Substrate and metal complexes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae provide new insights into the catalytic mechanism

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases are metal-dependent enzymes that catalyse the first committed step in the biosynthesis of aromatic amino acids in microorganisms and plants, the condensation of 2-phophoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to DAHP. The DAHP synthases are possible targets for fungicides and represent a model system for feedback regulation in metabolic pathways. To gain further insight into the role of the metal ion and the catalytic mechanism in general, the crystal structures of several complexes between the tyrosine-regulated form of DAHP synthase from Saccharomyces cerevisiae and different metal ions and ligands have been determined. The crystal structures provide evidence that the simultaneous presence of a metal ion and PEP result in an ordering of the protein into a conformation that is prepared for binding the second substrate E4P. The site and binding mode of E4P was derived from the 1.5A resolution crystal structure of DAHP synthase in complex with PEP, Co2+, and the E4P analogue glyceraldehyde 3-phosphate. Our data suggest that the oxygen atom of the reactive carbonyl group of E4P replaces a water molecule coordinated to the metal ion, strongly favouring a reaction mechanism where the initial step is a nucleophilic attack of the double bond of PEP on the metal-activated carbonyl group of E4P. Mutagenesis experiments substituting specific amino acids coordinating PEP, the divalent metal ion or the second substrate E4P, result in stable but inactive Aro4p-derivatives and show the importance of these residues for the catalytic mechanism.     

Exploring the ESCRTing machinery in eukaryotes

The profile of protein sorting into multivesicular bodies (MVBs) has risen recently with the identification of three heteromeric complexes known as ESCRT-I,-II,-III (Endosomal Sorting Complex Required for Transport). Genetic analyses in yeast have identified up to 15 soluble class E VPS (vacuolar protein sorting) proteins that have been assigned to the ESCRT machinery and function in cargo recognition and sorting, complex assembly, vesicle formation and dissociation. Despite their functional importance in yeast and mammalian cells, little is known about their presence and function in other organisms including plants. We have made use of the fully sequenced genomes of Arabidopsis thaliana and Oryza sativa, Drosophila melanogaster and Caenorhabditis elegans to explore the identity, structural characteristics and phylogenetic relationships of proteins assigned to the ESCRT machinery.     

Aliphatic alcohols and aldehydes of the honey bee cocoon induce arrestment behavior in Varroa jacobsoni (Acari: Mesostigmata), an ectoparasite of Apis mellifera

The ectoparasitic mite Varroa jacobsoni reproduces in the capped brood of the honey bees Apis cerana and Apis mellifera. Observations on the reproductive behavior of the mite have shown a well-structured spatial allocation of its activity using the bee or cell wall for different behaviors. The resulting advantages for the parasite of this subdivision of the concealed brood environment suggests an important role for chemostimuli in these substrates. Extracts of the European honey bee cocoons induce a strong arrestment response in the mite, as indicated by prolonged periods of walking on the extracts applied on a semipermeable membrane and by systematically returning to the stimulus after encountering the treatment borders. Two thin-layer chromatography fractions of the cocoon extract eliciting arrestment were found to contain saturated C₁₇ to C₂₂ primary aliphatic alcohols and C₁₉ to C₂₂ aldehydes. We analyzed extracts of the cocoon and different larvae, pupae, and adults of both worker and drone A. mellifera to determine the relative amounts of these chemostimuli in the different substrates employed by Varroa. Both aldehydes and alcohols were more abundant in the cocoon than in the cuticle of adult or developing bees. Mixtures of the aliphatic alcohols and aldehydes at the proportions found in the cocoons acted synergistically on the arrestment response, but this activity disappeared when mixed in equal amounts. When these oxygenated chemostimuli were mixed with C₁₉ to C₂₅ alkanes at the proportions found in the cocoon extract, we observed a significantly lower threshold for the chemostimulant mixture. These results indicate how Varroa may use mixtures of rarer products to differentiate between substrates and host stages during its developmental cycle within honey bee brood cells. Arch. Insect Biochem. Physiol. 37:129–145, 1998. © 1998 Wiley-Liss, Inc.     

