Active Transport of Membrane Components by Self-Organization of the Min Proteins

Heterogeneous distribution of components in the biological membrane is critical in the process of cell polarization. However, little is known about the mechanisms that can generate and maintain the heterogeneous distribution of the membrane components. Here, we report that the propagating wave patterns of the bacterial Min proteins can impose steric pressure on the membrane, resulting in transport and directional accumulation of the component in the membrane. Therefore, the membrane component waves represent transport of the component in the membrane that is caused by the steric pressure gradient induced by the differential levels of binding and dissociation of the Min proteins in the propagating waves on the membrane surface. The diffusivity, majorly influenced by the membrane anchor of the component, and the repulsed ability, majorly influenced by the steric property of the membrane component, determine the differential spatial distribution of the membrane component. Thus, transportation of the membrane component by the Min proteins follows a simple physical principle, which resembles a linear peristaltic pumping process, to selectively segregate and maintain heterogeneous distribution of materials in the membrane.

A High Frequency of Beneficial Mutations Across Multiple Fitness Components in Saccharomyces cerevisiae

Mutation-accumulation experiments are widely used to estimate parameters of spontaneous mutations affecting fitness. In many experiments only one component of fitness is measured. In a previous study involving the diploid yeast Saccharomyces cerevisiae, we measured the growth rate of 151 mutation-accumulation lines to estimate parameters of mutation. We found that an unexpectedly high frequency of fitness-altering mutations was beneficial. Here, we build upon our previous work by examining sporulation efficiency, spore viability, and haploid growth rate and find that these components of fitness also show a high frequency of beneficial mutations. We also examine whether mutation-acycumulation (MA) lines show any evidence of pleiotropy among accumulated mutations and find that, for most, there is none. However, MA lines that have zero fitness (i.e., lethality) for any one fitness component do show evidence for pleiotropy among accumulated mutations. We also report estimates of other parameters of mutation based on each component of fitness.ADAPTATION can occur from standing genetic variation or from newly arising mutations. The relative importance of these two sources of adaptive mutations is affected by a variety of factors, including those that alter standing levels of genetic variation (see Barrett and Schluter 2008) and those that generate new mutations. Predicting how quickly a population will adapt and the type of beneficial mutations that will fuel that adaptation requires estimates of the additive genetic variance in fitness and of the beneficial mutation rate and the distribution of beneficial effects. While additive genetic variance for fitness has been estimated in a variety of organisms (Mousseau and Roff 1987), the beneficial mutation rate and the distribution of beneficial effects have only been estimated in a few studies (Shaw et al. 2002; Joseph and Hall 2004; Perfeito et al. 2007; Dickinson 2008; Hall et al. 2008). Surprisingly, these studies estimate that between 6 (Joseph and Hall 2004) and 50% (Shaw et al. 2002) of fitness-altering mutations are beneficial. In contrast, most mutation-accumulation (MA) experiments identify few, if any, beneficial mutations. Such wildly different estimates have even been generated from studies of the same species in similar environments (Zeyl and Devisser 2001; Joseph and Hall 2004; Dickinson 2008; Hall et al. 2008). If these estimates are correct, then they would suggest that the genotypes used in these experiments have vastly different evolutionary potential with respect to their capacity to exhibit rapid adaptation from new mutations.A more likely scenario is that much of the variation in estimates of the beneficial mutation rate is due to methodological differences between studies. One possibility is the fitness component being analyzed. The beneficial mutation rate may be under- or overestimated if the fitness component is under stabilizing selection or subject to antagonistic pleiotropy. Analyses of mutation-accumulation data typically assume that selection is directional. As a result, analyses of phenotypes under stabilizing selection may falsely conclude that mutations that increase a phenotype are beneficial and mutations that lower values are deleterious (see Keightley and Lynch''s 2003 criticism of Shaw et al. 2002). Alternatively, the beneficial mutation rate may be over- (or under) estimated if mutations increase fitness in regard to one component, but lower fitness in regard to lifetime fitness or another fitness component (i.e., antagonistic pleiotropy). Here, we explore these possibilities by investigating whether the high beneficial mutation rates estimated from our previous experiments are specific to the fitness component that we examined.In two previous studies we accumulated mutations in 152 yeast, MA lines and used measures of their effects on diploid growth rate to estimate parameters of beneficial and deleterious mutations. In the first study we estimated that 6% of mutations accumulated during the first 1012 generations of accumulation improved diploid growth (Joseph and Hall 2004). To determine whether this high beneficial mutation rate was due to sampling error, we passaged the lines for an additional 1050 generations and found that 13% of mutations improved diploid growth (Hall et al. 2008). Similarly, another yeast MA experiment (Dickinson 2008) estimated an uncorrected frequency of beneficial mutations of 25%, although correction for within-colony selection reduces this estimate by approximately half. Together, these studies indicate that a substantial proportion of mutations accumulated in these yeast MA lines are beneficial for a single fitness component and that this observation cannot be explained by the chance sampling of a few beneficial mutations.In this study we return to our yeast MA lines (Joseph and Hall 2004) and examine whether the high beneficial mutation rate that we estimated after 1012 generations is an artifact of the fitness component that we examined. To test this hypothesis we examined whether our MA lines carry mutations that are beneficial across multiple fitness components: diploid growth, sporulation efficiency, spore viability, and haploid growth rate. If our previous results are due to us analyzing a fitness component that is either subject to stabilizing selection or antagonistic pleiotropy, then mutations accumulated in our lines will be conditionally beneficial and analyses of additional fitness components would yield different estimates of the beneficial mutation rate. We found that three of the four fitness components yield high estimates of the beneficial mutation rate. This suggests that multiple MA lines have accumulated beneficial mutations and that the high beneficial mutation rate that we previously estimated is not an artifact of the fitness component that we examined.Measuring multiple components of fitness also allowed us to examine the pleiotropic effects of beneficial and deleterious mutations. In general, we found that mutations altering one component of fitness have little effect on other components. However, lethal mutations were typically pleiotropic.

