The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively (Maurer and Dougherty J. Biol. Chem. 278(23):21076–21082, 2003). We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (I24C-SL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin–spin interaction with the neighboring I24C-SL residues in the MscL channel pentamer (Perozo et al.Nature 418:942–948, 2002). The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics     

Flexible N-terminal region of prion protein influences conformation of protease-resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.     

A peptide that antagonizes TCR-mediated reactions with both syngeneic and allogeneic agonists: functional and structural aspects

We identify and consider some characteristics of a peptide antagonist for the Ag-specific receptor on 2C cells (the 2C TCR). The peptide, GNYSFYAL (called GNY), binds to H-2K(b), and a very high-resolution crystal structure of the GNY-K(b) complex at 1.35 A is described. Although the GNY peptide does not bind to L(d), the potency of GNY-K(b) as an antagonist is evident from its ability to specifically inhibit 2C TCR-mediated reactions to an allogenic agonist complex (QLSPFPFDL-L(d)), as well as to a syngeneic agonist complex (SIYRYYGL-K(b)). The crystal structure and the activities of alanine-substituted peptide variants point to the properties of the peptide P4 side chain and the conformation of the Tyr-P6 side chain as the structural determinants of GNYSFYAL antagonist activity.     

Effects of the sea louse Lepeophtheirus salmonis on temporal changes in cortisol,sex steroids,growth and reproductive investment in Arctic charr Salvelinus alpinus

Groups of mature (5+ year old) Arctic charr Salvelinus alpinus held in sea water were exposed for 34 days to either a high (mean ±s.e . 0·15 ± 0·01 sea lice Lepeophtheirus salmonis g^?1 fish mass) (HI), medium (0·07 ± 0·00 sea lice g^?1 fish mass) (MI) or no [control (C)] sea‐lice infection during early stages of gonad development (June to July). Infection with sea lice resulted in increased plasma cortisol concentrations and this was related to intensity of infection; females tended to have higher cortisol concentrations than males at high infection intensities (HI group: female c. 130 ng ml^?1; male c. 80 ng ml^?1). Plasma osmolality (C c. 330, MI c. 350 and HI c. 415 mOsm) and chloride concentrations (C c. 135, MI c. 155 and HI c. 190 mM) increased significantly with infection intensity, indicating osmoregulatory problems in infected fish. A strong positive relationship between plasma osmolality and cortisol concentration was recorded. Plasma sex‐steroid concentrations were influenced negatively by sea‐lice infection, particularly in the HI group, and were inversely related to plasma cortisol concentrations. The most heavily infected fish postponed the initiation of reproductive development until exposed to fresh water and timing of ovulation tended to be delayed in these fish. Growth rate and condition were negatively influenced by sea‐lice infection and growth rate was inversely related to plasma cortisol concentrations. Sea‐lice infection resulted in mortality among females in the HI group, and the proportion of maturing females was lower in the MI group (46%) than in the controls (85%). Egg production in the MI and HI groups was c. 50 and 30% of the C group. Egg size, embryonic survival and fry mass did not differ across groups. Sea lice influence reproductive development and egg production in S. alpinus, and consequently these parasites may influence populations via sublethal effects on broodfish, affecting growth and condition, and their reproductive output.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

The essential fatty acid status in phenylketonuria patients under treatment

Phenylketonuric patients are on a special diet that lacks certain essential fatty acids. This study evaluates the essential fatty acid status of a group of phenylketonuric patients in the Netherlands undergoing dietary treatment. To this end, the essential fatty acid status of nine phenylketonuria patients was studied. On the basis of age and gender, two control subjects were selected for each patient. The essential fatty acid composition of duplicate food portions and the essential fatty acid status of plasma and erythrocytes were analyzed. Phenylketonuria subjects had a different essential fatty acid profile from their peers, especially concerning the n-3 fatty acids. N-6 and n-3 fatty long-chain polyenes were hardly consumed by phenylketonuria subjects, in contrast to the control subjects. Linoleic acid, on the other hand, was consumed in significantly higher amounts by phenylketonuria subjects and made up about 40% of their daily fat consumption. The essential fatty acid consumption pattern of the phenylketonuria subjects is mirrored by the essential fatty acid concentrations in blood. The essential fatty acid status of the phenylketonuric diet should be improved in order to prevent deficiency in n-3 fatty acids.     