Insights from modeling studies on how climate change affects invasive alien species geography

Climate change and biological invasions are threatening biodiversity and ecosystem services worldwide. It has now been widely acknowledged that climate change will affect biological invasions. A large number of studies have investigated predicted shifts and other changes in the geographic ranges of invasive alien species related to climate change using modeling approaches. Yet these studies have provided contradictory evidence, and no consensus has been reached. We conducted a systematic review of 423 modeling case studies included in 71 publications that have examined the predicted effects of climate change on those species. We differentiate the approaches used in these studies and synthesize their main results. Our results reaffirm the major role of climate change as a driver of invasive alien species distribution in the future. We found biases in the literature both regarding the taxa, toward plants and invertebrates, and the areas of the planet investigated. Despite these biases, we found for the plants and vertebrates studied that climate change will more frequently contribute to a decrease in species range size than an increase in the overall area occupied. This is largely due to oceans preventing terrestrial invaders from spreading poleward. In contrast, we found that the ranges of invertebrates and pathogens studied are more likely to increase following climate change. An important caveat to these findings is that researchers have rarely considered the effects of climate change on transport, introduction success, or the resulting impacts. We recommend closing these research gaps, and propose additional avenues for future investigations, as well as opportunities and challenges for managing invasions under climate change.     

A single-cell sequencing approach to the classification of large, vacuolated sulfur bacteria

The colorless, large sulfur bacteria are well known because of their intriguing appearance, size and abundance in sulfidic settings. Since their discovery in 1803 these bacteria have been classified according to their conspicuous morphology. However, in microbiology the use of morphological criteria alone to predict phylogenetic relatedness has frequently proven to be misleading. Recent sequencing of a number of 16S rRNA genes of large sulfur bacteria revealed frequent inconsistencies between the morphologically determined taxonomy of genera and the genetically derived classification. Nevertheless, newly described bacteria were classified based on their morphological properties, leading to polyphyletic taxa. We performed sequencing of 16S rRNA genes and internal transcribed spacer (ITS) regions, together with detailed morphological analysis of hand-picked individuals of novel non-filamentous as well as known filamentous large sulfur bacteria, including the hitherto only partially sequenced species Thiomargarita namibiensis, Thioploca araucae and Thioploca chileae. Based on 128 nearly full-length 16S rRNA-ITS sequences, we propose the retention of the family Beggiatoaceae for the genera closely related to Beggiatoa, as opposed to the recently suggested fusion of all colorless sulfur bacteria into one family, the Thiotrichaceae. Furthermore, we propose the addition of nine Candidatus species along with seven new Candidatus genera to the family Beggiatoaceae. The extended family Beggiatoaceae thus remains monophyletic and is phylogenetically clearly separated from other related families.     

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.     

Inhibition of alpha interferon signaling by hepatitis B virus

Alpha interferon (IFN-alpha) and pegylated IFN-alpha (pegIFN-alpha) are used for the treatment of chronic hepatitis B (CHB). Unfortunately, only a minority of patients can be cured. The mechanisms responsible for hepatitis B virus (HBV) resistance to pegIFN-alpha treatment are not known. pegIFN-alpha is also used to treat patients with chronic hepatitis C (CHC). As with chronic hepatitis B, many patients with chronic hepatitis C cannot be cured. In CHC, IFN-alpha signaling has been found to be inhibited by an upregulation of protein phosphatase 2A (PP2A). PP2A inhibits protein arginine methyltransferase 1 (PRMT1), the enzyme that catalyzes the methylation of the important IFN-alpha signal transducer STAT1. Hypomethylated STAT1 is less active because it is bound by its inhibitor, PIAS1. In the present work, we investigated whether similar molecular mechanisms are also responsible for the IFN-alpha resistance found in many patients with chronic hepatitis B. We analyzed the expression of PP2A, the enzymatic activity of PRMT1 (methylation assays), the phosphorylation and methylation of STAT1, the association of STAT1 with PIAS1 (via coimmunoprecipitation assays), the binding of activated STAT1 to interferon-stimulated response elements (via electrophoretic mobility shift assays), and the induction of interferon target genes (via real-time RT-PCR) in human hepatoma cells expressing HBV proteins as well as in liver biopsies from patients with chronic hepatitis B and from controls. We found an increased expression of PP2A and an inhibition of IFN-alpha signaling in cells expressing HBV proteins and in liver biopsies of patients with CHB. The molecular mechanisms involved are similar to those found in chronic hepatitis C.     

