Is the failing heart out of fuel or a worn engine running rich? A study of mitochondria in old spontaneously hypertensive rats

Hypertension now affects about 600 million people worldwide and is a leading cause of death in the Western world. The spontaneously hypertensive rat (SHR), provides a useful model to investigate hypertensive heart failure (HF). The SHR model replicates the clinical progression of hypertension in humans, wherein early development of hypertension is followed by a long stable period of compensated cardiac hypertrophy that slowly progresses to HF. Although the hypertensive failing heart generally shows increased substrate preference towards glucose and impaired mitochondrial function, the cause-and-effect relationship between these characteristics is incompletely understood. To explore these pathogenic processes, we compared cardiac mitochondrial proteomes of 20-month-old SHR and Wistar-Kyoto controls by iTRAQ-labelling combined with multidimensional LC/MS/MS. Of 137 high-scoring proteins identified, 79 differed between groups. Changes were apparent in several metabolic pathways, chaperone and antioxidant systems, and multiple subunits of the oxidative phosphorylation complexes were increased (complexes I, III and IV) or decreased (complexes II and V) in SHR heart mitochondria. Respiration assays on skinned fibres and isolated mitochondria showed markedly lower respiratory capacity on succinate. Enzyme activity assays often also showed mismatches between increased protein expression and activities suggesting elevated protein expression may be compensatory in the face of pathological stress.     

Regulation of the synthesis of cyclic glucan in Xanthomonas campestris by a diffusible signal molecule

The rpf gene cluster of Xanthomonas campestris pv. campestris is involved in the co-ordinate positive regulation of the production of extracellular enzymes and the extracellular polysaccharide xanthan. Several of the rpf genes are involved in a regulatory system involving the small diffusible molecule DSF (for diffusible signal factor). Synthesis of DSF requires RpfF, and a two-component sensory transduction system involving RpfC has been implicated in the perception of the signal and signal transduction. Here we show that mutations in both rpfF and rpfC lead to reductions in the levels of cyclic glucan. The levels of cyclic glucan synthetase in membrane preparations from rpfF and rpfC mutants were, however, unaltered from the wild-type. Similar alterations in the level of cyclic glucan without changes in cyclic glucan synthetase activity were seen when wild-type bacteria were exposed to osmotic stress. These results extend the range of cellular functions subject to regulation by the rpf genes and DSF system.     

