Topology and Growth of the Intracytoplasmic Membrane System of Rhodopseudomonas spheroides: Protein, Chlorophyll, and Phospholipid Insertion into Steady-State Anaerobic Cells

An equilibrium density gradient centrifugation study involving the separation of "old" and "new" membranes has been developed to determine the manner in which protein, lipid, and chlorophyll are incorporated into growing intracytoplasmic membranes (chromatophores) of Rhodopseudomonas spheroides. Chromatophores derived from cells grown in an H(2)O-medium had a density of 1.175 to 1.180 g/cm(3) and were readily separable from chromatophores having a density of 1.220 to 1.230 isolated from cells grown in a 70% D(2)O-medium. After a shift from "D(2)O-" to "H(2)O"-based media, only hybrid chromatophores derived from a combination of "heavy" (old) and "light" (new) chromatophore material could be detected. The experimentally determined, median density values for the growing intracytoplasmic membrane system followed a theoretically determined profile which was calculated from the density of full "heavy" and full "light" material assuming random, homogeneous incorporation of new material into old membrane. The distribution of the radioactive labels for protein (leucine) and chlorophyll (delta-aminolevulinic acid) were identical and showed a reproducible displacement of the "old" material to the heavy side of the optical density at 365 nm (OD(365)) absorbance and a displacement of the "new" material to the light side of the OD(365) absorbance profile. Specific phospholipid growth showed no displacement for either the "old" or "new" material from the median absorbance profile.     

GENERAL PATTERN, 35SO4-INCORPORATION AND INTRACELLULAR LOCALIZATION OF GLYCANS IN DEVELOPING SEA URCHIN (ANTHOCIDARIS) EMBRYOS

Glycosaminoglycan (GAG) prepared from sea urchin embryos ( Anthocidaris crassispina ) at various stages with or without pulse ³⁵SO₄-labelling was separated into various fractions by chromatography on DEAE-cellulose with a linear NaCl concentration gradient: fraction "P" (nonacidic) and fractions "A" through "F" (of increasing acidities). The ³⁵SO₄-radioactivity was negligible in "P" and "A", largest in "B" and "C", and decreased in the other fractions three alphabetical order. During development (hatched blastulae to gastrulae) the glycans in fractions "P" and "A" decreased in amount, whereas those in "E" and "F" increased. "E" contained heparin-like (AMPS-1) and dermatanpolysulfate-like (AMPS-2) GAG in addition to a sulfated fucogalactan-like (E₁) glycan. Another sulfated fucogalactan-like (F₁) glycan was found in "F". A sulfated polysialic acid-like (S₁) glycan was found in "C". An EDTA-extract of gastrulae gave AMPS-2, E₁ and F₁. The mitochondria-rich fraction gave AMPS-1, whereas the yolk granule-rich fraction gave S₁. Most of the other still unidentified components in "B", "C", and "D" appeared to be derived from glycoproteins and were mainly located in the crude yolk-mitochondrial and cytosol fractions.     

中国古本草书艾蒿类植物的初步考订

本文对中国古本草书记载的艾蒿类植物,如白蒿、艾、白艾、艾蒿、野艾蒿、苹、籟蒿、水蒿、萎蒿、柳叶蒿、(艹闾)蒿、艾叶、家艾、蕲艾、牛尾蒿、草蒿、青蒿、黄花蒿、香蒿、臭蒿、狃蒿、邪蒿、茵陈蒿)蓬高、野同蒿、紫香蒿、牡蒿、齐头蒿、水辣菜、奄闾子、庵(艹闾)、刘寄奴及金寄奴等作了初步考订,同时对一些名为"艾"或"蒿"但非菊科蒿属植物亦作一初步的考订。     

Hormone-like product of vascular tissue stimulating starch accumulation in pith explants of kale and endogenous cytokinins

