A new method for studying the genetic control of specific mitochondrial proteins in Paramecium aurelia

Summary Mitochondria from one syngen (or sub-species) of Paramecium aurelia have been introducted into a different syngen by preparing erythromycin-resistant mitochondria from syngen 1 and micro-injecting them into erythromycin-sensitive syngen 7 cells. The recipient sensitive cells were then placed in erythromycin to inhibit the replication of the sensitive mitochondria. Such selected clones contain a syngen 7 nucleus but a mitochondrial genome which is derived from syngen 1 erythromycin-resistant mitochondria.Using this method it has been shown that the mitochondrial enzyme fumarase is not coded by the mitochondrial genome, and by implication, is coded by the nuclear genome. The use of this technique as a method for determining if specific mitochondrial proteins are controlled by nuclear or mitochondrial genes is discussed.     

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D

1. The anaerobic coproporphyrinogenase activity in an extract of Rhodopseudomonas spheroides is inhibited by 1,10-phenanthroline, alphaalpha'-bipyridyl, flavins, 2,4-dinitrophenol and 1,4-naphthaquinone. These compounds have no effect on the aerobic coproporphyrinogenase activity. 2. On removal of small-molecular-weight material from a crude extract, the anaerobic system becomes very unstable; it can be stabilized by adding succinate. Now nicotinamide nucleotides, in addition to Mg(2+), ATP and methionine, are required for protoporphyrin to be formed. 3. A mechanism for the anaerobic reaction is proposed, based on the cofactor requirements and the effect of inhibitors. 4. The enzyme responsible for aerobic activity has been partially purified and some of its properties are reported. 5. A crude extract of Chromatium strain D also exhibits coproporphyrinogenase activity under anaerobic conditions in the presence of S-adenosylmethionine or ATP plus methionine. The requirement for other cofactors is variable.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

PINK1 drives production of mtDNA-containing extracellular vesicles to promote invasiveness

The cystine-glutamate antiporter, xCT, supports a glutathione synthesis program enabling cancer cells to cope with metabolically stressful microenvironments. Up-regulated xCT, in combination with glutaminolysis, leads to increased extracellular glutamate, which promotes invasive behavior by activating metabotropic glutamate receptor 3 (mGluR3). Here we show that activation of mGluR3 in breast cancer cells activates Rab27-dependent release of extracellular vesicles (EVs), which can transfer invasive characteristics to “recipient” tumor cells. These EVs contain mitochondrial DNA (mtDNA), which is packaged via a PINK1-dependent mechanism. We highlight mtDNA as a key EV cargo necessary and sufficient for intercellular transfer of invasive behavior by activating Toll-like receptor 9 in recipient cells, and this involves increased endosomal trafficking of pro-invasive receptors. We propose that an EV-mediated mechanism, through which altered cellular metabolism in one cell influences endosomal trafficking in other cells, is key to generation and dissemination of pro-invasive microenvironments during mammary carcinoma progression.     

Growth and carbon stock change in eucalypt woodlands in northeast Australia: ecological and greenhouse sink implications

Data from 57 permanent monitoring sites are used to document the growth in woody vegetation and estimate the carbon sink in 27 M ha of eucalypt woodlands (savannas), contained within c. 60 M ha of grazed woodlands in Queensland (northeast Australia). The study sites are shown to be representative of the environment and structure of the eucalypt woodlands in the defined study area. Mean basal area increment for all live woody plants in 30 long‐term sites, with an average initial basal area of 11.86 ± 1.38 (SE) m² ha^?1, was 1.06 m² ha^?1 over a mean 14 years timeframe. The majority of the measurement period, commencing between 1982 and 1988, was characterized by below‐average rainfall. The increase in live tree basal area was due primarily to growth of existing trees (3.12 m² ha^?1) rather than establishment of new plants (0.25 m² ha^?1) and was partly offset by death (2.31 m² ha^?1). A simple but robust relationship between stand basal area and stand biomass of all woody species was developed for the eucalypt dominant woodlands. Analysis of above‐ground carbon stocks in live and standing dead woody plants gave a mean net above‐ground annual carbon increment for all 57 sites of 0.53 t C ha^?1 y^?1, similar to values estimated elsewhere in world savannas. Published root : shoot ratios were used to infer C flux in woody root systems on these sites. This results in an estimated sink in above‐ and below‐ground biomass of 18 Mt C y^?1 over the eucalypt woodlands studied, and potentially up to 35 Mt C y^?1 if extended to all grazed woodlands in Queensland. It is suggested that introduction of livestock grazing and altered fire regimes have triggered the change in tree‐grass dominance in these woodlands. Thus, change in carbon stocks in the grazed woodlands of Queensland is identified as an important component of human‐induced greenhouse gas flux in Australia, equivalent in magnitude to c. 25% of the most recently published (1999) total estimated national net emissions. The latter inventory takes into account emissions from land clearing, but does not include the sink identified in the present study. This sequestration also represents a small but significant contribution to the global terrestrial carbon sink.     

The population genetics of Trypanosoma brucei and the origin of human infectivity

The African trypanosome, Trypanosoma brucei, is a zoonotic parasite transmitted by tsetse flies. Two of the three subspecies, T. brucei gambiense and T.b. rhodesiense, cause sleeping sickness in humans whereas the third subspecies, T.b. brucei, is not infective to humans. We propose that the key to understanding genetic relationships within this species is the analysis of gene flow to determine the importance of genetic exchange within populations and the relatedness of populations. T.brucei parasites undergo genetic exchange when present in infections of mixed genotypes in tsetse flies in the laboratory, although this is not an obligatory process. Infections of mixed genotype are surprisingly common in field isolates from tsetse flies such that there is opportunity for genetic exchange to occur. Population genetic analyses, taking into account geographical and host species of origin, show that genetic exchange occurs sufficiently frequently in the field to be an important determinant of genetic diversity, except where particular clones have acquired the ability to infect humans. Thus, T. brucei populations have an 'epidemic' genetic structure, but the better-characterized human-infective populations have a 'clonal' structure. Remarkably, the ability to infect humans appears to have arisen on multiple occasions in different geographical locations in sub-Saharan Africa. Our data indicate that the classical subspecies terminology for T. brucei is genetically inappropriate. It is an implicit assumption in most infectious disease biology that when a zoonotic pathogen acquires the capability to infect humans, it does so once and then spreads through the human population from that single-source event. For at least one major pathogen in tropical medicine, T. brucei, this assumption is invalid.