A comparison of the effects of NN′-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on energy-linked reactions in mitochondria and submitochondrial particles

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.     

Genetic Basis of Colicin E Susceptibility in Escherichia coli I. Isolation and Properties of Refractory Mutants and the Preliminary Mapping of Their Mutations

Colicin E-resistant mutants were isolated in Escherichia coli K-12 which, although still apparently possessing the E receptor and adsorbing colicin, were nevertheless insensitive (refractory) to its effect. Eight phenotypic groups were obtained, but some mutants from three of these groups were all shown to map at gal, whereas a second refractory locus, giving resistance to E1 alone, mapped close to thy. It is suggested that the successful fixation of any of the three distinct colicins of group E may involve a dual role for the cell surface "receptor," the first for the binding of the protein and the second for the correct orientation of the bound molecule relative to the cytoplasmic membrane. The majority of the refractory mutants isolated may derive from changes in components concerned with the second of these receptor functions. Two groups of mutants, however, refractory to only E1 or E2, probably reflect changes in the intracellular transmission systems which specifically mediate the effects of these two colicins, the changes not allowing transmission through the cytoplasmic membrane to the respective targets of the colicins. The E1 adsorption site was shown to be distinct from that for E2 and E3, indicating an early separation of the colicin E transmission systems.     

Detection of an unbalanced translocation (4;14) in a mildly retarded father and son by flow cytometry

Summary A child with impaired intelligence, minor dysmorphisms, obesity and genital hypoplasia was found to have an apparently balanced translocation, 46,XY,t(4;14)(q12;q13), following cytogenetic analysis. The same rearrangement was also detected in the child's father, who had similar phenotypic abnormalities to his son. Detailed study of flow karyotypes produced from lymphoblastoid cell lines established that in both patients the translocation was in fact unbalanced with approximately 11 million base pairs of DNA (corresponding to about 6.0% of chromosome 4 or 11.0% of chromosome 14) being lost.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Ovarian mesothelial and extramesothelial cells in interactive culture

Summary The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14±4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93±0.40 × 10⁶ cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%–300 U/ml) under periodical resuspension and gentle scraping of SC (1.40±0.25 × 10⁶/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4μg/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2′-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.     

Retrotransfer kinetics of R300B by pQKH6, a conjugative plasmid from river epilithon

Abstract The kinetics of conjugation, retrotransfer and mobilisation were studied at 5–15 min intervals between strains of Pseudomonas putida using plasmid pQKH6, isolated from river epilithon, and R300B. Transconjugants from the direct conjugation of pQKH6 and mobilisation of R300B by pQKH6 appeared rapidly, reaching maximum densities within 30–60 min of the start of both filter and liquid mating experiments. However, retrotransconjugants only appeared after a delay. This delay was short (approx. 45–60 min) in filter mating and much longer (2–5 h) for liquid mating experiments. Attempts at predicting the time course of retrotransconjugant development from (1) numbers of transconjugants from the conjugation and mobilisation experiments and (ii) mathematical models based on a mass action approach, both failed to reproduce the observed delay. It was concluded that retrotransfer did not proceed by either a one-step mechanism occuring early in conjugation or two separate conjugation and mobilisation steps. The clear demonstration of a delay in retrotransconjugant formation implies that a new mechanism must be sought. The likely importance of retrotransfer in the environment is discussed.     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.