Conclusions:

We find that for three of four fitness components examined, a high frequency of spontaneous, fitness-altering mutations in diploid yeast is beneficial. Further, we do not detect pleiotropy of small-effect mutations, while lethal mutations show high levels of pleiotropy. In most cases, pleiotropy is positive. Two lines show evidence of antagonistic pleiotropy, indicating trade-offs, although heterozygote advantage cannot be ruled out.     

Transgenic markers for mammalian chimeras

Summary We describe the construction of aggregation chimeras between normal and transgenic embryos containing multiple copies of mouse -globin genes. The transgenic component of the chimeras is then detected in tissue sections by a DNA-DNA in situ hybridization technique, using a biotinylated DNA -globin probe and an avidin-linked alkaline phosphatase detection system. The general advantages of transgenic markers for chimeras are discussed.     

Budding Yeast Centrosome Duplication Requires Stabilization of Spc29 via Mps1-mediated Phosphorylation

Protein phosphorylation plays an important role in the regulation of centrosome duplication. In budding yeast, numerous lines of evidence suggest a requirement for multiple phosphorylation events on individual components of the centrosome to ensure their proper assembly and function. Here, we report the first example of a single phosphorylation event on a component of the yeast centrosome, or spindle pole body (SPB), that is required for SPB duplication and cell viability. This phosphorylation event is on the essential SPB component Spc29 at a conserved Thr residue, Thr²⁴⁰. Mutation of Thr²⁴⁰ to Ala is lethal at normal gene dosage, but an increased copy number of this mutant allele results in a conditional phenotype. Phosphorylation of Thr²⁴⁰ was found to promote the stability of the protein in vivo and is catalyzed in vitro by the Mps1 kinase. Furthermore, the stability of newly synthesized Spc29 is reduced in a mutant strain with reduced Mps1 kinase activity. These results demonstrate the first evidence for a single phosphorylation event on an SPB component that is absolutely required for SPB duplication and suggest that the Mps1 kinase is responsible for this protein-stabilizing phosphorylation.Centrosomes are critical for organizing microtubules that make up the mitotic and meiotic spindles that segregate chromosomes during cell division. The duplication of these organelles must be tightly regulated to occur once and only once during each cell cycle to prevent the formation of monopolar or multipolar mitotic spindles that can cause chromosomal instability. The yeast centrosome is called the spindle pole body (SPB)³ and is one of the best characterized microtubule-organizing centers. Although the SPB and the centrosome are morphologically distinct, they share the common function of spindle organization. Many SPB components and regulators of SPB assembly and function are conserved throughout evolution (1). This has made the yeast SPB an excellent model in which to study the regulation of centrosome duplication.The regulation of centrosome function and duplication by phosphorylation is well documented (2–10). Although several yeast SPB components are phosphoproteins in vivo (11–16), little is known about the specific sites of phosphorylation or the roles these modifications play in the regulation of SPB duplication and function. The yeast cyclin-dependent kinase Cdc28 and the multifunctional Mps1 kinase have both been implicated in the regulation of SPB components by phosphorylation (17–20). Two essential SPB components, Spc42 and Spc110, are phosphorylated by both of these kinases. Prevention of modification by either kinase alone is not detrimental, but the two kinases work in concert with each other to produce a fully functional protein. These examples demonstrate that some SPB components are coordinately regulated by the actions of more than one protein kinase and that an accumulation of hyperphosphorylation, rather than specific individual phosphorylation events, is the predominant mechanism of phosphoregulation of SPB components.In this study, we demonstrate that a single phosphothreonine, phospho-Thr²⁴⁰, near the C terminus of the SPB component Spc29 is absolutely required for SPB duplication and mitotic progression. The modification promotes the stability of the Spc29 protein and appears to be catalyzed by the Mps1 kinase. These results reveal the first single phosphorylation event known to be essential for SPB duplication and elucidate a mechanism by which cells can achieve tight regulation of centrosome duplication through a cascade of phosphorylation-mediated protein stabilization wherein the yeast cyclin-dependent kinase stabilizes the Mps1 kinase by phosphorylation (19), and the Mps1 kinase in turn stabilizes the Spc29 protein by phosphorylation, ensuring adequate levels of this critical SPB component for the assembly of new spindle poles.     

Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation

Nijmegen breakage syndrome arises from hypomorphic mutations in the NBN gene encoding nibrin, a component of the MRE11/RAD50/nibrin (MRN) complex. In mammalian cells, the MRN complex localizes to the nucleus, where it plays multiple roles in the cellular response to DNA double-strand breaks. In the current study, sequences in mouse nibrin required to direct the nuclear localization of the MRN complex were identified by site-specific mutagenesis. Unexpectedly, nibrin was found to contain both nuclear localizing signal (NLS) sequences and a nuclear export signal (NES) sequence whose functions were confirmed by mutagenesis. Both nuclear import and export sequences were active in vivo. Disruption of either the NLS or NES sequences of nibrin significantly altered the cellular distribution of nibrin and Mre11 and impaired survival after exposure to ionizing radiation. Mutation of the NES sequence in nibrin slowed the turnover of phosphorylated nibrin after irradiation, indicating that nuclear export of nibrin may function, in part, to downregulate posttranslationally modified MRN complex components after DNA damage responses are complete.Exposure to ionizing radiation (IR) results in a spectrum of damage to cells that includes the induction of DNA double-strand breaks (DSBs). In mammalian cells, sensing of DNA DSBs is extremely rapid, occurring within seconds of exposure to IR, and very sensitive, responding to as little as a single DSB in a cell. The sensitivity and speed of this response require immediate access to genomic DNA and raise the possibility that nuclear localization of key components of the damage-sensing or signaling cascade could play an important regulatory role in the process.The earliest measurable cellular response to DNA DSBs is phosphorylation of the protein kinase ATM on serine 1981. ATM exists normally in cells as an inactive dimer which, upon the induction of DNA DSBs, undergoes a transphosphorylation reaction and dissociates into active monomers (1). ATM is recruited to the sites of DNA DSBs via an interaction with the C-terminal end of the nibrin protein, amino acids 735 to 754 (9, 23), and subsequently phosphorylates nibrin (7, 10, 17, 21, 24) and other substrates. Phosphorylated nibrin then plays two key roles, one as a transducer of signals necessary to activate the S-phase checkpoint and the other as a scaffold for the recruitment and phosphorylation of other ATM substrates.The MRE11/RAD50/nibrin (MRN) complex, of which nibrin is a component, has well-defined DNA repair functions, including DNA binding and nuclease activity. Consistent with these functions, hypomorphic mutations in nibrin and MRE11 result in radiation sensitivity disorders, Nijmegen breakage syndrome (NBS) and ataxia telangiectasia-like disorder, respectively. MRE11 interacts with a conserved binding site at the C-terminal end of nibrin, adjacent to the binding site for ATM (6, 9, 23). In NBS cells, where full-length nibrin is absent, MRE11 and RAD50 lose their nuclear localization and are distributed randomly throughout the cell, indicating a requirement for nibrin to maintain the correct subcellular localization of the MRN complex (3). Similar effects are observed in ataxia telangiectasia-like disorder cells, which have mutations in MRE11 that impair its binding to nibrin (20). Nibrin mutants lacking the C-terminal 100 amino acids that include the MRE11 binding site localize to the nucleus when expressed in NBS cells but fail to relocalize either MRE11 or RAD50 or to complement the cellular radiosensitivity associated with NBS (6, 15). These results suggest that sequences mediating nuclear localization of nibrin are located 5′ of the C-terminal 100 amino acids.Given the critical role that nuclear localization plays in the function of the MRN complex, and hence the mammalian DNA DSB response, in the current study we used in vitro mutagenesis to map and identify sequences in mouse nibrin that affect the nuclear localization of the MRN complex. We demonstrate that the nuclear localization of nibrin and MRE11 represents an equilibrium state in a dynamic process of active import and export mediated by specific sequences in nibrin. Maintenance of this equilibrium by nibrin-mediated shuttling between the cytoplasm and the nucleus is required for normal cellular responses to DNA DSBs and may play a role in downregulating responses after damage.     

Down-regulation of Mitochondrial Acyl Carrier Protein in Mammalian Cells Compromises Protein Lipoylation and Respiratory Complex I and Results in Cell Death

The objective of this study was to evaluate the physiological importance of the mitochondrial fatty acid synthesis pathway in mammalian cells using the RNA interference strategy. Transfection of HEK293T cells with small interfering RNAs targeting the acyl carrier protein (ACP) component reduced ACP mRNA and protein levels by >85% within 24 h. The earliest phenotypic changes observed were a marked decrease in the proportion of post-translationally lipoylated mitochondrial proteins recognized by anti-lipoate antibodies and a reduction in their catalytic activity, and a slowing of the cell growth rate. Later effects observed included a reduction in the specific activity of respiratory complex I, lowered mitochondrial membrane potential, the development of cytoplasmic membrane blebs containing high levels of reactive oxygen species and ultimately, cell death. Supplementation of the culture medium with lipoic acid offered some protection against oxidative damage but did not reverse the protein lipoylation defect. These observations are consistent with a dual role for ACP in mammalian mitochondrial function. First, as a key component of the mitochondrial fatty acid biosynthetic pathway, ACP plays an essential role in providing the octanoyl-ACP precursor required for the protein lipoylation pathway. Second, as one of the subunits of complex I, ACP is required for the efficient functioning of the electron transport chain and maintenance of normal mitochondrial membrane potential.Eukaryotes employ two distinct systems for the synthesis of fatty acids de novo. The bulk of fatty acids destined for membrane biogenesis and energy storage are synthesized in the cytosolic compartment by megasynthases in which the component enzymes are covalently linked in very large polypeptides; this system is referred to as the type I fatty acid synthase (FAS)² (1, 2). A second system localized in mitochondria is composed of a suite of discrete, freestanding enzymes that closely resemble their counterparts in prokaryotes (3–10), which are characterized as type II FASs (11). Most of the constituent enzymes of the mitochondrial fatty acid biosynthetic system have been identified and characterized in fungi and animals; all are nuclear-encoded proteins that are transported to the matrix compartment of mitochondria. Fungi with deleted mitochondrial FAS genes fail to grow on non-fermentable carbon sources, have low levels of lipoic acid and elevated levels of mitochondrial lysophospholipids (12, 13). These observations indicate that the mitochondrial FAS may serve to provide the octanoyl precursor required for the biosynthesis of lipoyl moieties de novo, as well as providing fatty acids that are utilized in remodeling of mitochondrial membrane phospholipids (14). The mitochondrial FAS system in animals is less well characterized. However, kinetic analysis of the β-ketoacyl synthase enzyme responsible for catalysis of the chain extension reaction in human mitochondria suggested that this system is uniquely engineered to produce mainly octanoyl moieties and has limited ability to form long-chain products (9). Indeed, studies with a reconstituted system from bovine heart mitochondrial matrix extracts confirmed that octanoyl moieties are the main product and are utilized for the synthesis of lipoyl moieties (15). One of the key components of the prokaryotic and mitochondrial FAS systems is a small molecular mass, freestanding protein, the ACP, that shuttles substrates and pathway intermediates to each of the component enzymes. The mitochondrial ACP is localized primarily in the matrix compartment (16), but a small fraction is integrated into complex I of the electron transport chain (17–23). As is the case with many of the other 45 subunits of complex I, the role of the ACP subunit is unclear (24). To clarify the physiological importance of the mitochondrial FAS, and the mitochondrial ACP in particular, in mammalian mitochondrial function we have utilized an RNA interference strategy to knockdown the mitochondrial ACP in cultured HEK293T cells.     

