Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13^th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.     

Evidence for a Slow-Turnover Form of the Ca2+-Independent Phosphoenolpyruvate Carboxylase Kinase in the Aleurone-Endosperm Tissue of Germinating Barley Seeds

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in aleurone-endosperm extracts of barley (Hordeum vulgare) seeds during germination, and specific anti-sorghum (Sorghum bicolor) C₄ PEPC polyclonal antibodies immunodecorated constitutive 103-kD and inducible 108-kD PEPC polypeptides in western analysis. The 103- and 108-kD polypeptides were radiolabeled in situ after imbibition for up to 1.5 d in ³²P-labeled inorganic phosphate. In vitro phosphorylation by a Ca²⁺-independent PEPC protein kinase (PK) in crude extracts enhanced the enzyme''s velocity and decreased its sensitivity to l-malate at suboptimal pH and [PEP]. Isolated aleurone cell protoplasts contained both phosphorylated PEPC and a Ca²⁺-independent PEPC-PK that was partially purified by affinity chromatography on blue dextran-agarose. This PK activity was present in dry seeds, and PEPC phosphorylation in situ during imbibition was not affected by the cytosolic protein-synthesis inhibitor cycloheximide, by weak acids, or by various pharmacological reagents that had proven to be effective blockers of the light signal transduction chain and PEPC phosphorylation in C₄ mesophyll protoplasts. These collective data support the hypothesis that this Ca²⁺-independent PEPC-PK was formed during maturation of barley seeds and that its presumed underlying signaling elements were no longer operative during germination.Higher-plant PEPC (EC 4.1.1.31) is subject to in vivo phosphorylation of a regulatory Ser located in the N-terminal domain of the protein. In vitro phosphorylation by a Ca²⁺-independent, low-molecular-mass (30–39 kD) PEPC-PK modulates PEPC regulation interactively by opposing metabolite effectors (e.g. allosteric activation by Glc-6-P and feedback inhibition by l-malate; Andreo et al., 1987), decreasing significantly the extent of malate inhibition of the leaf enzyme (Carter et al., 1991; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). These metabolites control the rate of phosphorylation of PEPC via an indirect target-protein effect (Wang and Chollet, 1993; Echevarría et al., 1994; Vidal and Chollet, 1997).Several lines of evidence support the view that this protein-Ser/Thr kinase is the physiologically relevant PEPC-PK (Li and Chollet, 1993; Chollet et al., 1996; Vidal et al., 1996; Vidal and Chollet, 1997). The presence and inducible nature of leaf PEPC-PK have been established further in various C₃, C₄, and CAM plant species (Chollet et al., 1996). In all cases, CHX proved to be a potent inhibitor of this up-regulation process so that apparent changes in the turnover rate of PEPC-PK itself or another, as yet unknown, protein factor were invoked to account for this observation (Carter et al., 1991; Jiao et al., 1991; Chollet et al., 1996). Consistent with this proposal are recent findings about PEPC-PK from leaves of C₃, C₄, and CAM plants that determined activity levels of the enzyme to depend on changes in the level of the corresponding translatable mRNA (Hartwell et al., 1996).Using a cellular approach we previously showed in sorghum (Sorghum bicolor) and hairy crabgrass (Digitaria sanguinalis) that PEPC-PK is up-regulated in C₄ mesophyll cell protoplasts following illumination in the presence of a weak base (NH₄Cl or methylamine; Pierre et al., 1992; Giglioli-Guivarc''h et al., 1996), with a time course (1–2 h) similar to that of the intact, illuminated sorghum (Bakrim et al., 1992) or maize leaf (Echevarría et al., 1990). This light- and weak-base-dependent process via a complex transduction chain is likely to involve sequentially an increase in pHc, inositol trisphosphate-gated Ca²⁺ channels of the tonoplast, an increase in cytosolic Ca²⁺, a Ca²⁺-dependent PK, and PEPC-PK.Considerably less is known about the up-regulation of PEPC-PK and PEPC phosphorylation in nongreen tissues. A sorghum root PEPC-PK purified on BDA was shown to phosphorylate in vitro both recombinant C₄ PEPC and the root C₃-like isoform, thereby decreasing the enzyme''s malate sensitivity (Pacquit et al., 1993). PEPC from soybean root nodules was phosphorylated in vitro and in vivo by an endogenous PK (Schuller and Werner, 1993; Zhang et al., 1995; Zhang and Chollet, 1997). A Ca²⁺-independent nodule PEPC-PK containing two active polypeptides (32–37 kD) catalyzed the incorporation of phosphate on a Ser residue of the target enzyme and was modulated by photosynthate transported from the shoots (Zhang and Chollet, 1997). Regulatory seryl phosphorylation of a heterotetrameric (α₂β₂) banana fruit PEPC by a copurifying, Ca²⁺-independent PEPC-PK was shown to occur in vitro (Law and Plaxton, 1997). Although phosphorylation was also detected in vivo and found to concern primarily the α-subunit, PEPC exists mainly in the dephosphorylated form in preclimacteric, climacteric, and postclimacteric fruit.In a previous study we showed that PEPC undergoes regulatory phosphorylation in aleurone-endosperm tissue during germination of wheat seeds (Osuna et al., 1996). Here we report on PEPC and the requisite PEPC-PK in germinating barley (Hordeum vulgare) seeds. PEPC was highly phosphorylated by a Ca²⁺-independent Ser/Thr PEPC-PK similar to that found in other plant systems studied previously (Chollet et al., 1996); however, the PK was already present in the dry seed and its activity did not require protein synthesis during imbibition.     