Historical biogeography of scarabaeine dung beetles

Abstract Aim (1) To review briefly global biogeographical patterns in dung beetles (Coleoptera: Scarabaeidae: Scarabaeinae), a group whose evolutionary history has been dominated by ecological specialization to vertebrate dung in warmer climates. (2) To develop hypotheses accounting for the evolution of these patterns. Location Six principal biogeographical regions: Palaearctic, Oriental, Afrotropical, Australasia, Neotropical, Nearctic and five outlying islands or island groups harbouring endemic genera: Caribbean, Madagascar, Mauritius, New Caledonia, New Zealand. Methods Major patterns of tribal, generic and species distribution are investigated using cluster analysis, ordination, parsimony analysis of endemism and track analysis. Attempts are made to resolve biogeographical patterns with findings in the fields of plate tectonics, fossil and evolutionary history, plus phylogeny of both mammals and dung beetles. Results Because of conflict between published findings, it is uncertain at what point in time density of dinosaur dung, mammal dung or both became sufficiently great to select for specialized habits in dung beetles. However, biogeographical evidence would suggest a Mesozoic origin followed by further taxonomic radiation during the Cenozoic, possibly in response to the increasing size and diversity of mammalian dung types in South America and Afro‐Eurasia. Proportional generic distribution in fourteen tribes and subtribes showed four principal biogeographical patterns: (1) southerly biased Gondwanaland distribution, (2) Americas or (3) Madagascar endemism, and (4) northerly biased, Afro‐Eurasian‐centred distribution with limited numbers of genera also widespread in other regions. Proportional composition of faunas in eleven geographical regions indicated three principal distributional centres, East Gondwanaland fragments, Afro‐Eurasia and the Americas. These patterns probably result from three principal long‐term range expansion and vicariance events (Mesozoic: Gondwanaland interchange and fragmentation, Cenozoic: Afro‐Eurasian/Nearctic interchange and the Great American interchange). It is suggested that old vicariance caused by the Mesozoic fragmentation of Gondwanaland leads to a high degree of regional endemism at generic or tribal level across one or more Gondwanaland tracks. In contrast, it is suggested that the more recent Cenozoic range expansions occurred primarily towards northern regions leading to endemism primarily at species level. These Cenozoic radiations were facilitated by the re‐linking of continents, either because of tectonic plate movements (Africa to Eurasia in Miocene), climatically induced sea‐level change (Afro‐Eurasia to Nearctic in Miocene and Pleistocene), or similar coupled with orogenics (Nearctic to Neotropical in Pliocene). Speciation has followed vicariance either because of climatic change or physical barrier development. These recent range expansions probably occurred principally along an Afro‐Eurasian land track to the Nearctic and Neotropical and an Americas land track northwards from the Neotropics to the Nearctic, with limited dispersal from Eurasia to Australia, probably across a sea barrier. This accounts for the overall, spatially constrained, biogeographical pattern comprising large numbers of species‐poor genera endemic to a single biogeographical region and fewer more species‐rich genera, many of which show wider biogeographical distributions. In most southerly regions (Australasia, Madagascar, Neotropical), faunal composition and generic endemism is primarily dominated by elements with Gondwanaland ancestry, which is consistent with the Gondwanaland origin claimed for Scarabaeinae. In Afro‐Eurasia (Palaearctic, Oriental, Afrotropical), generic endemism of monophyletically derived Afro‐Eurasian and widespread lineages is centred in the Afrotropical region and faunal composition is numerically dominated by Afro‐Eurasian and widespread elements. In the Nearctic region, the fauna is jointly dominated by widespread elements, derived from Afro‐Eurasia, and Gondwanaland and Americas elements derived from the Neotropical region. Main conclusions Global biogeographical patterns in scarabaeine dung beetles primarily result from Mesozoic and Cenozoic range expansion events followed by vicariance, although recent dispersal to Australia may have occurred across sea barriers. Detailed phylogenetics research is required to provide data to support dispersal/vicariance hypotheses.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Low molecular weight GTP-binding proteins in human neutrophil granule membranes

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.     

Activation of the MAPK module from different spatial locations generates distinct system outputs

The Ras/Raf/MEK/ERK (MAPK) pathway directs multiple cell fate decisions within a single cell. How different system outputs are generated is unknown. Here we explore whether activating the MAPK module from different membrane environments can rewire system output. We identify two classes of nanoscale environment within the plasma membrane. The first, which corresponds to nanoclusters occupied by GTP-loaded H-, N- or K-Ras, supports Raf activation and amplifies low Raf kinase input to generate a digital ERKpp output. The second class, which corresponds to nanoclusters occupied by GDP-loaded Ras, cannot activate Raf and therefore does not activate the MAPK module, illustrating how lateral segregation on plasma membrane influences signal output. The MAPK module is activated at the Golgi, but in striking contrast to the plasma membrane, ERKpp output is analog. Different modes of Raf activation precisely correlate with these different ERKpp system outputs. Intriguingly, the Golgi contains two distinct membrane environments that generate ERKpp, but only one is competent to drive PC12 cell differentiation. The MAPK module is not activated from the ER. Taken together these data clearly demonstrate that the different nanoscale environments available to Ras generate distinct circuit configurations for the MAPK module, bestowing cells with a simple mechanism to generate multiple system outputs from a single cascade.     

Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

New species of the myrmecophile Polyplocotes Westwood (Coleoptera: Ptinidae) from South Australia

Six new species of the Australian myrmecophilous ptinid genus Polyplocotes are described from South Australia. Three are from the deserts of central Australia, one from the Franklin Islands in the Great Australian Bight, one from Eyre Peninsula and one from the Riverland region. Morphologically, the majority of these new species are conventional Polyplocotes , but two are less typical. The characters uniting the genus are explored in the discussion, and comparisons are made to related genera. Although the six new species described here have not been observed in the field, the species of this genus are known to be myrmecophilous, and ant – beetle interactions similar to those seen in other spider beetles might occur between these new species and their host ants.