Novel EGFR inhibitors prepared by combination of dithiocarbamic acid esters and 4-anilinoquinazolines

On the basis of combination strategy, a novel series of EGFR inhibitors were designed and synthesized by combination of dithiocarbamic acid esters and 4-anilinoquinazolines. The effect of the synthesized compounds on cell proliferation was evaluated by MTT assay in three human cancer cell lines: MDA-MB-468, SK-BR-3 and HCT-116. Two compounds (11d and 11f) were found more potent against all three cell lines and five compounds (11a, 11d-11g) were found more potent against both MDA-MB-468 and SK-BR-3 than Lapatinib. SAR studies revealed that the substituents on C6 and C7 positions of quinazoline, the amine component of dithiocarbamate moiety and the linker greatly affected the activity. This work provides a promising new strategy for the preparation of potent tyrosine kinase inhibitors.     

Chemical communication in heliothine moths

The chemical and temporal features of the sex-pheromone emitted by Heliothis virescens females are encoded by a diverse array of output pathways from the male-specific macroglomerular complex (MGC) in the antennal lobe. Most output neurons (29 out of 32) were activated by antennal stimulation with the principal component of the sex-pheromone blend of this species, (Z)-11-hexadecenal. Six neurons were excited solely by this component, 8 neurons also responded to the second essential blend component, (Z)-9-tetradecenal, and 14 neurons displayed equivalent responses to the two. Many neurons also effectively encoded the onset and duration of the stimulus. In one additional neuron, a prolonged excitatory response (synergism) was evoked only by the blend of the two components, indicating that some MGC neurons function as blend detectors.In contrast to the situation in Helicoverpa zea, none of the MGC neurons in H. virescens responded selectively to (Z)-9-tetradecenal, suggesting that these two noctuid species employ different neural strategies to encode information about their respective pheromone blends.Three MGC-output neurons responded selectively to (Z)-11-hexadecenyl acetate, an odorant released by some sympatric species that disrupts normal upwind flight to pheromones. Thus, changes in the attractant and deterrent chemical signals, as well as the physical features of these odor plumes, are encoded in the MGC across a diverse parallel array of output pathways to the protocerebrum.Abbreviations AL antennal lobe - AN antennal nerve - 16:AL hexadecanal - MGC macroglomerular complex - 14:AL tetradecanal - Z11-16:AL (Z) 11-hexadecenal - Z11-16:AC (Z) 11-hexadecenyl acetate - Z9-14:AL (Z) 9-tetradecenal - Z9-14:FO (Z) 9-tetradecenyl formate     

Contribution of a Xylan-Binding Module to the Degradation of a Complex Cellulosic Substrate by Designer Cellulosomes