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.     

Evidence for a dual pattern of cranial venous sinuses on the endocranial cast of Taung (Australopithecus africanus)

According to published accounts, an enlarged occipital-marginal sinus system is absent in Australopithecus africanus, although it occurs in high frequencies in A. robustus, A. Boisei, and Hadar hominids commonly designated A. afarensis. In this report, we describe, for the first time, an enlarged occipital-marginal sinus system on the endocranial cast of the Taung specimen, which is part of the holotype of A. africanus. In addition, well-developed right transverse and sigmoid sinuses are represented on the Taung endocast. The various components of the dual venous sinus system on the Taung endocast are measured, and the system is compared to those of other fossil hominids. The compresence of a lateral sinus system and enlarged occipital and marginal sinuses occurs in two Hadar specimens, 2 specimens of A. robustus crassidens, 1 A. boisei specimen, and several early H. sapiens crania. Hence, the presence of strong transverse sinus impressions in a fragmentary specimen may not be interpreted as an indication that an enlarged occipital-marginal sinus system was not present in the original specimen. Conversely, lack of transverse sinus grooves in a fragmentary specimen does provide indirect evidence than an enlarged occipital-marginal system would probably have been present in the whole specimen, as in 2 specimens of A. boisei. Including Taung, enlarged occipital and marginal sinuses occur in 1 out of 5, or 20%, of A. africanus specimens. This figure compares well with the range of mean frequencies in modern human cranial series (1.5 to 28%), but is much lower than are the frequencies for A. boisei, A. robustus, and the Hadar hominids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

In Vivo Evidence for Platelet-Induced Physiological Angiogenesis by a COX Driven Mechanism