When stem explants of kale (Brassica oleracea L. var.medullosa), containing pith parenchyma and a strip of vascular tissue, were cultured on simple sucrose medium, a hormone-like factor was transported from the vascular tissue to the adjacent pith, where it stimulated accumulation of starch. Similarly, up to a sevenfold increase of starch content in explants could be induced by cytokinins added to the culture medium. The relative stimulatory effect of several cytokinins (5×10^?6 M) and hormone-like product of vascular tissue (HPVT) in a typical experiment were: control (1.0), trans-zeatin (6.7), HPVT (6.2), N⁶-[2-isopentenyl]adenine (5.4), transzeatin riboside (5.2), N⁶-[2-isopentenyl]adenosine (5.4), kinetin (3.6), 6-benzylaminopurine (3.5), and adenine (2.1). Concentration of endogenous cytokinins was determined using ELISA (trans-zeatin, N⁶-[2-isopentenyl]adenine and their ribosides) andAmaranthus bioassay (total cytokinins). No effect of vascular tissue on the level of endogenous cytokinins in explants was found. The results support the conclusions of previous experiments that the HPVT stimulating starch accumulation is not a cytokinin.     

Morphological variability and degenerative evolution of human hepatic hydatid cysts

The findings are presented of a macro and microscopic investigation of 89 hydatid hepatic cysts removed intact from 59 patients by total pericystectomy. Detailed analysis revealed significant morphostructural variability and cysts grouped into 10 types were characterized, providing useful clinical indications. Only 30 cysts resulted fertile (33.7%), probably due to mean age of sample; 7 of these were "classic" cysts, 1 "septated" and 22 "multivesicular" packed with daughter cysts (DC), of varying turgidity or collapsed. Among the remaining 59 sterile cysts, 52 were degenerated and classified as "hyperlaminated" cysts due to the presence of large convoluted sheets of laminar tissue (SLT) surrounded by varying amounts of caseous (40 specimens), granular (6) or gelatinous (6) matrix. Moreover, "multivesicular", "acephalocyst", "caseous" and "serous" cysts were also recovered among the sterile specimens. Some "multivesicular" cysts (14) appeared as "transitional forms" towards the various types of "hyperlaminated" cysts containing all different forms of DC and large SLT intermingled with a variously degenerated matrix. The comprehensive study allows to hypothesize the train of events leading, over the years, to the gradual transformation and degeneration of the larval form Echinococcus granulosus in the human liver.     

Induction and Inhibition of Type I Interferon Responses by Distinct Components of Lymphocytic Choriomeningitis Virus

