Evidence for Geographic Isolation and Signs of Endemism within a Protistan Morphospecies

The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of “Spumella-like” flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Microbial metabolomics: toward a platform with full metabolome coverage

Achieving metabolome data with satisfactory coverage is a formidable challenge in metabolomics because metabolites are a chemically highly diverse group of compounds. Here we present a strategy for the development of an advanced analytical platform that allows the comprehensive analysis of microbial metabolomes. Our approach started with in silico metabolome information from three microorganisms-Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae-and resulted in a list of 905 different metabolites. Subsequently, these metabolites were classified based on their physicochemical properties, followed by the development of complementary gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry methods, each of which analyzes different metabolite classes. This metabolomics platform, consisting of six different analytical methods, was applied for the analysis of the metabolites for which commercial standards could be purchased (399 compounds). Of these 399 metabolites, 380 could be analyzed with the platform. To demonstrate the potential of this metabolomics platform, we report on its application to the analysis of the metabolome composition of mid-logarithmic E. coli cells grown on a mineral salts medium using glucose as the carbon source. Of the 431 peaks detected, 235 (=176 unique metabolites) could be identified. These include 61 metabolites that were not previously identified or annotated in existing E. coli databases.     

Cross-talk between TCR and CCR7 signaling sets a temporal threshold for enhanced T lymphocyte migration

Lymphocyte homing to, and motility within, lymph nodes is regulated by the chemokine receptor CCR7 and its two ligands CCL19 and CCL21. There, lymphocytes are exposed to a number of extracellular stimuli that influence cellular functions and determine the cell fate. In this study, we assessed the effect of TCR engagement on CCR7-mediated cell migration. We found that long-term TCR triggering of freshly isolated human T cells through CD3/CD28 attenuated CCR7-driven chemotaxis, whereas short-term activation significantly enhanced CCR7-mediated, but not CXCR4-mediated, migration efficiency. Short-term activation most prominently enhanced the migratory response of naive T cells of both CD4 and CD8 subsets. We identified distinct roles for Src family kinases in modulating CCR7-mediated T cell migration. We provide evidence that Fyn, together with Ca(2+)-independent protein kinase C isoforms, kept the migratory response of naive T cells toward CCL21 at a low level. In nonactivated T cells, CCR7 triggering induced a Fyn-dependent phosphorylation of the inhibitory Tyr505 of Lck. Inhibiting Fyn in these nonactivated T cells prevented the negative regulation of Lck and facilitated high CCR7-driven T cell chemotaxis. Moreover, we found that the enhanced migration of short-term activated T cells was accompanied by a synergistic, Src-dependent activation of the adaptor molecule linker for activation of T cells. Collectively, we characterize a cross-talk between the TCR and CCR7 and provide mechanistic evidence that the activation status of T cells controls lymphocyte motility and sets a threshold for their migratory response.     

Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Molecular Mechanisms of Ischemia–Reperfusion Injury in Brain: Pivotal Role of the Mitochondrial Membrane Potential in Reactive Oxygen Species Generation

Stroke and circulatory arrest cause interferences in blood flow to the brain that result in considerable tissue damage. The primary method to reduce or prevent neurologic damage to patients suffering from brain ischemia is prompt restoration of blood flow to the ischemic tissue. However, paradoxically, restoration of blood flow causes additional damage and exacerbates neurocognitive deficits among patients who suffer a brain ischemic event. Mitochondria play a critical role in reperfusion injury by producing excessive reactive oxygen species (ROS) thereby damaging cellular components, and initiating cell death. In this review, we summarize our current understanding of the mechanisms of mitochondrial ROS generation during reperfusion, and specifically, the role the mitochondrial membrane potential plays in the pathology of cerebral ischemia/reperfusion. Additionally, we propose a temporal model of ROS generation in which posttranslational modifications of key oxidative phosphorylation (OxPhos) proteins caused by ischemia induce a hyperactive state upon reintroduction of oxygen. Hyperactive OxPhos generates high mitochondrial membrane potentials, a condition known to generate excessive ROS. Such a state would lead to a “burst” of ROS upon reperfusion, thereby causing structural and functional damage to the mitochondria and inducing cell death signaling that eventually culminate in tissue damage. Finally, we propose that strategies aimed at modulating this maladaptive hyperpolarization of the mitochondrial membrane potential may be a novel therapeutic intervention and present specific studies demonstrating the cytoprotective effect of this treatment modality.     

Brain‐derived neurotrophic factor selectively regulates dendritogenesis of parvalbumin‐containing interneurons in the main olfactory bulb through the PLCγ pathway

Molecular mechanisms of neurotrophin signaling on dendrite development and dynamics are only partly understood. To address the role of brain‐derived neurotrophic factor (BDNF) in the morphogenesis of GABAergic neurons of the main olfactory bulb, we analyzed mice lacking BDNF, mice carrying neurotrophin‐3 (NT3) in the place of BDNF, and TrkB signaling mutant mice with a receptor that can activate phospholipase Cγ (PLCγ) but is unable to recruit the adaptors Shc/Frs2. BDNF deletion yielded a compressed olfactory bulb with a significant loss of parvalbumin (PV) immunoreactivity in GABAergic interneurons of the external plexiform layer. Dendrite development of PV‐positive interneurons was selectively attenuated by BDNF since other Ca²⁺‐binding protein‐containing neuron populations appeared unaffected. The deficit in PV‐positive neurons could be rescued by the NT3/NT3 alleles. The degree of PV immunoreactivity was dependent on BDNF and TrkB recruitment of the adaptor proteins Shc/Frs2. In contrast, PLCγ signaling from the TrkB receptor was sufficient for dendrite growth in vivo and consistently, blocking PLCγ prevented BDNF‐dependent dendrite development in vitro. Collectively, our results provide genetic evidence that BDNF and TrkB signaling selectively regulate PV expression and dendrite growth in a subset of neurochemically‐defined GABAergic interneurons via activation of the PLCγ pathway. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006