We sought to determine a role for platelets in in vivo angiogenesis, quantified by changes in the capillary to fibre ratio (C∶F) of mouse skeletal muscle, utilising two distinct forms of capillary growth to identify differential effects. Capillary sprouting was induced by muscle overload, and longitudinal splitting by chronic hyperaemia. Platelet depletion was achieved by anti-GPIbα antibody treatment. Sprouting induced a significant increase in C∶F (1.42±0.02 vs. contralateral 1.29±0.02, P<0.001) that was abolished by platelet depletion, while the significant C∶F increase caused by splitting (1.40±0.03 vs. control 1.28±0.03, P<0.01) was unaffected. Granulocyte/monocyte depletion showed this response was not immune-regulated. VEGF overexpression failed to rescue angiogenesis following platelet depletion, suggesting the mechanism is not simply reliant on growth factor release. Sprouting occurred normally following antibody-induced GPVI shedding, suggesting platelet activation via collagen is not involved. BrdU pulse-labelling showed no change in the proliferative potential of cells associated with capillaries after platelet depletion. Inhibition of platelet activation by acetylsalicylic acid abolished sprouting, but not splitting angiogenesis, paralleling the response to platelet depletion. We conclude that platelets differentially regulate mechanisms of angiogenesis in vivo, likely via COX signalling. Since endothelial proliferation is not impaired, we propose a link between COX1 and induction of endothelial migration.     

Kinematic Validation of a Multi-Kinect v2 Instrumented 10-Meter Walkway for Quantitative Gait Assessments

Walking ability is frequently assessed with the 10-meter walking test (10MWT), which may be instrumented with multiple Kinect v2 sensors to complement the typical stopwatch-based time to walk 10 meters with quantitative gait information derived from Kinect’s 3D body point’s time series. The current study aimed to evaluate a multi-Kinect v2 set-up for quantitative gait assessments during the 10MWT against a gold-standard motion-registration system by determining between-systems agreement for body point’s time series, spatiotemporal gait parameters and the time to walk 10 meters. To this end, the 10MWT was conducted at comfortable and maximum walking speed, while 3D full-body kinematics was concurrently recorded with the multi-Kinect v2 set-up and the Optotrak motion-registration system (i.e., the gold standard). Between-systems agreement for body point’s time series was assessed with the intraclass correlation coefficient (ICC). Between-systems agreement was similarly determined for the gait parameters’ walking speed, cadence, step length, stride length, step width, step time, stride time (all obtained for the intermediate 6 meters) and the time to walk 10 meters, complemented by Bland-Altman’s bias and limits of agreement. Body point’s time series agreed well between the motion-registration systems, particularly so for body points in motion. For both comfortable and maximum walking speeds, the between-systems agreement for the time to walk 10 meters and all gait parameters except step width was high (ICC ≥ 0.888), with negligible biases and narrow limits of agreement. Hence, body point’s time series and gait parameters obtained with a multi-Kinect v2 set-up match well with those derived with a gold standard in 3D measurement accuracy. Future studies are recommended to test the clinical utility of the multi-Kinect v2 set-up to automate 10MWT assessments, thereby complementing the time to walk 10 meters with reliable spatiotemporal gait parameters obtained objectively in a quick, unobtrusive and patient-friendly manner.     

Molecular Evidence for High Frequency of Multiple Paternity in a Freshwater Shrimp Species Caridina ensifera

Background

Molecular genetic analyses of parentage provide insights into mating systems. Although there are 22,000 members in Malacostraca, not much has been known about mating systems in Malacostraca. The freshwater shrimp Caridina ensifera blue, is a new species belonging to Malacostraca which was discovered recently in Sulawesi, Indonesia. Due to its small body size and low fecundity, this species is an ideal species to study the occurrence and frequency of multiple paternity and to understand of how the low fecundity species persist and evolve.

Methodology/Principal Findings

In this study, we developed four polymorphic microsatellites from C. ensifera and applied them to investigate the occurrence and frequency of multiple paternity in 20 C. ensifera broods caught from Lake Matano, Sulawesi. By genotyping the mother and all offspring from each brood we discovered multiple paternity in all 20 broods. In most of the 20 broods, fathers contributed skewed numbers of offspring and there was an apparent inverse correlation between reproductive success of sires and their relatedness to mothers.

Conclusions/Significance

Our results in combination with recent reports on multiple paternity in crayfish, crab and lobster species suggests that multiple paternity is common in Malacostraca. Skewed contribution of fathers to the numbers of offspring and inverse correlation between reproductive success of sires and their relatedness to mothers suggest that sperm competition occurred and/or pre- and postcopulatory female choice happen, which may be important for avoiding the occurrence of inbreeding and optimize genetic variation in offspring and for persistence and evolution of low fecundity species.     

