Crystal Structure of the Avian Astrovirus Capsid Spike

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.     

Chlorite dismutases, DyPs, and EfeB: 3 microbial heme enzyme families comprise the CDE structural superfamily

Heme proteins are extremely diverse, widespread, and versatile biocatalysts, sensors, and molecular transporters. The chlorite dismutase family of hemoproteins received its name due to the ability of the first-isolated members to detoxify anthropogenic ClO₂⁻, a function believed to have evolved only in the last few decades. Family members have since been found in 15 bacterial and archaeal genera, suggesting ancient roots. A structure- and sequence-based examination of the family is presented, in which key sequence and structural motifs are identified, and possible functions for family proteins are proposed. Newly identified structural homologies moreover demonstrate clear connections to two other large, ancient, and functionally mysterious protein families. We propose calling them collectively the CDE superfamily of heme proteins.     

DINITROGEN FIXATION BY CYANOBACTERIA AND ASSOCIATIVE RHIZOSPHERE BACTERIA IN THE ARAPAHO PRAIRIE IN THE SAND HILLS OF NEBRASKA

Free-living and lichenized cyanobacterial (cyanolichen) colonies occupy 12–29% of the surface area in the Sand Hills grasslands on the Arapaho Prairie in southwestern Nebraska. During brief periods of favorable conditions (i.e., moderate temperature and high moisture), these diazotrophs are capable of high rates of dinitrogen fixation. Maximum rates of acetylene reduction measured during 1981 and 1982 were 2,237 μg N m^-2 hr^-l. The duration of dinitrogen fixation activity is dependent upon moisture conditions; rates of acetylene reduction were shown to decline 90% in as little as three hr. Desiccated colonies resumed acetylene reduction activity within one hr and achieved equilibrium rates within four hr following rehydration. Associative, heterotrophic diazotrophs were isolated from the rhizospheres of several grass species. These bacteria exhibited a range of culturing characteristics which may reflect differing plant-bacterial interaction potentials important to the establishment and maintenance of the plant species. None of the field assays for heterotrophic dinitrogen fixation performed over the two-yr period indicated a capacity of this group to provide ecologically significant levels of N in this grassland. Our gross estimate of the annual contribution was 3.5 kg N ha^-1 from cyanolichens and 0.8 kg N ha^-1 from associative heterotrophs.     

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.     

Evidence for a Shared Nuclear Pore Complex Architecture That Is Conserved from the Last Common Eukaryotic Ancestor