Conversion of components of the Thermobifida fusca free-enzyme system to the cellulosomal mode using the designer cellulosome approach can be employed to discover the properties and inherent advantages of the cellulosome system. In this article, we describe the conversion of the T. fusca xylanases Xyn11A and Xyn10B and their synergistic interaction in the free state or within designer cellulosome complexes in order to enhance specific degradation of hatched wheat straw as a model for a complex cellulosic substrate. Endoglucanase Cel5A from the same bacterium and its recombinant dockerin-containing chimera were also studied for their combined effect, together with the xylanases, on straw degradation. Synergism was demonstrated when Xyn11A was combined with Xyn10B and/or Cel5A, and ∼1.5-fold activity enhancements were achieved by the designer cellulosome complexes compared to the free wild-type enzymes. These improvements in activity were due to both substrate-targeting and proximity effects among the enzymes contained in the designer cellulosome complexes. The intrinsic cellulose/xylan-binding module (XBM) of Xyn11A appeared to be essential for efficient substrate degradation. Indeed, only designer cellulosomes in which the XBM was maintained as a component of Xyn11A achieved marked enhancement in activity compared to the combination of wild-type enzymes. Moreover, integration of the XBM in designer cellulosomes via a dockerin module (separate from the Xyn11A catalytic module) failed to enhance activity, suggesting a role in orienting the parent xylanase toward its preferred polysaccharide component of the complex wheat straw substrate. The results provide novel mechanistic insight into the synergistic activity of designer cellulosome components on natural plant cell wall substrates.Thermobifida fusca is an aerobic thermophilic soil bacterium with strong cellulolytic activity (52). The T. fusca enzyme system is an extensively studied free cellulase system in which nearly all of the cellulolytic enzymes have been fully characterized, from the individual enzyme sequences to the three-dimensional structures, as well as the biochemical activities of the native and recombinant proteins. The genome sequence has been published (36), and the number and types of carbohydrate-active enzymes produced by the organism are known. This actinomycete produces six different cellulases that have been well studied (29, 31, 32, 50, 52). T. fusca also has the ability to grow on xylan and produces several enzymes involved in xylan degradation, such as xylanases, β-xylosidase, α-l-arabinofuranosidase, and acetylesterases (1, 21).Previous research has suggested that the multienzyme cellulosome complex from Clostridium thermocellum is far more efficient than free cellulase systems that were tested in degrading polysaccharides (33). The cellulosome system is characterized by the strong bimodular interaction between the cohesin and dockerin modules that integrates the various enzymes into the complex (5, 35, 55). Scaffoldin subunits (nonenzymatic protein components) contain the cohesin modules that incorporate the enzymes into the complex via their resident dockerins. The primary scaffoldin subunit also includes a carbohydrate (cellulose)-binding module (CBM) through which the complex recognizes and binds to the cellulosic substrate (42, 46).In order to evaluate the reasons for the apparent advantage of cellulosomes over free enzymes, it is interesting to compare the properties of the best-characterized free-enzyme systems for degradation of polysaccharides with those of the best-studied cellulosome system. We have initiated a program to convert the free-enzyme system of T. fusca into an artificial designer cellulosome (11-13). The designer cellulosome concept is based on the very high affinity (20, 44) and specific interaction (37, 43, 55) between a cohesin and a dockerin module from the same species. Since the various scaffoldin-borne cohesins of a given species essentially show the same specificity of binding for the enzyme-borne dockerins, designer cellulosomes are constructed from recombinant chimeric scaffoldins containing divergent cohesins from different species, for which matching dockerin-containing enzyme hybrids are prepared, as a platform for promoting synergistic action among enzyme components (5). Free cellulases from the T. fusca system were converted to the cellulosomal mode by replacing their native CBM with a desired dockerin module, and in some cases, the resultant “designer cellulosomes” exhibited enhanced synergistic activity on crystalline cellulosic substrates compared to that of the mixture of wild-type enzymes (11).In this study, we incorporated xylanolytic enzymes into designer cellulosomes and investigated their hydrolytic effects on purified xylans and on a native, complex cellulosic substrate (hatched wheat straw). We focused on T. fusca xylanases 11A and 10B (Xyn11A and Xyn10B), which are the most abundant xylanases produced during growth on xylan (34). Xyn11A and Xyn10B function as endoxylanases (28, 34); Xyn11A contains a C-terminal family 2 CBM that binds both cellulose and xylan, whereas Xyn10B lacks a CBM. In some experiments, one of the previously converted (dockerin-containing) T. fusca endoglucanases, f-5A (11), was also introduced into the designer cellulosomes in order to evaluate cooperation between xylanases and cellulases in hydrolysis of a natural substrate. This study contributes primary information concerning a major feature of cellulosomes that had not been suitably addressed in earlier research: although xylanases are integral components of cellulosomes, their synergistic action in the cellulosome mode has yet to be examined experimentally. The xylan-binding CBM (termed XBM for the purposes of this report) was found to contribute to the activity of the parent Xyn11A enzyme.     

Propagation of viral genomes by replicating ammonia-oxidising archaea during soil nitrification

Ammonia-oxidising archaea (AOA) are a ubiquitous component of microbial communities and dominate the first stage of nitrification in some soils. While we are beginning to understand soil virus dynamics, we have no knowledge of the composition or activity of those infecting nitrifiers or their potential to influence processes. This study aimed to characterise viruses having infected autotrophic AOA in two nitrifying soils of contrasting pH by following transfer of assimilated CO₂-derived ¹³C from host to virus via DNA stable-isotope probing and metagenomic analysis. Incorporation of ¹³C into low GC mol% AOA and virus genomes increased DNA buoyant density in CsCl gradients but resulted in co-migration with dominant non-enriched high GC mol% genomes, reducing sequencing depth and contig assembly. We therefore developed a hybrid approach where AOA and virus genomes were assembled from low buoyant density DNA with subsequent mapping of ¹³C isotopically enriched high buoyant density DNA reads to identify activity of AOA. Metagenome-assembled genomes were different between the two soils and represented a broad diversity of active populations. Sixty-four AOA-infecting viral operational taxonomic units (vOTUs) were identified with no clear relatedness to previously characterised prokaryote viruses. These vOTUs were also distinct between soils, with 42% enriched in ¹³C derived from hosts. The majority were predicted as capable of lysogeny and auxiliary metabolic genes included an AOA-specific multicopper oxidase suggesting infection may augment copper uptake essential for central metabolic functioning. These findings indicate virus infection of AOA may be a frequent process during nitrification with potential to influence host physiology and activity.Subject terms: Microbial ecology, Stable isotope analysis