Type I interferons (IFNs) play a critical role in the host defense against viruses. Lymphocytic choriomeningitis virus (LCMV) infection induces robust type I IFN production in its natural host, the mouse. However, the mechanisms underlying the induction of type I IFNs in response to LCMV infection have not yet been clearly defined. In the present study, we demonstrate that IRF7 is required for both the early phase (day 1 postinfection) and the late phase (day 2 postinfection) of the type I IFN response to LCMV, and melanoma differentiation-associated gene 5 (MDA5)/mitochondrial antiviral signaling protein (MAVS) signaling is crucial for the late phase of the type I IFN response to LCMV. We further demonstrate that LCMV genomic RNA itself (without other LCMV components) is able to induce type I IFN responses in various cell types by activation of the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5. We also show that expression of the LCMV nucleoprotein (NP) inhibits the type I IFN response induced by LCMV RNA and other RIG-I/MDA5 ligands. These virus-host interactions may play important roles in the pathogeneses of LCMV and other human arenavirus diseases.Type I interferons (IFNs), namely, alpha interferon (IFN-α) and IFN-β, are not only essential for host innate defense against viral pathogens but also critically modulate the development of virus-specific adaptive immune responses (6, 8, 28, 30, 36, 50, 61). The importance of type I IFNs in host defense has been demonstrated by studying mice deficient in the type I IFN receptor, which are highly susceptible to most viral pathogens (2, 47, 62).Recent studies have suggested that the production of type I IFNs is controlled by different innate pattern recognition receptors (PRRs) (19, 32, 55, 60). There are three major classes of PRRs, including Toll-like receptors (TLRs) (3, 40), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (25, 48, 51), and nucleotide oligomerization domain (NOD)-like receptors (9, 22). TLRs are a group of transmembrane proteins expressed on either cell surfaces or endosomal compartments. RLRs localize in the cytosol. Both TLRs and RLRs are involved in detecting viral pathogens and controlling the production of type I IFNs (52, 60). In particular, the endosome-localized TLRs (TLR3, TLR7/8, and TLR9) play important roles in detecting virus-derived double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), and DNA-containing unmethylated CpG motifs, respectively. In contrast, RIG-I detects virus-derived ssRNA with 5′-triphosphates (5′-PPPs) or short dsRNA (<1 kb), whereas melanoma differentiation-associated gene 5 (MDA5) is responsible for recognizing virus-derived long dsRNA as well as a synthetic mimic of viral dsRNA poly(I):poly(C) [poly(I·C)] (24, 60). Recognition of viral pathogen-associated molecular patterns (PAMPs) ultimately leads to the activation and nuclear translocation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB), which, in turn, switches on a cascade of genes controlling the production of both type I IFNs and other proinflammatory cytokines (10, 11, 60).Lymphocytic choriomeningitis virus (LCMV) infection in its natural host, the mouse, is an excellent system to study the impact of virus-host interactions on viral pathogenesis and to address important issues related to human viral diseases (1, 45, 49, 67). LCMV infection induces type I IFNs as well as other proinflammatory chemokines and cytokines (6, 41). Our previous studies have demonstrated that TLR2, TLR6, and CD14 are involved in LCMV-induced proinflammatory chemokines and cytokines (66). The mechanism by which LCMV induces type I IFN responses, however, has not been clearly defined (7, 8, 31, 44). The role of the helicase family members RIG-I and MDA5 in virus-induced type I IFN responses has been recently established. RIG-I has been found to be critical in controlling the production of type I IFN in response to a number of RNA viruses, including influenza virus, rabies virus, Hantaan virus, vesicular stomatitis virus (VSV), Sendai virus (SeV), etc. In contrast, MDA5 is required for responses to picornaviruses (15, 25, 63).In the present study, we demonstrated that LCMV genomic RNA strongly activates type I IFNs through a RIG-I/MDA5-dependent signaling pathway. Our present study further demonstrated that the LCMV nucleoprotein (NP) blocks LCMV RNA- and other viral ligand-induced type I IFN responses.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Arsenic Speciation in Phloem and Xylem Exudates of Castor Bean