The nuclear pore complex (NPC) is a macromolecular assembly embedded within the nuclear envelope that mediates bidirectional exchange of material between the nucleus and cytoplasm. Our recent work on the yeast NPC has revealed a simple modularity in its architecture and suggested a common evolutionary origin of the NPC and vesicle coating complexes in a progenitor protocoatomer. However, detailed compositional and structural information is currently only available for vertebrate and yeast NPCs, which are evolutionarily closely related. Hence our understanding of NPC composition in a full evolutionary context is sparse. Moreover despite the ubiquitous nature of the NPC, sequence searches in distant taxa have identified surprisingly few NPC components, suggesting that much of the NPC may not be conserved. Thus, to gain a broad perspective on the origins and evolution of the NPC, we performed proteomics analyses of NPC-containing fractions from a divergent eukaryote (Trypanosoma brucei) and obtained a comprehensive inventory of its nucleoporins. Strikingly trypanosome nucleoporins clearly share with metazoa and yeast their fold type, domain organization, composition, and modularity. Overall these data provide conclusive evidence that the majority of NPC architecture is indeed conserved throughout the Eukaryota and was already established in the last common eukaryotic ancestor. These findings strongly support the hypothesis that NPCs share a common ancestry with vesicle coating complexes and that both were established very early in eukaryotic evolution.Nearly all eukaryotic cells possess an extensive endomembrane system that is principally responsible for protein targeting and modification (1). The nucleus, the defining eukaryotic feature, is separated from the cytoplasm by a double bilayered nuclear envelope (NE)¹ that is contiguous with the rest of this endomembrane system via connections to the endoplasmic reticulum. Nuclear pore complexes (NPCs) fenestrate the NE, serving as the exclusive sites mediating exchange between the nucleoplasmic and cytoplasmic compartments. Macromolecules are chaperoned through the NPC by numerous transport factors. It has been proposed that the endomembrane system and nucleus have an autogenous origin (i.e. evolving from invaginations of an ancestral plasma membrane) and were established early in eukaryotic evolution (2).The composition of the NPC has been cataloged at ∼30 distinct nucleoporins (Nups) (3) for the yeast Saccharomyces cerevisiae (4) and vertebrates (5), two members of the Opisthokonta (animals, fungi, and closely related protists). Ultrastructural studies have identified objects morphologically similar (at a first approximation) to opisthokont NPCs in the other major eukaryote supergroups (6–8). However, very few data are available concerning the detailed NPC molecular composition and architecture for nearly all eukaryotic lineages, leaving a relatively narrow view of the “typical” NPC and its origins. A few examples of potential Nup orthologs beyond the opisthokonts have been reported, leading to the suggestion that substantial portions of the NPC may have an ancient, pre-last common eukaryotic ancestor (LCEA) origin (9). However, a more extensive study has concluded that LCEA possessed a primitive ancestral NPC that passed few components to its modern descendants (10).In yeast and vertebrates, the NPC consists of an eight-spoked core surrounding a central tube that serves as the conduit for macromolecular exchange. Each spoke can be divided into two similar nucleoplasmic and cytoplasmic halves. The eight spokes connect to form several coaxial rings: the membrane rings, the two outer rings at the nucleoplasmic and cytoplasmic periphery, and the two adjacent inner rings (11). Groups of Nups that we term “linker Nups” are attached between both sets of outer and inner rings. Another group of related proteins, collectively termed phenylalanine-glycine (FG) Nups, are largely exposed on the inner surface of the spokes and anchored either to the inner rings or to the linker Nups (11).Opisthokont Nups can be grouped into three structural classes (11, 12). The first class comprises membrane-bound proteins that anchor the NPC into the NE. The second class is the core scaffold Nups; these proteins constitute the bulk of the NPC mass, form the central tube, and provide the scaffold for the deployment of the third class of Nups across both faces of the NPC. The core scaffold Nups are remarkably restricted at the structural level and contain only three distinct arrangements of 2-fold types: proteins dominated by an α-solenoid fold (also termed a helix-turn-helix repeat domain), proteins consisting of a β-propeller fold, and finally proteins composed of an amino-terminal β-propeller fold followed by a carboxyl-terminal α-solenoid fold (which we here term a β-α structure) (12). FG Nups comprise the third class. These Nups carry multiply repeated degenerate “Phe-Gly” motifs (FG repeats) separated by hydrophilic or charged residues that form large unstructured domains. Each FG Nup also contains a small structured domain (often a coiled coil motif) that serves as the anchor site for interaction with the remainder of the NPC.Many transport factors belong to a structurally related protein family collectively termed karyopherins (Kaps) (13, 14). Transport across the NPC depends on the interactions between Kaps, cargo molecules, and the disordered repeat domains of FG Nups; the latter are thought to form the selective barrier for nucleocytoplasmic transport, guiding the Kap·cargo complexes (and other transport factors) through the central tube while excluding other macromolecules (for reviews, see Refs. 3 and 15–22).Significantly we have previously noted that the fold composition and arrangement of many of the core scaffold Nups are shared with proteins that form coating structures that participate in the generation and transport of vesicles between different endomembrane compartments; significantly many vesicle coating complex proteins and NPC scaffold Nups share an α-solenoid fold, β-propeller fold, or β-α structure (12, 23–28). These similarities gave rise to the “protocoatomer hypothesis,” which suggests a common ancestry for the NPC and these vesicle coat complexes. However, it is unclear how many, if any, of these particular core scaffold Nups are widely conserved, and hence it is unclear how general this potential relationship is throughout the Eukaryota. Thus, two scenarios are possible. The first is that the coatomer-like proteins are only found in a subset of the eukaryotes (including the opisthokonts), indicating that they are a relatively recent acquisition of only some eukaryotes and are not a general feature of all NPCs. The second is that the coatomer-like proteins are conserved in all eukaryotes, providing strong support to the protocoatomer hypothesis. To directly address this issue we characterized the NPC of Trypanosoma brucei, a highly divergent but experimentally tractable organism, using proteomics. The resulting data indicate an ancient origin for the majority of the NPC components and shed light on the origin of LCEA itself.     

Absolutes und relatives Wachstum bei Ammonoideen

The following basic growth parameters of the ammonoid shell are analysed: radius of the coiling spiral; whorl width; plane of cross-section; umbilical spiral. The absolute growth of these parameters follows a general growth formula which is based on the probability theory (time-dependent stochastic processes). The in crease of all basic parameters is ruled by analogous formulas which are derived from the general growth formula; the increase of the coiling spiral results in the geometrical figure of a logarithmic spiral, because this is the only one that meets the conditions of the general growth formula. The relative growth can be described as a morphogenetic program that is subdivided in presumably three formally connected subprograms. Each subprogram is defined by specific allometry and integration constants.
Folgende Grundparameter des Wachstums von Ammonoideen-Gehäusen werden analysiert: Radius der äuβeren Spirale, Windungsbreite, Querschnittsfläche, Nabelspirale. Das absolute Wachstum dieser Parameter folgt einer allgemeinen Wachstumsformel, die sich aus der Wahrscheinlichkeitstheorie (zeitabhängige stochastische Prozesse) ergibt. Die Zunahme aller Grundparameter folgt analogen Formeln, die sich aus der allgemeinen Wachstumsformel ableiten lassen; das Anwachsen des äuβeren RadiS führt zur geometrischen Figur einer logarith-mischen Spirale, da nur diese der allgemeinen Wachstumsformel genügt. Das relative Wachstum läβt sich als ein morphogenetisches Wachstumsprogramm beschreiben, das sich in vermutlich drei formal miteinander verknüpfte Unter-programme aufteilt. Jedes Unterprogramm ist durch spezifische Allometrie- und Integrationskonstanten festgelegt.     