Microbially mediated oxidation of ammonia to nitrate during nitrification is a central component of the global nitrogen (N) cycle. It is also responsible for major losses of applied fertiliser N in soil, generating atmospheric pollution via direct and indirect production of nitrous oxide (N₂O) as well as nitrate (NO₃^-) pollution of groundwater [1]. Autotrophic ammonia-oxidising archaea (AOA) of the class Nitrososphaeria are a ubiquitous component of soil microbial communities and often dominate ammonia oxidation and nitrification-associated N₂O emissions when ammonia is supplied at low rates via organic matter mineralisation [2], slow-release fertilisers [3] or in acidic soils [4]. Integrated temperate viruses (proviruses) and other virus-associated protein-encoding genes are found in most AOA genomes suggesting frequent interaction (see Supplementary Text). While viruses infecting marine AOA have been characterised through metagenomic approaches [5] and cultivation [6], those infecting soil AOA or other nitrifier groups are currently uncharacterised.Virus infection can influence biogeochemical cycling by augmenting host activity or causing cell mortality and subsequent release of nutrients [7]. Recent advances have demonstrated that soil virus communities are dynamic in a wide range of soils [e.g. 8, 9] and augmenting virus loads modulate C and N fluxes [10, 11]. Nevertheless, identifying active interactions with specific populations or functional groups in soil remains challenging due to structural complexity and the vast diversity of hosts and viruses. Recent use of stable-isotope approaches has investigated whole community host-virus dynamics [12, 13] or interactions between individual host-virus populations specific to a functional process and substrate [14]. The aim of this study was to utilise the latter approach with ¹³CO₂-based DNA-SIP to focus on nitrification-associated interactions and to test the hypothesis that viruses are a dynamic component of soil AOA activity.     

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 ± 4% of the GABAergic synapses and 25 ± 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.We have recently developed a method for the preparation of a brain fraction enriched in GABAergic postsynaptic complex (1). This fraction, insoluble in Triton X-100, was enriched in Gray''s type-II postsynaptic densities (type-II PSDs)² and in the postsynaptic GABAergic markers GABA_A receptors (GABA_ARs) and gephyrin. Here we report that septin 11 is a major component of the type-II PSD fraction.Septins are a family of proteins with GTPase activity that form heterooligomeric filaments and ringlike structures that act as diffusion barriers and scaffolds. Septins are involved in cytokinesis, positioning of the mitotic spindle, cellular morphology, vesicle trafficking, apoptosis, neurodegeneration, and neoplasia (2–5). In mammals, 14 septin genes have been identified. Each septin gene is expressed in several spliced forms. Although most septins are highly expressed in the brain (6), only recently is their role in neuronal function (7–9) and in neuropathology (10–14) is beginning to be addressed for some septins.Septin 11 is expressed in various tissues, including the brain (15), but little is known about the role of septin 11 in the brain. Septins 3, 5, 6, and 7 are localized in the presynaptic terminals, frequently associated with synaptic vesicles (6, 16, 17). In neurons, septin 11 forms heterooligomeric complexes with septin 7 and septin 5 (9, 18). Nevertheless, the regional and developmental distribution of septin 11 in the brain and in hippocampal cultures is not identical to that of septin 7 or septin 5 (8). These results and other heterooligomerization studies show that septin 11 is not always associated with septin 7 and septin 5 (7, 15, 19). Thus, septin 11 is expected to have functional properties both similar to and different from those of septin 7 and other septins that heterooligomerize with septin 11. In the present paper, we show that septin 11 is associated with the GABAergic synapses, particularly with the postsynapse, and concentrates at the neck of dendritic spines in the intact brain. Others have recently shown that another septin (septin 7) accumulates at the base of dendritic protrusions of cultured neurons (8, 9). However, it is not known whether septins also accumulate at the base of the dendritic spines in the brain. To the best of our knowledge, this is the first time that (i) a septin has been shown to be associated with GABAergic synapses and (ii) a septin has been shown to concentrate at the neck of dendritic spines and dendritic branching points in the intact brain.     

Genetic deletion of the mitochondrial phosphate carrier desensitizes the mitochondrial permeability transition pore and causes cardiomyopathy