How arsenic (As) is transported in phloem remains unknown. To help answer this question, we quantified the chemical species of As in phloem and xylem exudates of castor bean (Ricinus communis) exposed to arsenate [As(V)], arsenite [As(III)], monomethylarsonic acid [MMA(V)], or dimethylarsinic acid. In the As(V)- and As(III)-exposed plants, As(V) was the main species in xylem exudate (55%–83%) whereas As(III) predominated in phloem exudate (70%–94%). The ratio of As concentrations in phloem to xylem exudate varied from 0.7 to 3.9. Analyses of phloem exudate using high-resolution inductively coupled plasma-mass spectrometry and accurate mass electrospray mass spectrometry coupled to high-performance liquid chromatography identified high concentrations of reduced and oxidized glutathione and some oxidized phytochelatin, but no As(III)-thiol complexes. It is thought that As(III)-thiol complexes would not be stable in the alkaline conditions of phloem sap. Small concentrations of oxidized glutathione and oxidized phytochelatin were found in xylem exudate, where there was also no evidence of As(III)-thiol complexes. MMA(V) was partially reduced to MMA(III) in roots, but only MMA(V) was found in xylem and phloem exudate. Despite the smallest uptake among the four As species supplied to plants, dimethylarsinic acid was most efficiently transported in both xylem and phloem, and its phloem concentration was 3.2 times that in xylem. Our results show that free inorganic As, mainly As(III), was transported in the phloem of castor bean exposed to either As(V) or As(III), and that methylated As species were more mobile than inorganic As in the phloem.Arsenic (As) is an environmental and food chain contaminant that has attracted much attention in recent years. Soil contamination with As may lead to phytotoxicity and reduced crop yield (Panaullah et al., 2009). Food crops are also an important source of inorganic As, a class-one carcinogen, in human dietary intake, and there is a need to decrease the exposure to this toxin (European Food Safety Authority, 2009). Paddy rice (Oryza sativa) is particularly efficient in As accumulation, which poses a potential risk to the population based on a rice diet (Meharg et al., 2009; Zhao et al., 2010a). Other terrestrial food crops generally do not accumulate as much As as paddy rice; however, where soils are contaminated, relatively high concentrations of As in wheat (Triticum aestivum) grain have been reported (Williams et al., 2007; Zhao et al., 2010b). On the other hand, some fern species in the Pteridaceae family are able to tolerate and hyperaccumulate As in the aboveground part to >1,000 mg kg⁻¹ dry weight (e.g. Ma et al., 2001; Zhao et al., 2002); these plants offer the possibility for remediation of As-contaminated soil or water (Salido et al., 2003; Huang et al., 2004). A better understanding of As uptake and long-distance transport, metabolism, and detoxification is needed for developing strategies for mitigating As contamination, through either decreased As accumulation in food crops or enhanced As accumulation for phytoremediation.The pathways of As uptake by plant roots differ between different As species; arsenate [As(V)] enters plant cells via phosphate transporters, whereas arsenite [As(III)] is taken up via some aquaporins (for review, see Zhao et al., 2009). In rice, a silicic acid efflux protein also mediates As(III) efflux toward stele for xylem loading (Ma et al., 2008). Methylated As species, such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)], which may be present in the environment as products of microbial or algal methylation of inorganic As or from past uses of methylated As pesticides, are taken up by rice roots partly through the aquaporin NIP2;1 (for nodulin 26-like intrinsic protein; also named Lsi1; Li et al., 2009). Once inside plant cells, As(V) is reduced to As(III), possibly catalyzed by As(V) reductase(s) such as the plant homologs of the yeast (Saccharomyces cerevisiae) ACR2 (Bleeker et al., 2006; Dhankher et al., 2006; Ellis et al., 2006; Duan et al., 2007). As(III) has a high affinity to thiol (-SH) groups and is detoxified by complexation with thiol-rich phytochelatins (PCs; Pickering et al., 2000; Schmöger et al., 2000; Raab et al., 2005; Bluemlein et al., 2009; Liu et al., 2010). As(III)-PC complexation in roots was found to result in reduced mobility for efflux and for long-distance transport, possibly because the complexes are stored in the vacuoles (Liu et al., 2010). Excess As(III) causes cellular toxicity by binding to the vicinal thiol groups of enzymes, such as the plastidial lipoamide dehydrogenase, which has been shown to be a sensitive target of As toxicity (Chen et al., 2010). The As hyperaccumulating Pteris species differ from nonhyperaccumulating plants because of enhanced As(V) uptake (Wang et al., 2002; Poynton et al., 2004), little As(III)-thiol complexation (Zhao et al., 2003; Raab et al., 2004), and efficient xylem loading of As(III) (Su et al., 2008). Recently, an As(III) efflux transporter, PvACR3, has been found to play an important role in As(III) detoxification by transporting As(III) into vacuoles in Pteris vittata (Indriolo et al., 2010).With the exception of As hyperaccumulators, most plant species have a limited root-to-shoot translocation of As (Zhao et al., 2009). The chemical species of As in xylem exudate have been determined in a number of plant species. As(III) was found to be the predominant species (80%–100%) in the xylem sap of rice, tomato (Solanum lycopersicum), cucumber (Cucumis sativus), and P. vittata even when these plants were fed As(V) (Mihucz et al., 2005; Xu et al., 2007; Ma et al., 2008; Su et al., 2010), suggesting that As(V) is reduced in roots before being loaded into the xylem. In other plant species, such as Brassica juncea (Pickering et al., 2000), wheat, and barley (Hordeum vulgare; Su et al., 2010), As(V) accounted for larger proportions (40%–50%) of the total As in the xylem sap. Studies using HPLC-inductively coupled plasma (ICP)-mass spectrometry (MS) coupled with electrospray (ES)-MS showed no evidence of As(III)-thiol complexation in the xylem sap of sunflower (Helianthus annuus; Raab et al., 2005). When rice plants were exposed to MMA(V) or DMA(V), both As species were found in the xylem sap (Li et al., 2009). Generally, methylated As species are taken up by roots at slower rates than inorganic As, but they are more mobile during the xylem transport from roots to shoots (Marin et al., 1992; Raab et al., 2007; Li et al., 2009).It has been shown that phloem transport contributes substantially to As accumulation in rice grain (Carey et al., 2010). However, little is known about how As is transported in phloem (Zhao et al., 2009). There are no reports on the chemical species of As in phloem exudate. The speciation of As in phloem is important because it dictates how As is loaded in the source tissues and unloaded in the sink tissues, such as grain. Questions with regard to the oxidation state, methylation, and complexation of As in phloem sap remain to be answered. Unlike xylem sap, phloem sap is much more difficult to obtain in sufficient quantities for analysis. In this study, we investigated As speciation in phloem and xylem exudates of castor bean (Ricinus communis), which is widely used as a model plant to investigate phloem transport of solutes (e.g. Hall et al., 1971; Hall and Baker, 1972; Allen and Smith, 1986; Bromilow et al., 1987).     