Sex in Cheese: Evidence for Sexuality in the Fungus Penicillium roqueforti

Although most eukaryotes reproduce sexually at some moment of their life cycle, as much as a fifth of fungal species were thought to reproduce exclusively asexually. Nevertheless, recent studies have revealed the occurrence of sex in some of these supposedly asexual species. For industrially relevant fungi, for which inoculums are produced by clonal-subcultures since decades, the potentiality for sex is of great interest for strain improvement strategies. Here, we investigated the sexual capability of the fungus Penicillium roqueforti, used as starter for blue cheese production. We present indirect evidence suggesting that recombination could be occurring in this species. The screening of a large sample of strains isolated from diverse substrates throughout the world revealed the existence of individuals of both mating types, even in the very same cheese. The MAT genes, involved in fungal sexual compatibility, appeared to evolve under purifying selection, suggesting that they are still functional. The examination of the recently sequenced genome of the FM 164 cheese strain enabled the identification of the most important genes known to be involved in meiosis, which were found to be highly conserved. Linkage disequilibria were not significant among three of the six marker pairs and 11 out of the 16 possible allelic combinations were found in the dataset. Finally, the detection of signatures of repeat induced point mutations (RIP) in repeated sequences and transposable elements reinforces the conclusion that P. roqueforti underwent more or less recent sex events. In this species of high industrial importance, the induction of a sexual cycle would open the possibility of generating new genotypes that would be extremely useful to diversify cheese products.     

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro

Interleukin-10 (IL-10) has been shown to exert anti-inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL-10 results in a significant dose-dependent increase in both Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E) colony growth in both serum-containing and serum-free murine cultures in vitro. IL-10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU-E than in BFU-E, and was similar when IL-10 was added to BFU-E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU-E colony number was noted when IL-10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti-IL-10 antibody up to 7 days later. The increases in BFU-E by IL-10 addition were not the result of prolongation of BFU-E colony lifespan, which was not significantly different in IL-10 treated and control cultures, respectively. Rather IL-10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL-10-induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage-containing and depleted cultures. Studies to determine if the IL-10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL-10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL-10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo. © 1996 Wiley-Liss, Inc.     