Freeze-Recovery Physiology of Nitrogenase Activity in Terrestrial Nostoc sp. Colonies

Nostoc sp. colonies from field collections were cultured and propagated on silica sand with aqueous N-free BG-11 medium. Laboratory experiments were conducted to characterize the in vivo freeze-recovery physiology of nitrogenase activity. Nitrogenase activity was monitored by the acetylene reduction technique. Frozen Nostoc sp. colonies were thawed and warmed to 10, 15, 20, 25, or 30°C. At 25 and 30°C, nitrogenase activity was detected within 6 h after thawing. At 20°C or lower, nitrogenase activity was not detected until 12 h after thawing. Optimum thawing temperature with respect to the recovery of nitrogenase activity was 25°C. In subsequent experiments, laboratory-grown Nostoc colonies were used along with the following conditions: prefreezing treatment of 3 days of exposure to light or darkness, freezing, and then thawing to 25°C in light or darkness with or without metabolic inhibitors [3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), monofluoroacetate, or chloramphenicol]. Approximately 30% of the energy in the initial recovery of nitrogenase activity (to 12 h after thawing) appeared to be supplied via the utilization of carbon compounds stored before freezing. Photosynthetic conditions (i.e., light and without DCMU) were necessary for maximum recovery of nitrogenase activity. In the presence of the protein synthesis inhibitor chloramphenicol, nitrogenase activity was still detected at 12 to 48 h after thawing. Although damage may occur to nitrogenase, some of the enzyme was capable of surviving the freeze-thaw period in vivo. However, complete recovery of nitrogenase activity (equal to prefreezing activity) may entail some de novo synthesis of nitrogenase.     

Analytical detection of 9(4)-O-acetylated sialoglycoproteins and gangliosides using influenza C virus.

The unique glycoprotein of influenza C virus, designated hemagglutinin (HEF), exhibits three functions: hemagglutination, esterase activity, and fusion factor. As the virus uses 9-O-acetylated sialic acid as a high-affinity receptor determinant for attachment to cells, its binding activity was used to reveal O-acetylated sialic acid residues after polyacrylamide gel electrophoresis and transfer onto nitrocellulose sheets of proteins and thin-layer chromatography of lipids. The specificity of the binding for O-acetylated sialoglycoconjugates was investigated. Our results showed that influenza C virus could detect the different forms of the two murine glycophorins which are known to be O-acetylated sialoglycoconjugates. The virus also bound to O-acetylated gangliosides isolated from embryonic chicken brain such as purified O-acetylated NeuAc alpha (2-8)NeuAc alpha (2-8)NeuAc alpha (2-3)Gal beta (1-4)Glc beta (1-1)ceramide (GT3). The esterase activity of the HEF protein of influenza C virus was used to unmask the sialic acid. After its deacetylation by the virus enzyme, the O-acetylated GT3 was recognized by a monoclonal antibody which binds only to the nonacetylated derivative. The results presented here show that influenza C virus is a discriminating analytical probe for identifying O-acetylated sialoglycoconjugates directly after Western blotting of proteins and thin-layer chromatography of lipids, thus providing a new analytical tool.     

Invertebrate colonization of a new, man-made stream in southern Sweden

SUMMARY.

• 1 The invertebrate colonization of a man-made stream, Flugströmmen, in southern Sweden was monitored for 18 months in 1988 and 1989. Benthic samples were taken on twelve occasions from three sites (upstream, middle and downstream) and community structure was compared with that at ten natural, permanent reference sites nearby.
• 2 The number of species colonizing increased rapidly during the first 3 months. The increase was most rapid upstream but levelled off during the second year at this site, while numbers continued to increase downstream.
• 3 Simuliid species were the earliest colonizers and reached high densities at upstream and middle sites during the first year. Ephemeropteran and plecopteran species also occurred early on, whereas Coleoptera, Odonata and Trichoptera were, on average, slower to colonize. Blackfly densities decreased upstream after the first year and hydropsychids became numerically dominant.
• 4 The colonization order of functional feeding groups was as predicted: filter feeders first, grazers/collectors intermediate, predators and shredders last.
• 5 After a year, the community structure in Flugströmmen closely resembled that in lake-outlet streams situated in the area, although communities at the three sites within the stream were most similar to one another.
• 6 The possible role of competitive and predatory processes in determining the observed successional patterns are discussed.