogestational, that oestrogen is necessary for induction of full hypothalamic-hypophysial responsiveness to progesterone and that a hypothalamic dopaminergic pathway may have a non-essential role in the control of ovulation possibly associated with increasing hypophysial sensitivity to LH-RH.     

Regulation of RNA synthesis in yeast III

Summary Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse-long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.     

Reduced color polymorphism in a population of Bunodosoma granulifera

The proportions of carotenoids responsible for column coloration were determined for a population of the sea anemone Bunodosoma granulifera from Grand Cayman using TLC. This population exhibited a very low degree of polymorphism for these proportions, compared to previously sampled populations from Puerto Rico and Curaçao. In addition to the carotenoid pigment system a second system possessing pigments similar to bilins was found for the first time in this genus. This population was also assayed electrophoretically for enzymes encoded by 12 loci. Analysis of these data suggested that the reduced polymorphism for column color may be the result of selection. Although the proportions of carotenoids varied little, the amount of carotenoid per unit of epidermal tissue varied almost four-fold from 0.05 to 0.19 μg/mg dry wt.     

The combining site of the dinitrophenyl-binding immunoglobulin A myeloma protein MOPC 315

Magnetic-resonance techniques are used to refine the model of the combining site of the Fv fragment of the dinitrophenyl-binding mouse myeloma protein MOPC 315 constructed by Padlan, Davies, Pecht, Givol & Wright (1976) (Cold Spring Harbor Symp. Quant. Biol. 41, in the press). Light-absorption studies indicate a dinitrophenyl–tryptophan interaction in the Fv fragment of the type occurring in free solution. The Dnp-aspartate–tryptophan complex is therefore used as a starting point for the n.m.r. (nuclear-magnetic-resonance) analysis of the dinitrophenyl–Fv fragment interaction. Ring-current calculations are used to determine the geometry of the complex. The specificity of complex-formation between dinitrophenyl and tryptophan is confirmed by the lack of ring-current shifts of the dinitrophenyl resonances when tryptophan is replaced by any other aromatic amino acid. Proton n.m.r. difference spectra (at 270MHz), resulting from the addition of a variety of haptens to the Fv fragment, show that the combining site is highly aromatic in nature. Calculations on the basis of ring-current shifts define the geometry of the combining site, which involves a dinitrophenyl ring in van der Waals contact with four aromatic amino acid residues on the protein. The observation of a nuclear Overhauser effect on the H₍₃₎ resonance of the dinitrophenyl ring provides additional constraints on the relative geometry of the H₍₃₎ proton and an aromatic amino acid residue on the Fv fragment. The specificity of the Fv fragment for dinitrophenyl ligands arises from a stacking interaction of the dinitrophenyl ring with tryptophan-93_L, in an `aromatic box' of essentially tryptophan-93_L, phenylalanine-34_H and tyrosine-34_L; asparagine-36_L and tyrosine-34_L also contribute by forming hydrogen bonds with the nitro groups on the dinitrophenyl ring. The n.m.r. results also confirm that the antibody–hapten reaction may be visualized as a single encounter step. An Appendix shows the method of calculation of ring currents for the four aromatic amino acids and their use in calculating structures.     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.