Diversity and plasticity of the intracellular plant pathogen and insect symbiont "Candidatus Liberibacter asiaticus" as revealed by hypervariable prophage genes with intragenic tandem repeats

"Candidatus Liberibacter asiaticus" is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of "Ca. Liberibacter" associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyv(I) and hyv(II)) were identified in the prophage regions of the Psy62 "Ca. Liberibacter asiaticus" genome. Sequence analyses of the hyv(I) and hyv(II) genes in 35 "Ca. Liberibacter asiaticus" DNA isolates collected globally revealed that the hyv(I) gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv(II) contains up to 2 NITRs and 4 partial repeats and shares homology with hyv(I). Frequent deletions or insertions of these repeats within the hyv(I) and hyv(II) genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of "Ca. Liberibacter asiaticus" bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single "Ca. Liberibacter asiaticus"-infected sample. This is the first evidence of different "Ca. Liberibacter asiaticus" populations coexisting in a single HLB-affected sample. The Florida "Ca. Liberibacter asiaticus" isolates contain both hyv(I) and hyv(II), while all other global "Ca. Liberibacter asiaticus" isolates contain either one or the other. Interclade assignments of the putative Hyv(I) and Hyv(II) proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple "Ca. Liberibacter asiaticus" populations in the world and a multisource introduction of the "Ca. Liberibacter asiaticus" bacterium into Florida.     

Comparison of the Pseudorabies Virus Us9 Protein with Homologs from Other Veterinary and Human Alphaherpesviruses

