Interaction of Cu(II)with His-Val-Gly-Asp and of Zn(II) with His-Val-His,Two Peptides at the Active Site of Cu,Zn-Superoxide Dismutase

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). We have already studied the interaction of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II). As a continuation of this work and for comparison purposes we have also studied the interaction of Zn(II) with His-Val-His and Cu(II) with His-Val-Gly-Asp using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. Histamine type of coordination is observed for/ZnAH/²⁺, /ZnA/⁺, /ZnA²H/⁺ and/ZnA²/ in acidic pH while deprotonation of coordinated water molecules is observed at higher pH. /CUB/ species is characterized by the formation of a macrochelate and histamine type coordination. Its stability results in the suppression of amide deprotonation which occurs at high pH resulting in the formation of the highly distorted from square planar geometry 4N complex/CuBH_-3/³.     

Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.     

Influence of diet and photoperiod on development and reproduction of European populations of Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae)

The current study examines the effect of photoperiod (16:08 or 12:12 h L:D) and diet (eggs of Ephestia kuehniella Zeller (Lepidoptera: Phycitidae) or the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae)) on the development and reproduction of the multicoloured Asian lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae). A long-term laboratory population of H. axyridis (since 1998) and a melanic and non-melanic population originating from field collected individuals of H. axyridis in Belgium were used in this study. Long day conditions (16 h photoperiod) shortened development of the field populations with 2–3 days when compared with short day conditions (12 h photoperiod). Oviposition in the field populations was delayed by 1–3 months when reared at a 12 h photoperiod. Dissections indicated that the females were in reproductive diapause. As compared with live pea aphids, a diet consisting of E. kuehniella eggs yielded heavier adult body weights (up to 12%) and increased the number of egg laying days (by 45–169%) for both field populations at a 16 h photoperiod and lengthened adult life span (by 45–92%) under both light regimens. The morph types differed in their response to the foods offered in terms of developmental rate, pre-oviposition period and number of oviposition days. The laboratory and field strains responded differentially to regimens of food and photoperiod. The study indicated a greater nutritional plasticity of the non-melanic morphs which may offer them a competitive advantage that may in part explain the predominance of non-melanic morphs in newly colonized areas.     

Rapid mapping and identification of mutations in Caenorhabditis elegans by restriction site-associated DNA mapping and genomic interval pull-down sequencing

Forward genetic screens provide a powerful approach for inferring gene function on the basis of the phenotypes associated with mutated genes. However, determining the causal mutation by traditional mapping and candidate gene sequencing is often the rate-limiting step, especially when analyzing many mutants. We report two genomic approaches for more rapidly determining the identity of the affected genes in Caenorhabditis elegans mutants. First, we report our use of restriction site-associated DNA (RAD) polymorphism markers for rapidly mapping mutations after chemical mutagenesis and mutant isolation. Second, we describe our use of genomic interval pull-down sequencing (GIPS) to selectively capture and sequence megabase-sized portions of a mutant genome. Together, these two methods provide a rapid and cost-effective approach for positional cloning of C. elegans mutant loci, and are also applicable to other genetic model systems.     

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T‐DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, α‐tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus‐end marker GFP–EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell‐to‐cell movement of TMV encoding GFP‐tagged movement protein (MP‐GFP) is reduced in ATER2 accompanied by a reduced association of MP‐GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.     

The function of communities in protein interaction networks at multiple scales

Background  

If biology is modular then clusters, or communities, of proteins derived using only protein interaction network structure should define protein modules with similar biological roles. We investigate the link between biological modules and network communities in yeast and its relationship to the scale at which we probe the network.     

A large plant beta-tubulin family with minimal C-terminal variation but differences in expression

Tubulins, as the major structural component of microtubules (MT), are highly conserved throughout the entire eukaryotic kingdom. They consist of alpha/beta heterodimers. Both monomers, at least in multicellular organisms, are encoded by gene families. In higher plants up to eight beta-tubulin isotypes, mostly differing in their very C-termini, have been described. These variable beta-tubulin C-termini have been discussed in the context of functional microtubule diversity. However, in plants, in contrast to vertebrates, functional isotype specificity remains yet to be demonstrated. Unlike higher plants, unicellular green algae in general do not exhibit isotypic variations. The moss Physcomitrella patens is a phylogenetic intermediate between higher plants and green algae. We isolated six beta-tubulin genes from Physcomitrella, named PpTub1 to 6. We show that the exon/intron structure, with the exception of one additional intron in PpTub6, is identical with that of higher plants, and that some members of the family are differentially expressed. Moreover, we find that all Physcomitrella isotypes are highly conserved and, most strikingly, are almost identical within their C-terminal amino acids (aa). This evolutionary ancient and large beta-tubulin gene family without significant isotypic sequence variation points to a role of differential regulation in the evolution of plant tubulin isotypes.     

PicU, a second serine protease autotransporter of uropathogenic Escherichia coli

Escherichia coli is the major aetiological agent of urinary tract infections (UTI). Like diarrhoeagenic strains of E. coli, uropathogenic isolates possess virulence determinants that distinguish them from commensal strains and allow them to produce the clinical manifestations associated with UTI. Several autotransporter proteins have been associated with the ability of E. coli, and other Gram-negative bacteria, to cause disease. Recently, we described the existence within uropathogenic E. coli (UPEC) strains of Sat, a toxin of the serine protease autotransporter of Enterobacteriaceae (SPATE) subfamily. Using features common to proteins secreted via the autotransporter pathway we have identified nine additional autotransporter proteins from the genomic sequence data of UPEC CFT073. Surprisingly, two additional members of the SPATE subfamily were identified. One protein, designated PicU, was homologous to the Pic protein identified in Shigella flexneri and enteroaggregative E. coli. The PicU protein was expressed and investigated for functional activity.     

New semidominant mutations that affect mouse development

Dominantly acting mutations that produce visible phenotypes are frequently recovered, either during routine maintenance of colonies or from mutagenesis experiments. We have studied 12 dominant mouse mutations that cause a tail dysmorphology, a coat spotting phenotype, or a combination of these. The majority of these mutations act in a semidominant manner with the homozygous state associated with embryonic lethality and a visible phenotype at or before midgestation. The homozygous phenotypes include axis truncation and neural crest cell defects, as may be expected from the heterozygous phenotypes. The majority of mutations, however, also produced other phenotypes that include neural tube closure defects and aberrant heart looping. In one coat spotting mutant the homozygous condition is lethal before neural crest cell production commences. The mutated genes often function in processes additional to those alluded to by the heterozygous phenotype.