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca²⁺ challenge and led to a greater Ca²⁺ uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca²⁺ overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy.The mitochondrial oxidative phosphorylation (OXPHOS) system is the primary source of cellular energy production. Defects in OXPHOS occur with a frequency of 1 in 5000 live births¹ and underlie a wide range of mitochondrial disorders that often affect multiple organ systems and tissues with high oxidative energy demands, such as brain, skeletal muscle, and heart.² Cardiac phenotypes associated with mitochondrial disease are diverse, and can range from cardiomyopathies to cardiac conduction defects.^{3, 4, 5}The mitochondrial phosphate carrier (PiC) is a member of the solute carrier 25A family that has a critical role in OXPHOS, serving as the primary route for inorganic phosphate (Pi) import into the mitochondrial matrix.^{6, 7} PiC, together with the adenine nucleotide translocator (ANT) and the ATP synthase, forms the ATP synthasome whereby all of the metabolites needed to generate ATP are within one immediate microdomain.^{8, 9} The importance of PiC in facilitating energy production is highlighted by the profound disease phenotype observed in patients presenting with mutations in the skeletal muscle-specific isoform of this gene.^{10, 11} Such patients present with a multisystemic disorder characterized by muscle hypotonia, lactic acidosis, severe hypertrophic cardiomyopathy, and shortened lifespan.^{10, 11} Similarly, patients with SLC25A4 (ANT1 protein) deficiency present with cardiomyopathy,¹² as do mice lacking the Slc25a4 gene,¹³ likely due to a similar molecular defect in the efficiency of ATP production within the mitochondria.In addition to its role in mitochondrial energy metabolism, PiC has been implicated in regulating cell death by serving either as a modulator or a direct component of the mitochondrial permeability transition pore (MPTP).^{14, 15, 16} The MPTP is a non-selective channel that forms in response to Ca²⁺ overload and oxidant stress that allows inner-membrane permeability to solutes up to 1500 Da in size, leading to loss of mitochondrial membrane potential, mitochondrial swelling and rupture, and eventually cell death through necrosis.¹⁵ Structurally, the MPTP complex has been proposed to be comprised of the ATP synthase^{17, 18} and to be regulated by cyclophilin D (CypD),^{19, 20} ANT,²¹ and the pro-apoptotic proteins Bax and Bak in the outer mitochondrial membrane.^{22, 23} PiC has also been suggested to be an inner-membrane component of the MPTP because it can form nonspecific channels in lipid membranes and because the MPTP is known to be activated by Pi.^{24, 25, 26, 27} Finally, PiC directly interacts with CypD in the mitochondrial matrix, which is a verified regulator and component of the MPTP.¹⁶ Saccharomyces cerevisiae lacking PiC have altered MPTP characteristics with a smaller pore size, suggesting it might directly participate in the mitochondrial permeability pore.²⁸ However, partial reduction of PiC by siRNAs in cultured cells had no effect on mitochondrial permeability activity, suggesting that PiC is not required for MPTP function.²⁷ Definitive genetic proof of PiC''s involvement in MPTP formation/function is currently lacking.In the present study, we tested the role of PiC in MPTP regulation and cell death in vivo using a mouse model with inducible cardiomyocyte-specific deletion of the Slc25a3 gene (encodes PiC). We found that cardiac mitochondria depleted of PiC were able to undergo permeability transition, suggesting that PiC is not a requisite component of the MPTP. However, the extent of Ca²⁺-induced MPTP opening was blunted, suggesting that PiC serves to regulate this activity. Furthermore, Slc25a3 deletion produced a unique mouse model of mitochondrial-driven hypertrophic cardiomyopathy that recapitulates features observed in human patients with phosphate carrier deficiency and metabolic cardiomyopathy.     

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine–arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.DNA double-stranded breaks (DSBs)³ can be produced in physiological and genotoxic processes. Improper repair or failure to repair DSBs can lead to gene deletions, duplications, translocations, and missegregation of large chromosome fragments, which may result in gene dosage imbalance, cancer development, or cell death (1–3). Historically, two distinct pathways have been described which ensure that DSBs are repaired: nonhomologous end joining (NHEJ) and homologous recombination (HR). During HR, the damaged chromosome interacts via synapsis with an undamaged DNA molecule with which it shares extensive sequence homology, usually its sister chromatid (4, 5). HR is most active in the late S and G₂ phases of the cell cycle. In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6, 7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8, 9).Two types of end-joining reactions can be defined operationally. The first one, which may be called conservative NHEJ (C-NHEJ), is characterized by the precise joining of short, overhanging, complementary ends. Proteins including Ku70/Ku80 and XRCC4 (10–12) are associated with this highly efficient pathway, whereby most ends are rejoined successfully without any alteration of the DNA sequence (8). The alternative pathway for NHEJ is the highly mutagenic and deletional NHEJ (D-NHEJ), which results in short deletions after use of imperfect microhomology of about 1–10 bp at the repair junctions. D-NHEJ activity has been demonstrated in the budding yeast Saccharomyces cerevisiae. In addition, D-NHEJ is independent of Rad52, Rad1, or Ku80 but depends on Mre11 in yeast (13, 14). However, the genetic determinants of this subpathway have not been well established in mammalian cells.Mre11 is the core subunit of the Mre11·Rad50·Nbs1 complex (called the MRN complex), which is conserved throughout all kingdoms of life. The MRN complex is a central player in most aspects of the cellular response to DSBs, including HR, NHEJ, telomere maintenance, and DNA damage checkpoints (15–17). Loss of Mre11 results in increased radiosensitivity and chromosomal instability (17). Patients with germ line mutations of Mre11 have clinical presentations similar to those of ataxia telangiectasia patients (ataxia telangiectasia-like disorder) (18).After DNA damage, the MRN complex is recruited to the sites of damage via zinc hooks at the ends of the long, flexible arms of Rad50 (19, 20). Mre11 contains both single-stranded DNA endonuclease and 3′-5′ exonuclease activities in vitro, but in vivo Mre11 is also implicated in 5′-3′ DSB resection. The MRN complex also interacts with BRCA1 and CtIP, which may be essential for DSB end resection to generate 3′ overhanging single-stranded DNA during initiation of HR (21, 22).Mre11 has an N-terminal nuclease domain, which contains five phosphoesterase motifs, and a C-terminal glycine–arginine-rich domain (GAR). Arthur et al. (23) showed that an H85L mutation completely abrogated exonuclease activity, whereas binding to Rad50 and Nbs1 was retained. Complementation of ataxia telangiectasia-like disorder cells with this mutant, called Mre11-3, restored the localization of the MRN complex to DSBs in IR-induced foci (23, 24). Methylation of the GAR region has also been shown to be important for the DNA binding and exonuclease activity of Mre11 in vitro (25, 26). Both the crystal structure of yeast Mre11 and data from conditional knock-out mice (Mre11^H129N/Δ) reveal that the nuclease activity of Mre11 is required for HR repair of DSBs (22, 27). However, the role of Mre11 in NHEJ is not well defined (27, 28). Most recently, Mre11 was reported to support NHEJ in mammalian cell (29–31). However, whether Mre11 regulates both NHEJ subpathways or only D-NHEJ is controversial, and the mechanisms by which Mre11 is involved in NHEJ remain to be established.To address these questions, we have established a system that can analyze the accuracy and efficiency of rejoining of two adjacent DSB ends at chromosomal level in human embryonic kidney 293 (HEK293) cells. We show here that Mre11 siRNA knockdown in these cells results in significant reduction of the overall NHEJ efficiency. Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-NHEJ by ∼10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of C-NHEJ. Mutation of Mre11 in either the phosphoesterase domain (Mre11-3) or the GAR region (Mre11-R/A) to produce abnormal exonuclease activity impaired the D-NHEJ pathway only. The D-NHEJ deficiency is significantly more severe in cells with Mre11-R/A than that in cells with Mre11-3. Therefore, our data suggest that Mre11 is required specifically for D-NHEJ repair of DNA DSBs and that its exonuclease activity is at least one of the important mechanisms for this DNA end joining subpathway.     