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.Alphaherpesviruses are classified by their variable host range, short reproductive cycle, and ability to establish latency in the peripheral nervous system (PNS) (36, 37). Commonly studied pathogens of this subfamily include herpes simplex virus (HSV) and varicella-zoster virus (VZV), as well as the veterinary pathogens pseudorabies virus (PRV), equine herpesvirus (EHV), and bovine herpesvirus (BHV). Initial infection begins with the virus entering the host mucosal surfaces and spreading between cells of the mucosal epithelium. Invariably, virus enters the PNS through the infection of peripheral nerves that innervate this region. The virus establishes a latent infection in PNS neurons that can be reactivated and that persists for the life of the host (36). In most natural infections, virus replication in the PNS never spreads to the central nervous system (CNS). However, on rare occasions, invasion of the CNS does occur, resulting in devastating encephalitis (46). Trafficking of virus particles from infected epithelial cells into the axon and subsequent transport to neuronal cell bodies is known as retrograde spread of infection. Trafficking of virus particles that are assembled in the neuronal cell body and subsequently sorted into axons for transport to epithelial cells at the initial site of infection (upon reactivation from latency) is known as anterograde spread of infection.Though the natural host of PRV is swine, the virus infects a wide variety of animals, including rodents, cats, dogs, rabbits, cattle, and chicken embryos, but not higher primates (1, 30, 47). In contrast to the well-contained spread of PRV within its natural host, infection of other mammals is usually lethal. Instead of stopping in the PNS, infection continues on to second-order and third-order neurons in the CNS (reviewed in reference 35). This facet of PRV infection makes it a useful tracer of neuronal connections (18). Work in our lab has identified three PRV proteins, Us9 and the gE/gI heterodimer, which are critical for efficient anterograde spread of infection in vivo (i.e., spread from presynaptic to postsynaptic neurons) (6, 45). The molecular mechanism by which these proteins function has been further elucidated in vitro using primary neuronal cultures of superior cervical ganglion (SCG) harvested from embryonic rat pups. PRV Us9 and, to a lesser extent, gE/gI are required for efficient axonal targeting of viral structural proteins, a necessary step for subsequent anterograde, transneuronal spread (10, 11, 27, 28, 42).PRV Us9 is a type II, tail-anchored (TA) membrane protein that is highly enriched in lipid raft microdomains and resides predominantly in or near the trans-Golgi network (TGN) inside infected cells (5-7, 27). It has homologs in most of the alphaherpesviruses, including VZV (16), HSV-1 (22), HSV-2 (17), EHV-1 (21, 40), EHV-4 (41), BHV-1 (25), and BHV-5 (14). Though several studies have examined individually the Us9 proteins encoded by VZV (16), HSV-1 (4, 22, 34, 39), BHV-1 (13), and BHV-5 (14), several gaps in our understanding of Us9 biology remain, namely, whether all of the PRV Us9 homologs are type II membrane proteins, if the proteins localize to similar subcellular compartments within different cell types, and if they can functionally substitute for the loss of PRV Us9 in axonal sorting and anterograde spread of infection. The aim of this study is to examine PRV Us9 in parallel assays with its homologs from VZV, HSV-1, EHV-1, and BHV-1 to identify potential similarities and differences between these highly conserved alphaherpesvirus proteins.     

Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (K_D = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (K_av) measured with gel permeation chromatography, a high hydrodynamic radius (R_h) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional ¹⁵N-¹H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding.Leptospira interrogans is a pathogenic spirochete that causes leptospirosis throughout the world, especially in developing countries but also in regions of the United States where it has reemerged (1). Weil''s syndrome, a severe form of this disease, is an acute febrile illness associated with multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (1), and has a 15% mortality rate if not treated (2). The molecular pathogenesis of leptospirosis is poorly understood, and the bacterial virulence factors involved are largely unknown. Recently, several potential Leptospira virulence factors have been described, including sphingomyelinases, serine proteases, zinc-dependent proteases, and collagenase (3); LipL32 (4); lipopolysaccharide (5); a novel factor H, laminin, and Fn-binding protein (Lsa24 or Len) (6–8); Loa 22 (9); and Lig (Leptospiral immunoglobulin-like) proteins (10–12).Lig proteins, including LigA, LigB, and LigC, contain multiple immunoglobulin-like repeat domains (13 in LigA, 12 in LigB and LigC) (10–12). Interestingly, the first 630 residues, from the N terminus to the first half of the seventh immunoglobulin-like domain, are conserved between LigA and LigB, but the rest of the immunoglobulin-like domains are variable (10–12) between the two proteins. Also, a non-immunoglobulin-like repeat region found on the C-terminal tail of LigB is not found in LigA (10–12). Lig proteins are categorized as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² due to their ability to bind to eukaryotic cells (13) through their interactions with extracellular matrix components, including fibronectin (Fn), laminin, collagens, elastin, and tropoelastin (13, 14, 45). Previously, a high affinity Fn-binding region was localized to LigBCen2, which includes the partial eleventh and complete twelfth immunoglobulin-like repeat region and the first 47 amino acids of the non-repeat regions of LigB (15). LigBCen2 was shown to bind to both the N-terminal domain (NTD) and the gelatin binding domain (GBD) of Fn. The addition of calcium induces a conformational change in LigBCen2 and enhances binding between LigBCen2 and the NTD of Fn (15).The first step in the process of bacterial infection is cellular adhesion, mediated by bacterial adhesins interacting with various components of the extracellular matrix (16). Known interaction modes between Fn and bacterial Fn-binding proteins include the β-zipper (17, 18) and the cationic cradle (19). It was recently discovered that the Fn-binding domains in certain Fn-binding proteins are disordered and extended but gain structure upon binding to the NTD of Fn (20–22).We have performed a fine-mapping study of the NTD-binding site on LigBCen2 and identified this site as LigBCen2NR, a portion of the non-repeat region (amino acids 1119–1165). The addition of NTD promotes the folding of LigBCen2NR from a disordered and extended structure to a folded structure. This finding is notable, since LigBCen2NR is located in the non-immunoglobulin-like region of LigB, as compared with other Fn-binding proteins, such as Staphylococcus aureus FnbpA and FnbpB (23), Streptococcus dysgalactiae FnBB (17), and Streptococcus pyogenes SfbI and SfbII (24). Thus, the binding mode appears to be similar to the known β-zipper mechanism but unique in sequence-specific interactions. This finding provides the fundamental groundwork for the development of a therapeutic agent to target this interaction in order to prevent or treat Leptospira infection.     