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii

We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc₃Man₅GlcNAc₂) and the host 14-sugar precursor (Glc₃Man₉GlcNAc₂) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.Animals, fungi, and plants synthesize Asn-linked glycans (N-glycans) by means of a lipid-linked precursor containing 14 sugars (dolichol-PP-Glc₃Man₉GlcNAc₂) (26). Recently we used bioinformatics and experimental methods to show that numerous protists are missing sets of glycosyltransferases (Alg1 to Alg14) and so make truncated N-glycan precursors containing 0 to 11 sugars (46). For example, Entamoeba histolytica, which causes dysentery, makes N-glycan precursors that contain seven sugars (Man₅GlcNAc₂) (33). Giardia lamblia, a cause of diarrhea, makes N-glycan precursors that contain just GlcNAc₂ (41). N-glycan precursors may be identified by metabolic labeling with radiolabeled mannose (Entamoeba) or glucosamine (Giardia) (46). Unprocessed N-glycans of each protist may be recognized by wheat germ agglutinin 1 (WGA-1) (GlcNAc₂ of Giardia) or by the antiretroviral lectin cyanovirin-N (Man₅GlcNAc₂ of Entamoeba) (2, 33, 41).N-glycans are transferred from lipid-linked precursors to sequons (Asn-Xaa-Ser or Asn-Xaa-Thr, where Xaa cannot be Pro) on nascent peptides by an oligosaccharyltransferase (OST) (28). For the most part, transfer of N-glycans by the OST is during translocation, although there are human and Trypanosoma OSTs that transfer N-glycans after translocation (34, 45).N-glycan-dependent quality control (QC) systems for protein folding and endoplasmic reticulum (ER)-associated degradation (ERAD), which are present in most eukaryotes, are missing from Giardia and a few other protists that make truncated N-glycans (5, 26, 53). There is positive Darwinian selection for sequons (sites of N-glycans) that contain Thr in secreted and membrane proteins of organisms that have N-glycan-dependent QC (12). This selection occurs for the most part by an increased probability that Asn and Thr will be present in sequons rather than elsewhere in secreted and membrane proteins. In contrast, there is no selection on sequons that contain Ser, and there is no selection on sequons in the secreted proteins of organisms that lack N-glycan-dependent QC.For numerous reasons, we are interested in the N-glycans of Plasmodium falciparum and Toxoplasma gondii, which cause severe malaria and disseminated infections, respectively.(i) There has been controversy for a long time as to whether Plasmodium makes N-glycans. While some investigators identified a 14-sugar Plasmodium N-glycan resembling that of the human host (29), others identified no N-glycans (6, 22).(ii) There is also controversy concerning whether the N-glycans of Toxoplasma, after removal of Glc by glucosidases in the ER lumen, contain either 7 sugars (Man₅GlcNAc₂), like Entamoeba (32, 33), or 11 sugars (Man₉GlcNAc₂), like the human host (16, 19, 26). If it is Man₅GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan predicted by its set of Alg enzymes (32, 46). If it is Man₉GlcNAc₂, then Toxoplasma uses the dolichol-PP-linked glycan of the host cell (16, 19, 26).(iii) Both Plasmodium and Toxoplasma are missing proteins involved in N-glycan-dependent QC of protein folding (5).(iv) We hypothesize that there may be negative selection against N-glycans in Plasmodium and Toxoplasma, because the N-glycans added in the ER lumen during translocation will likely interfere with threading of nucleus-encoded apicoplast proteins into a nonphotosynthetic, chloroplast-derived organelle called the apicoplast (21, 35, 37, 48, 52, 54). Nucleus-encoded apicoplast proteins have a bipartite signal at the N terminus, which targets proteins first to the lumen of the ER and second to lumen of the apicoplast. This bipartite signal has been used in transformed plasmodia where green fluorescent protein (GFP) is targeted to the apicoplast with the bipartite signal of the acyl carrier protein (ACP_leader-GFP), to the secretory system with the signal sequence only (ACP_signal-GFP), and to the cytosol with the organelle-targeting transit peptide only (ACP_transit-GFP) (55). Similar constructs have been used to characterize signals that target nucleus-encoded proteins of Toxoplasma to the apicoplast (11, 25).Here we use a combination of bioinformatic, biochemical, and morphological methods to characterize the N-glycans of Plasmodium and Toxoplasma and to test our hypothesis that there is negative selection against N-glycans in protists with apicoplasts.     

Yersinia pestis: New Evidence for an Old Infection

The successful reconstruction of an ancient bacterial genome from archaeological material presents an important methodological advancement for infectious disease research. The reliability of evolutionary histories inferred by the incorporation of ancient data, however, are highly contingent upon the level of genetic diversity represented in modern genomic sequences that are publicly accessible, and the paucity of available complete genomes restricts the level of phylogenetic resolution that can be obtained. Here we add to our original analysis of the Yersinia pestis strain implicated in the Black Death by consolidating our dataset for 18 modern genomes with single nucleotide polymorphism (SNP) data for an additional 289 strains at over 600 positions. The inclusion of this additional data reveals a cluster of Y. pestis strains that diverge at a time significantly in advance of the Black Death, with divergence dates roughly coincident with the Plague of Justinian (6^th to 8^th century AD). In addition, the analysis reveals further clues regarding potential radiation events that occurred immediately preceding the Black Death, and the legacy it may have left in modern Y. pestis populations. This work reiterates the need for more publicly available complete genomes, both modern and ancient, to achieve an accurate understanding of the history of this bacterium.     