MaleHeliothis virescens maintain upwind flight in response to experimentally pulsed filaments of their sex pheromone (Lepidoptera: Noctuidae)

Aspects of the intermittent fine structure of an odor plume were mimicked and experimentally modified in the wind tunnel using an air-pulsing device. Filaments of a behaviorally active blend of six sex-pheromone components created by the device in a temporally regular fashion elicited sustained upwind flight and source location in male Heliothis virescens.Males did not exhibit sustained upwind flight in significant numbers until a frequency of 4 filaments/s was delivered, at a loading of 1 g of the major component, Z11-16: Ald, with the other components loaded at their appropriate ratios. A loading of 10 g Z11-16: Ald was found to be optimal at this filament delivery rate. Electroantennogram recordings to different filament delivery rates of the complete blend indicated that a stationary male antenna can respond to the pulse rates used in this study. Importantly, when a main component necessary for upwind flight, Z9-14:Ald, was isolated into its own filaments and pulsed alternately against filaments of the five other components (including the other component essential for upwind flight, Z11-16: Ald), upwind flight to the source was significantly reduced (9%) compared to upwind flight and source location in response to filaments composed of the entire blend (30%), indicating that the complete pheromone blend must arrive on the antenna simultaneously for optimal evocation of sustained upwind progress. Neurophysiological evidence from other studies suggests that higher-order interneurons whose phasic response is enhanced when the entire blend is presented simultaneously may be of importance in explaining this behavioral difference stemming from synchronous vsasynchronous arrival of the components.     

Proteose-peptone fractions of human and bovine milk

• 1.1. Human milk proteose-peptone fraction contained an average of 45% carbohydrate compared to about 11% carbohydrate present in the bovine milk fraction.
• 2.2. The human milk proteose-peptone fraction contained Lactobacillus bifidus var. Penn. growthpromoting factors, whereas there was very little such growth-promoting activity in the bovine milk material.
• 3.3. Polyacrylamide gel electrophoresis showed that human milk proteose-peptone fraction contained 4 major protein components with the respective mol. wts of approx 100,000, 70,000, 30,000, and 13,000. The latter was the most abundant component of human milk proteose-peptone fraction. There was, in addition a very low mol. wt carbohydrate-containing component. Bovine milk proteose-peptone fraction had 3 major protein components with mol. wts of 30,000, 18,000 and 12,000.
• 4.4. The human milk proteose-peptone component with the mol. wt of 13,000 was identified as α-lactalbumin, the component with the mol. wt of 30,000 was identified as a temperature-sensitive protein probably similar to galactothermin, and the components having mol. wts of 100,000 and 70,000 were closely associated to give a protein of a very high mol. wt. This complex also contained carbohydrate and was thus glycoprotein in nature.
• 5.5. The low-mol. wt carbohydrate-containing material was identified as being similar if not identical to glycoprotein B previously described.

     

Post-translational processing of 7S and 11S components of soybean storage proteins

The synthesis and processing of the major storage proteins in soybean cotyledons was studied both in vivo and in vitro. The and subunits of 7S as well as the 11S proteins are synthesized as higher molecular weight-precursors on membrane-bound polysomes. The initial translation products of the 7S are proteolytically cleaved during translation suggesting the removal of a signal peptide as evidenced by the presence of 2 and 2 peptides immunoreactive with 7S antibody in the in vitro chain completion products of the membrane-bound polysomes. This is followed or accompanied by cotranslational glycosylation, which increases their size equivalent to that of initially-synthesized precursors. In vivo pulse-labelled 7S and products are of slightly higher molecular weights than the immunoprecipitable chain-completion products, indicating further post-translational modifications. A slow post-translational processing during a period of 1.5 to 16 h yields the final 7S and glycoproteins.Acidic and basic subunits of the 11S protein appear to be synthesized from common large molecular weight (60K-59K) precursors. Antibodies to the 11S acidic component recognize both acidic and basic domains in the precursor while those raised against basic subunits appear to be specific for that region only. The processing of the 11S precursor is also very slow and occurs post-translationally. This slow rate of processing, coupled with a temporal difference in the synthesis of 7S and 11S components, suggests a highly coordinated mechanism for synthesis and packaging of these proteins into protein bodies during seed development.