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3,4-diethoxyphenyl)-1,3(dihydroxy)-2-(4'-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial , bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the , bond of 1-(3-4-diethoxyphenyl)-1-(hydroxy)-2-(4'-methoxyphenyl)ethane (XI) also occurred, indicating that a hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2,2(dihydroxy)-2-(4'-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the , bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MS mass spectrometry     

Investigation of Clade B New World Arenavirus Tropism by Using Chimeric GP1 Proteins

Clade B of the New World arenaviruses contains both pathogenic and nonpathogenic members, whose surface glycoproteins (GPs) are characterized by different abilities to use the human transferrin receptor type 1 (hTfR1) protein as a receptor. Using closely related pairs of pathogenic and nonpathogenic viruses, we investigated the determinants of the GP1 subunit that confer these different characteristics. We identified a central region (residues 85 to 221) in the Guanarito virus GP1 that was sufficient to interact with hTfR1, with residues 159 to 221 being essential. The recently solved structure of part of the Machupo virus GP1 suggests an explanation for these requirements.Arenaviruses are bisegmented, single-stranded RNA viruses that use an ambisense coding strategy to express four proteins: NP (nucleoprotein), Z (matrix protein), L (polymerase), and GP (glycoprotein). The viral GP is sufficient to direct entry into host cells, and retroviral vectors pseudotyped with GP recapitulate the entry pathway of these viruses (5, 13, 24, 31). GP is a class I fusion protein comprising two subunits, GP1 and GP2, cleaved from the precursor protein GPC (4, 14, 16, 18, 21). GP1 contains the receptor binding domain (19, 28), while GP2 contains structural elements characteristic of viral membrane fusion proteins (8, 18, 20, 38). The N-terminal stable signal peptide (SSP) remains associated with the mature glycoprotein after cleavage (2, 39) and plays a role in transport, maturation, and pH-dependent fusion (17, 35, 36, 37).The New World arenaviruses are divided into clades A, B, and C based on phylogenetic relatedness (7, 9, 11). Clade B contains the human pathogenic viruses Junin (JUNV), Machupo (MACV), Guanarito (GTOV), Sabia, and Chapare, which cause severe hemorrhagic fevers in South America (1, 10, 15, 26, 34). Clade B also contains the nonpathogenic viruses Amapari (AMAV), Cupixi, and Tacaribe (TCRV), although mild disease has been reported for a laboratory worker infected with TCRV (29).Studies with both viruses and GP-pseudotyped retroviral vectors have shown that the pathogenic clade B arenaviruses use the human transferrin receptor type 1 (hTfR1) to gain entry into human cells (19, 30). In contrast, GPs from nonpathogenic viruses, although capable of using TfR1 orthologs from other species (1), cannot use hTfR1 (1, 19) and instead enter human cells through as-yet-uncharacterized hTfR1-independent pathways (19). In addition, human T-cell lines serve as useful tools to distinguish these GPs, since JUNV, GTOV, and MACV pseudotyped vectors readily transduce CEM cells, while TCRV and AMAV GP vectors do not (27; also unpublished data). These properties of the GPs do not necessarily reflect a tropism of the pathogenic viruses for human T cells, since viral tropism is influenced by many factors and T cells are not a target for JUNV replication in vivo (3, 22, 25).     