Direct Magnetic Resonance Evidence for Peroxymonocarbonate Involvement in the Cu,Zn-Superoxide Dismutase Peroxidase Catalytic Cycle

Cu,Zn-superoxide dismutase (SOD1) is a copper- and zinc-dependent enzyme. The main function of SOD1 is believed to be the scavenging and detoxification of superoxide radicals. Nevertheless, the last 30 years have seen a rapid accumulation of evidence indicating that SOD1 may also act as a peroxidase, an alternative function that was implicated in the onset and progression of familial amyotrophic lateral sclerosis. Although SOD1 peroxidase activity and its dependence on carbon dioxide have been well described, the molecular basis of the SOD1 peroxidase cycle remains obscure, because none of the proposed catalytic intermediates have so far been identified. In view of recent observations, we hypothesized that the SOD1 peroxidase cycle relies on two steps: 1) reduction of SOD-Cu(II) by hydrogen peroxide followed by 2) oxidation of SOD-Cu(I) by peroxymonocarbonate, the product of the spontaneous reaction of bicarbonate with hydrogen peroxide, to produce SOD-Cu(II) and carbonate radical anion. This hypothesis has been investigated through electron paramagnetic resonance and nuclear magnetic resonance to provide direct evidence for a peroxycarbonate-driven, SOD1-catalyzed carbonate radical production. The results gathered herein indicate that peroxymonocarbonate () is a key intermediate in the SOD1 peroxidase cycle and identify this species as the precursor of carbonate radical anions.Cytosolic Cu,Zn-superoxide dismutase (SOD1)² is a metal-dependent enzyme capable of accelerating the rate of spontaneous superoxide dismutation into O₂ and H₂O₂ through the redox cycling of its copper ion (1, 2). SOD1 is widely distributed in mammalian cells and tissues and has been demonstrated to be located in the cytosol and in the intermembrane space of the mitochondria (see Ref. 3 and references therein). Because of that, SOD1 is believed to be a major player in the first line defense against reactive oxygen species, in particular superoxide anion.In addition to its dismutase activity, SOD1 possesses a well described but incompletely understood peroxidase activity which is dependent on hydrogen peroxide and markedly stimulated by small oxidizable anions such as nitrite and the ubiquitous carbon dioxide (3-12). The peroxidase activity of SOD1 has been proposed to impact the onset and progression of familial amyotrophic lateral sclerosis, a severely debilitating fatal disease characterized by the selective death of motor neurons (13-18). Although several reports exist in the literature indicating the formation of SOD1 aggregates and accumulation as a potential cause in the pathology progression, conflicting hypotheses are still under debate concerning the mechanisms that lead to the formation of SOD1-protein aggregates (19-21). Although some support the suggestion that free radical-induced covalent cross-links among SOD1 amino acids play a fundamental role in aggregate formation (22, 23), others support the view that metal loss from the enzyme structure leads to an unstable apo-form of SOD1 with increased capacity to form aggregates (24, 25). A detailed understanding of the SOD1 peroxidase cycle is essential to unraveling the mechanisms through which SOD1 aggregates are produced.The SOD1 peroxidase cycle is initiated when SOD1-Cu(II) is reduced by H₂O₂ or its deprotonated form (12), the peroxide anion (HOO^-), to SOD1-Cu(I). This latter species is subsequently oxidized to a hypervalent intermediate (proposed to be either SOD1-Cu(III), SOD1-Cu(II)-·_OH, or SOD1-Cu=O) (8, 9) that remains to be characterized. The reduction of this hypervalent intermediate by small anions is supposed to close the cycle, leading to the native enzyme and diffusible highly reactive radicals derived from the anionic substrates (6, 10).During its peroxidase cycle, a considerable fraction of SOD1 is inactivated due to the oxidation of the copper-binding histidines to oxohistidine, presumably by the hypervalent intermediate, in a process that can be prevented by the presence of reducing substrates and, in their absence, unavoidably leads to copper loss (26). Although this process is well described