Stimulus-Induced Selective Association of Actin-Associated Proteins (α-Actinin) and Protein Kinase C Isoforms with the Cytoskeleton of Human Neutrophils

We report a selective, differential stimulus-dependent enrichment of the actin-associated protein α-actinin and of isoforms of the signaling enzyme protein kinase C (PKC) in the neutrophil cytoskeleton. Chemotactic peptide, activators of PKC, and cell adhesion all induce a significant increase in the amount of cytoskeletal α-actinin and actin. Increased association of PKCβI and βII with the cytoskeletal fraction of stimulated cells was also observed, with phorbol ester being more effective than chemotactic peptide. A fraction of phosphatase 2A was constitutively associated with the cytoskeleton independent of cell activation. None of the stimuli promoted association of vinculin or myosin II with the cytoskeleton. Phosphatase inhibitors okadaic acid and calyculin A prevented increases in cytoskeletal actin, α-actinin, and PKCβII induced by phorbol ester, suggesting the requirement for phosphatase activity in these events. Increases in cytoskeletal α-actinin and PKCβII showed differing sensitivity to agents that prevent actin polymerization (cytochalasin D, latrunculin A). Latrunculin A (1 μM) completely blocked PMA-induced increases in cytoskeletal α-actinin but reduced cytoskeletal recruitment of PKCβII only by 16%. Higher concentrations of latrunculin A (4 μM), which almost abolished the cytoskeletal actin pool, reduced cytoskeletal PKCβII by 43%. In conclusion, a selective enrichment of cytoskeletal and signaling proteins in the cytoskeleton of human neutrophils is induced by specific stimuli.     

A high risk phenotype of hypertrophic cardiomyopathy associated with a compound genotype of two mutated β-myosin heavy chain genes

Hypertrophic cardiomyopathy (HCM) is a genetically and clinically heterogeneous myocardial disease that is in most cases familial and transmitted in a dominant fashion. The most frequently affected gene codes for the cardiac (ventricular) β-myosin heavy chain. We have investigated the genetic cause of an isolated case of HCM, which was marked by an extremely severe phenotype and a very early age of onset. HCM is normally not a disease of small children. The proband was a boy who had suffered cardiac arrest at the age of 6.5years (resuscitation by cardioconversion). Upon screening of the β-myosin heavy chain gene as a candidate, two missense mutations, one in exon19 (Arg719Trp) and a second in exon12 (Met349Thr), were identified. The Arg719Trp mutation was de novo, as it was not found in the parents. In contrast, the Met349Thr mutation was inherited through the maternal grandmother. Six family members were carriers of this mutation but only the proband was clinically affected. Segregation and molecular analysis allowed us to assign the Met349Thr mutation to the maternal and the Arg719Trp de novo mutation to the paternal β-myosin allele. Thus, the patient has no normal myosin. We interpret these findings in terms of compound heterozygosity of a dominant (Arg719Trp) and a recessive (Met349Thr) mutation. Whereas a single mutated Arg719Trp allele would be sufficient to cause HCM, the concurrent Met349Thr mutation alone does not apparently induce the disease. Nevertheless, it conceivably contributes to the particularly severe phenotype. Received: 15 September 1997 / Accepted: 26 November 1997