Enzymic glycosphingolipid synthesis on polymer supports. III. Synthesis of GM3, its analog [NeuNAcα(2-3)Galβ(1-4)Glcβ(1-3)Cer] and their lyso-derivatives

Two water-soluble polymers, carrying 0.24 meq g–1 of lactosyl-(1-1)-sphingosine (7) and 0.13 meq g–1 of lactosyl-(1-3)-sphingosine (8) were prepared. The polymers served as acceptors in the -(2-3)-sialyltransferase reaction (up to 55.3 and 38.5% transfer yields, respectively). Subsequent photolysis, released compounds 11 (lyso-GM3) and 12 (lyso-GM3 analog), respectively; acylation and chromatography afforded (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-1)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (13, GM3) and (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-3)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (14, GM3 analogue), respectively, thus presenting a route to glycosphingolipids possessing the unusual glycosyl-(1-3)-spingosine linkage.     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

The C-Terminal Domain of Cernunnos/XLF Is Dispensable for DNA Repair In Vivo

The core nonhomologous end-joining DNA repair pathway is composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos). Although Cernunnos and X4 are structurally related and participate in the same complex together with L4, they have distinct functions during DNA repair. L4 relies on X4 but not on Cernunnos for its stability, and L4 is required for optimal interaction of Cernunnos with X4. We demonstrate here, using in vitro-generated Cernunnos mutants and a series of functional assays in vivo, that the C-terminal region of Cernunnos is dispensable for its activity during DNA repair.Nonhomologous end joining (NHEJ) represents the main pathway for solving DNA double-strand breaks (DSB) in mammals. The core of the NHEJ pathway is composed of seven proteins: Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, XRCC4 (X4), DNA ligase IV (L4), and Cernunnos/XLF (Cernunnos) (reviewed in reference 18). Briefly, the Ku70-Ku80 heterodimer bound to broken DNA recruits the serine/threonine kinase DNA-PKcs. DNA-PK phosphorylates downstream effectors such as the nuclease Artemis. The X4-L4 complex carries out the final joining of synapsed DNA ends in association with Cernunnos (2, 6). Cernunnos was identified through cDNA functional complementation of a fibroblast cell line obtained from a human patient with immune deficiency and microcephaly (5). The same factor, called XLF, was identified through a yeast two-hybrid screen with X4 as a bait (2).Cernunnos is structurally related to X4 and consists of a globular head domain followed by a coiled-coil region and an unstructured C-terminal domain (2, 6, 12). One major difference between the structures of X4 and Cernunnos appears in the coiled-coil region. While this region is linear in X4, a hinge in the middle of the coiled-coil of Cernunnos folds back the end of the domain toward the head (3, 14).Cernunnos interacts with the X4-L4 complex in vivo and in vitro (2, 6). Cernunnos and X4 both appear to interact directly with L4, but the Cernunnos-L4 interaction seems to be very weak (7). In addition, purified Cernunnos associates with DNA in a sequence-independent manner (20) but in a DNA length-dependent manner, like X4 (15). Although the X4-L4 complex can ligate DNA in vitro (10), Cernunnos further improves this activity (11, 15, 16, 20). Cernunnos seems important, in particular, for the ligation of mismatched or noncohesive DNA ends, but not for that of compatible DNA ends, in vitro (10, 20).Cernunnos is therefore a “core” NHEJ component, but limited information is available about its precise function during DNA repair in vivo. We show here that although X4 and Cernunnos share sequence and structural homologies, their functions are distinct. We also demonstrate that Cernunnos requires L4 for its association with X4. Lastly, the Cernunnos C terminus is dispensable for DNA repair following ionizing radiation (IR) and V(D)J recombination.