Modification of PATase by L/F‐transferase generates a ClpS‐dependent N‐end rule substrate in Escherichia coli

The N‐end rule pathway is conserved from bacteria to man and determines the half‐life of a protein based on its N‐terminal amino acid. In Escherichia coli, model substrates bearing an N‐degron are recognised by ClpS and degraded by ClpAP in an ATP‐dependent manner. Here, we report the isolation of 23 ClpS‐interacting proteins from E. coli. Our data show that at least one of these interacting proteins—putrescine aminotransferase (PATase)—is post‐translationally modified to generate a primary N‐degron. Remarkably, the N‐terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl‐tRNA‐protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N‐terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N‐end rule pathway, through the ClpAPS‐mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N‐degron, which is required for substrate delivery to ClpA.     

Dye to use with virus challenge for testing barrier materials.

Can FD&C Blue no. 1 dye photoinactivate bacteriophages phi X174, T7, PRD1, and phi 6 under laboratory lighting conditions? At high levels of light, the dye (500 microM) photoinactivated only phi 6. Thus, this dye can be used at concentrations up to 500 microM with bacteriophages phi X174, T7, and PRD1 to test barrier material integrity.     

Transmissibility of caprine scrapie in ovine transgenic mice

ABSTRACT: BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.     

Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype

Three clonal strain types (I, II, and III) of Toxoplasma gondii predominate worldwide. The outcome of infection in mice is highly dependent on the parasite genotype with type I strains being uniformly virulent, while types II and III are nonvirulent. Interactions with the innate immune response play a major role in determining the outcome of infection in the murine model. To identify key early differences in the innate immune response that contribute to pathogenesis, we examined the cytokine production of macrophages after in vitro infection with parasites of virulent type I and nonvirulent type II genotypes. Infection with type II strain parasites stimulated the production of proinflammatory cytokines, and particularly high levels of the Th1-polarizing cytokine, IL-12. Infection with type II strain parasites stimulated NF-kappaB nuclear translocation at early time points and led to the up-regulation of mRNA levels of IL-12 and other proinflammatory cytokines that was dependent on the myeloid differentiation factor 88 signaling pathway. Induction of IL-12 required active invasion by live parasites and was not blocked by infection with virulent type I strain parasites, arguing against an active inhibition of signaling. Our findings suggest that early induction of high levels of IL-12 by macrophages infected with type II strain parasites may contribute to more effective control.     

The reduction potential of the beta-carotene.+ /beta-carotene couple in an aqueous micro-heterogeneous environment

There is a resurgence of interest in the role of electron transfer reactions involving beta-carotene in photosynthesis. There is also current debate on the health benefits of dietary carotenoids and the possible deleterious effects on certain sub-populations such as smokers. The impact of dietary carotenoids on health may well be also related to radical reactions. A key parameter in biological systems is therefore the one-electron reduction potential of the carotenoid radical cation, now reported for the first time in a model biological aqueous environment. The value obtained is 1.06+/-0. 01 V and is sufficiently high to oxidise cell membrane proteins, but is low enough to repair P(680).+ in the photosynthetic reaction centre.     

Modeling the Interruption of the Transmission of Soil-Transmitted Helminths by Repeated Mass Chemotherapy of School-Age Children

Background

The control or elimination of neglected tropical diseases has recently become the focus of increased interest and funding from international agencies through the donation of drugs. Resources are becoming available for the treatment of soil-transmitted helminth (STH) infection through school-based deworming strategies. However, little research has been conducted to assess the impact of STH treatment that could be used to guide the design of efficient elimination programs.

Methodology

We construct and analyse an age-structured model of STH population dynamics under regular treatment. We investigate the potential for elimination with finite rounds of treatment, and how this depends on the value of the basic reproductive number R₀ and treatment frequency.

Principal findings

Analysis of the model indicates that its behaviour is determined by key parameter groupings describing the basic reproduction number and the fraction of it attributable to the treated group, the timescale of material in the environment and the frequency and efficacy of treatment. Mechanisms of sexual reproduction and persistence of infectious material in the environment are found to be much more important in the context of elimination than in the undisturbed baseline scenario. For a given rate of drug use, sexual reproduction dictates that less frequent, higher coverage treatment is more effective. For a given treatment coverage level, the lifespan of infectious material in the environment places a limit on the effectiveness of increased treatment frequency.

Conclusions

Our work suggests that for models to capture the dynamics of parasite burdens in populations under regular treatment as elimination is approached, they need to include the effects of sexual reproduction among parasites and the dynamics infectious material in the reservoir. The interaction of these two mechanisms has a strong effect on optimum treatment strategies, both in terms of how frequently to treat and for how long.     

Photocytotoxicity of lipofuscin in human retinal pigment epithelial cells.

Lipofuscin accumulates with age in a variety of highly metabolically active cells, including the retinal pigment epithelium (RPE) of the eye, where its photoreactivity has the potential for cellular damage. The aim of this study was to assess the phototoxic potential of lipofuscin in the retina. RPE cell cultures were fed isolated lipofuscin granules and maintained in basal medium for 7 d. Control cells lacking granules were cultured in an identical manner. Cultures were either maintained in the dark or exposed to visible light (2.8 mWcm2) at 37 degrees C for up to 48 h. Cells were subsequently assessed for alterations in cell morphology, cell viability, lysosomal stability, lipid peroxidation, and protein oxidation. Exposure of lipofuscin-fed cells to short wavelength visible light (390-550 nm) caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nonenal), protein oxidation (protein carbonyl formation), loss of lysosomal integrity, cytoplasmic vacuolation, and membrane blebbing culminating in cell death. This effect was wavelength-dependent because light exposure at 550 to 800 nm had no adverse effect on lipofuscin-loaded cells. These results confirm the photoxicity of lipofuscin in a cellular system and implicate it in cell dysfunction such as occurs in ageing and retinal diseases.     

Conserved residues in the N-domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.     

Photocontrol of Hook Opening in Cuscuta gronovii Willd

Hook opening in seedlings of Cuscuta gronovii Willd. occurred only after prolonged exposures to blue, red, or far red light. Prolonged far red exposure was less effective than prolonged exposure to red or blue light. Brief far red irradiation inhibited the inductive effect of red light. The far red inhibition was in turn reversed by brief red irradiation. These effects suggest the involvement of two photosystems in the control of hook opening in Cuscuta gronovii Willd.: a phytochrome-mediated system and a separate high energy requirement.