for the heme-dependent peroxidase cycle, current literature data (9, 27-30) and the fact that the proposed SOD1-bound hypervalent copper states (Cu(III), Cu(II)=O, and Cu(II)/^._OH) have never been characterized suggested to us that an alternative mechanism may take place, leading to production from and H₂O₂ by the enzyme, a process that does not involve copper oxidation beyond the thermodynamically stable Cu(II) form. In the presence of , a significant fraction of H₂O₂ is promptly converted to through the perhydration of CO₂ (27, 28, 31, 32). The peroxo bond in peroxymonocarbonate can be cleaved by reduced metals to produce and H₂O (30 33) (Reactions 1-5),where Reactions 1 and 2 represent SOD1 reduction, Reactions 3 and 4 represent SOD1 oxidation, and Reaction 5 represents peroxycarbonate formation.Interestingly, studies employing molecular modeling of the SOD1 active site indicate that and H₂O₂ gain access to the SOD1 active site, where they react to produce in close proximity to the copper ion (29). This interaction of with Cu(I) may result in production.On the basis of these new data, we hypothesized that SOD1-Cu(I), which is the predominant form of SOD1 exposed to excess hydrogen peroxide (8, 9), is oxidized back to the native form of the enzyme by more efficiently than by H₂O₂ itself or HOO^-. The latter two oxidations would slowly produce ^.OH radicals (or the equivalent SOD1-Cu(III), SOD1-Cu(II)=O, or Cu(II)/·_OH) in the enzyme active site, leading to the observed inactivation of SOD1 (see Scheme 1). Here we present data that strongly support this hypothesis; they indicate that is a key substrate for reduced SOD1, which mediates SOD1-Cu(I) reoxidation back to the resting SOD1-Cu(II) severalfold faster than H₂O₂ itself and, in doing so, serves as the carbonate radical anion precursor.Open in a separate windowSCHEME 1.Schematic representation of the peroxidase catalytic cycle of Cu,Zn-SOD in the presence of /CO₂. Native Cu,Zn-SOD is reduced by the peroxide anion, which gains access to the copper through the enzyme''s anion channel. Reduced Cu,Zn-SOD is reoxidized by the peroxycarbonate anion (), which is in equilibrium with H₂O₂//CO₂, leading to carbonate radical anion production. Superoxide anion (), the product of HOO^--induced SOD1 reduction, can oxidize SOD1-Cu(I) back to its resting SOD-Cu(II) state at diffusion-limited rates; however, it can alternatively reduce another molecule of SOD1-Cu(II) to SOD1-Cu(I), considerably accelerating the rate of SOD1-Cu(II) reduction by H₂O₂. Whether will act as a reductant of SOD1-Cu(II) or an oxidant of SOD1-Cu(I) will depend on the ratio of SOD1-Cu(II)/SOD1-Cu(I) at a given time, because the rate constants for the reaction of with both SOD1 states are close to the diffusion limits.     

Evidence of a novel allergenic protein Narcin in the bulbs of Narcissus tazetta

Several plant-derived allergens have been identified which result in the formation of immunoglobulin E antibodies. Primarily, these allergens belong to the protein families including seed storage proteins, structural proteins and pathogenesis-related proteins. Several allergens are also reported from flower bulbs which cause contact dermatitis. Such symptoms are highly common with the bulb growers handling different species of Narcissus. Narcissus toxicity is also reported if the bulbs are consumed accidentally. The present study aimed to characterize the protein from the bulbs of Narcissus tazetta responsible for its allergenic response. A 13 kDa novel allergenic protein, Narcin was isolated from the bulbs of Narcissus tazetta. The protein was extracted using ammonium sulfate fractionation. The protein was further purified by anion exchange chromatography followed by gel filtration chromatography. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation. The allergenicity of the protein was measured by cytokine production using flow cytometry in peripheral blood mononuclear cells. Further estimation of total IgE was performed by ELISA method. This novel protein was found to induce pro-inflammatory cytokines and thus induce allergy by elevating total IgE level. The novel protein, Narcin isolated from Narcissus tazetta was found to exhibit allergenic properties.