RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : IV. Chemical and Cytological Characterization and Biosynthetic Capabilities of Fragments Obtained by Mild Procedures

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.     

Fc receptors on cultured myeloma and hybridoma cells

The specificity of the Fc gamma receptors on the X63.Ag8.653 nonproducing myeloma cell line has been examined for binding to IgG1-, IgG2a-, and IgG2b-containing antigen-antibody complexes. Complexes containing each of these subclasses bind, and the binding of each is inhibited by the others. Trypsin treatment did not inhibit the binding of any of these subclasses. Furthermore, the monoclonal anti-Fc receptor antibody 2.4G2 inhibits the binding of all three subclasses. These results, together with those of other investigators, suggest that there is a single FcR for IgG1, IgG2a, and IgG2b on mouse B cells which differs in its specificity from the macrophage Fc gamma R. This is confirmed by the fact that a mutant IgG2b myeloma protein which binds to the macrophage Fc gamma 1/gamma 2b receptor does not bind to the Fc gamma R on X63.Ag8.653.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : II. Effects of Fatty Acids and Albumin on Respiration

Fatty acids inhibited the ability of Escherichia coli membrane-envelope fragments to catalyze the oxidation of succinate and nicotinamide adenine dinucleotide, reduced form (NADH) and also inhibited the response of the Clark oxygen electrode to nonenzymatic oxygen uptake. In all cases, unsaturated fatty acids were much more inhibitory than saturated fatty acids. Albumin afforded complete protection from inhibition in the nonenzymatic oxygen-uptake experiments but only partial protection for the respiratory activities of the membrane fragments. The succinoxidase activity was totally inhibited by bovine serum albumin at concentrations that inhibited succinate dehydrogenase only slightly and NADH oxidase not at all. The E. coli acellular preparation showed no dehydrogenase or oxidase activity for any of the fatty acids under a variety of conditions. These conditions included variations of pH, concentration of fatty acids, and the presence or absence of albumin, CoA, ATP, NAD, cysteine, succinate, and carnitine. It thus appears that E. coli grown in the absence of fatty acid can not use fatty acids as an energy source.     

Egg-oviduct interaction initiates reproductive behavior

The experiments reported in this paper provide evidence that eggs must pass through the oviducts in order for receptivity to occur after ovulation in the female frog, Rana pipiens. In one experiment, oviductectomized frogs remained unreceptive after ovulation was induced by administration of exogenous pituitary glands, while sham-operates became receptive within 48 hr. Another experiment had four groups of subjects: ovariectomized females, females with oviducts ligated at the ostial end, females with openings in the uteri that prevented eggs from accumulating there, and sham-operated females. Only the last two groups, groups in which eggs could pass through the oviducts, became receptive. In these experiments, receptivity was indicated by absence of the release call during manual clasping of the trunk. Earlier experiments have shown that eggs have to pass through the oviducts in order to become fertilizable. Thus, the passage of eggs through the oviducts provides a mechanism which links the onset of reproductive behavior to the availability of fertilizable gametes.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

MSH2/MSH6 Complex Promotes Error-Free Repair of AID-Induced dU:G Mispairs as well as Error-Prone Hypermutation of A:T Sites

Mismatch repair of AID-generated dU:G mispairs is critical for class switch recombination (CSR) and somatic hypermutation (SHM) in B cells. The generation of a previously unavailable Msh2^−/−Msh6^−/− mouse has for the first time allowed us to examine the impact of the complete loss of MutSα on lymphomagenesis, CSR and SHM. The onset of T cell lymphomas and the survival of Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice are indistinguishable from Msh2^−/− mice, suggesting that MSH2 plays the critical role in protecting T cells from malignant transformation, presumably because it is essential for the formation of stable MutSα heterodimers that maintain genomic stability. The similar defects on switching in Msh2^−/−, Msh2^−/−Msh6^−/− and Msh2^−/−Msh6^−/−Msh3^−/− mice confirm that MutSα but not MutSβ plays an important role in CSR. Analysis of SHM in Msh2^−/−Msh6^−/− mice not only confirmed the error-prone role of MutSα in the generation of strand biased mutations at A:T bases, but also revealed an error-free role of MutSα when repairing some of the dU:G mispairs generated by AID on both DNA strands. We propose a model for the role of MutSα at the immunoglobulin locus where the local balance of error-free and error-prone repair has an impact in the spectrum of mutations introduced during Phase 2 of SHM.     

Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Many experiments require early determination of offspring''s sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick''s down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.     

Identification of cis-elements conferring high levels of gene expression in non-green plastids

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research.