Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.     

Toll-like receptor 3 mediates a more potent antiviral response than Toll-like receptor 4

We have recently described an IFN regulatory factor 3-mediated antiviral gene program that is induced by both Toll-like receptor (TLR)3 and TLR4 ligands. In our current study, we show that activation of IFN/viral response gene expression in primary macrophage cells is stronger and prolonged with TLR3 stimulation compared with that of TLR4. Our data also reveal that the cytoplasmic tails of both TLR3 and TLR4 can directly interact with myeloid differentiation factor 88 (MyD88). However, although Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like is able to associate with TLR4, we were unable to detect any interaction between Toll/IL-1 receptor homology domain-containing adaptor protein/MyD88 adaptor-like and TLR3. By using quantitative real-time PCR assays, we found that TLR3 expression is inducible by both TLR3 and TLR4 ligands, while TLR4 expression is not inducible by these same stimuli. Furthermore, using cells derived from mice deficient in the IFN-alphabetaR, we show that both TLR3 and TLR4 require IFN-beta autocrine/paracrine feedback to induce TLR3 expression and activate/enhance genes required for antiviral activity. More specifically, a subset of antiviral genes is initially induced independent of IFN-beta, yet the cytokine further enhances expression at later time points. This was in contrast to a second set of genes (including TLR3) that is induced only after IFN-beta production. Taken together, our data argue that, despite both TLR3 and TLR4 being able to use IFN-beta to activate/enhance antiviral gene expression, TLR3 uses multiple mechanisms to enhance and sustain the antiviral response more strongly than TLR4.     

Cutting edge: bacterial lipoprotein induces endotoxin-independent tolerance to septic shock

Tolerance to bacterial cell wall components is an adaptive host response. Endotoxin/LPS tolerance is characterized by a survival advantage against subsequent lethal LPS challenge. However, it is uncertain whether LPS tolerance can afford protection against other septic challenges. In this study, we show that tolerance induced by bacterial lipoprotein (BLP) protects mice against not only BLP-induced lethality, but also LPS-, live bacteria-, and polymicrobial sepsis-induced lethality. In contrast, LPS tolerance offers no survival benefit against the latter two challenges. Furthermore, induction of BLP tolerance results in overexpression of complement receptor type 3 and FcgammaIII/IIR on neutrophils (polymorphonuclear neutrophils) and peritoneal macrophages, with increased bacterial recognition and bactericidal activity, whereas LPS-tolerized mice exhibit an impaired ability to ingest and to kill bacteria. These results indicate that BLP tolerance is a novel adaptive host response associated with a unique protective effect during septic shock.     

Colony structure in a plant-ant: behavioural,chemical and genetic study of polydomy in<Emphasis Type="Italic"> Cataulacus mckeyi</Emphasis> (Myrmicinae)

Social organisation of colonies of obligate plant-ants can affect their interaction with myrmecophyte hosts and with other ants competing for the resources they offer. An important parameter of social organisation is whether nest sites of a colony include one or several host individuals. We determined colony boundaries in a plant-ant associated with the rainforest understorey tree Leonardoxa africana subsp. africana, found in coastal forests of Cameroon (Central Africa). This myrmecophyte is strictly associated with two ants, Petalomyrmex phylax and Cataulacus mckeyi. Plants provide food and nesting sites for P. phylax, which protects young leaves against insect herbivores. This mutualism is often parasitised by C. mckeyi, which uses but does not protect the host. The presence of C. mckeyi on a tree excludes the mutualistic ant. Because Petalomyrmex -occupied trees are better protected, their growth and survival are superior to those of Cataulacus -occupied trees, giving P. phylax an advantage in occupation of nest sites. C. mckeyi often colonises trees that have lost their initial associate P. phylax, as a result of injury to the tree caused by disturbance. Polydomy may allow C. mckeyi to occupy small clumps of trees, without the necessity of claustral colony foundation in each tree. Investigating both the proximate (behavioural repertoire, colony odour) and the ultimate factors (genetic structure) that may influence colony closure, we precisely defined colony boundaries. We show that colonies of C. mckeyi are monogynous and facultatively polydomous, i.e. a colony occupies one to several Leonardoxa trees. Workers do not produce males. Thus, the hypothesis that polydomy allows workers in queenless nests to evade queen control for their reproduction is not supported in this instance. This particular colony structure may confer on C. mckeyi an advantage in short-distance dispersal, and this could help explain its persistence within the dynamic Leonardoxa system.     

Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres

C-reactive protein (CRP) is a positive, acute-phase protein. Plasma levels rise dramatically in response to tissue injury or inflammation and fall rapidly after recovery or treatment. Antibody-based human CRP test systems do not readily detect CRP from other animals due to the species specificity of antibodies directed against human CRP. Thus, generic systems for CRP detection, based solely on the interaction between CRP and phosphocholine (PC), have been developed. PC-bovine serum albumin (PC-BSA) conjugates were produced and either labeled with horseradish peroxidase to facilitate CRP detection in a CRP enzyme-linked sorbent assay (ELSA) or coupled to carboxy-modified microspheres to facilitate the nonenzymatic, turbidimetric detection of CRP. The CRP-ELSA is a competitive assay, where the total assay time is 45 min, the assay sensitivity is 1.06 mg/L CRP, and the dynamic range of the assay is 0-500 mg/L. When PC-BSA conjugate is covalently coupled to carboxylated microspheres, agglutination occurs in the presence of CRP, the extent of which depends on the quantity of CRP present in the sample. Total assay time is 5 min with a dynamic range of 25-500 mg/L. Both assay formats are capable of accurately detecting human CRP and the CRP-ELSA can detect canine CRP as a disease state indicator.     

The origin and evolution of Eragrostis tef (Poaceae) and related polyploids: evidence from nuclear waxy and plastid rps16

Tef (Eragrostis tef; Poaceae) is an allotetraploid (2n = 4x = 40) cereal crop whose origin within the large genus Eragrostis is unknown. Previous studies have suggested a total of 14 wild Eragrostis species as potential progenitors. Phylogenetic analysis of sequence data from the nuclear gene waxy and the plastid locus rps16 strongly supports the widely held hypothesis of a close relationship between tef and E. pilosa, a wild allotetraploid. Eragrostis heteromera, another previously proposed progenitor, is shown by the waxy data to be a close relative of one of the tef genomes. Other putative progenitors included in the taxon sample are not supported as closely related to tef. Plastid sequences from five varieties of tef and four E. pilosa accessions are identical and therefore are uninformative with respect to the question of multiple origins of these polyploids. The waxy phylogeny also resolves the relationships among other allopolyploids, supporting a close relationship between the morphologically similar allotetraploids E. macilenta, E. minor, and E. mexicana. Eragrostis cilianensis, another morphologically similar allopolyploid, appears to have shared one diploid progenitor with these species but derived its other genome from an unrelated diploid.     

Chemical complementation: small-molecule-based genetic selection in yeast

Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

High-resolution crossover maps for each bivalent of Zea mays using recombination nodules

Recombination nodules (RNs) are closely correlated with crossing over, and, because they are observed by electron microscopy of synaptonemal complexes (SCs) in extended pachytene chromosomes, RNs provide the highest-resolution cytological marker currently available for defining the frequency and distribution of crossovers along the length of chromosomes. Using the maize inbred line KYS, we prepared an SC karyotype in which each SC was identified by relative length and arm ratio and related to the proper linkage group using inversion heterozygotes. We mapped 4267 RNs on 2080 identified SCs to produce high-resolution maps of RN frequency and distribution on each bivalent. RN frequencies are closely correlated with both chiasma frequencies and SC length. The total length of the RN recombination map is about twofold shorter than that of most maize linkage maps, but there is good correspondence between the relative lengths of the different maps when individual bivalents are considered. Each bivalent has a unique distribution of crossing over, but all bivalents share a high frequency of distal RNs and a severe reduction of RNs at and near kinetochores. The frequency of RNs at knobs is either similar to or higher than the average frequency of RNs along the SCs. These RN maps represent an independent measure of crossing over along maize bivalents.     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.