Ammonia inactivation of Ascaris ova in ecological compost by using urine and ash

Viable ova of Ascaris lumbricoides, an indicator organism for pathogens, are frequently found in feces-derived compost produced from ecological toilets, demonstrating that threshold levels of time, temperature, pH, and moisture content for pathogen inactivation are not routinely met. Previous studies have determined that NH(3) has ovicidal properties for pathogens, including Ascaris ova. This research attempted to achieve Ascaris inactivation via NH(3) under environmental conditions commonly found in ecological toilets and using materials universally available in an ecological sanitation setting, including compost (feces and sawdust), urine, and ash. Compost mixed with stored urine and ash produced the most rapid inactivation, with significant inactivation observed after 2 weeks and with a time to 99% ovum inactivation (T(99)) of 8 weeks. Compost mixed with fresh urine and ash achieved a T(99) of 15 weeks, after a 4-week lag phase. Both matrices had relatively high total-ammonia concentrations and pH values of >9.24 (pK(a) of ammonia). In compost mixed with ash only, and in compost mixed with fresh urine only, inactivation was observed after an 11-week lag phase. These matrices contained NH(3) concentrations of 164 to 173 and 102 to 277 mg/liter, respectively, when inactivation occurred, which was below the previously hypothesized threshold for inactivation (280 mg/liter), suggesting that a lower threshold NH(3) concentration may be possible with a longer contact time. Other significant results include the hydrolysis of urea to ammonia between pH values of 10.4 and 11.6, above the literature threshold pH of 10.     

Distribution of microbial biomass and potential for anaerobic respiration in Hanford Site 300 Area subsurface sediment

Subsurface sediments were recovered from a 52-m-deep borehole cored in the 300 Area of the Hanford Site in southeastern Washington State to assess the potential for biogeochemical transformation of radionuclide contaminants. Microbial analyses were made on 17 sediment samples traversing multiple geological units: the oxic coarse-grained Hanford formation (9 to 17.4 m), the oxic fine-grained upper Ringold formation (17.7 to 18.1 m), and the reduced Ringold formation (18.3 to 52 m). Microbial biomass (measured as phospholipid fatty acids) ranged from 7 to 974 pmols per g in discrete samples, with the highest numbers found in the Hanford formation. On average, strata below 17.4 m had 13-fold less biomass than those from shallower strata. The nosZ gene that encodes nitrous oxide reductase (measured by quantitative real-time PCR) had an abundance of 5 to 17 relative to that of total 16S rRNA genes below 18.3 m and <5 above 18.1 m. Most nosZ sequences were affiliated with Ochrobactrum anthropi (97 sequence similarity) or had a nearest neighbor of Achromobacter xylosoxidans (90 similarity). Passive multilevel sampling of groundwater geochemistry demonstrated a redox gradient in the 1.5-m region between the Hanford-Ringold formation contact and the Ringold oxic-anoxic interface. Within this zone, copies of the dsrA gene and Geobacteraceae had the highest relative abundance. The majority of dsrA genes detected near the interface were related to Desulfotomaculum spp. These analyses indicate that the region just below the contact between the Hanford and Ringold formations is a zone of active biogeochemical redox cycling.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

The Effect of Badger Culling on Breakdown Prolongation and Recurrence of Bovine Tuberculosis in Cattle Herds in Great Britain

Bovine tuberculosis is endemic in cattle herds in Great Britain, with a substantial economic impact. A reservoir of Mycobacterium bovis within the Eurasian badger (Meles meles) population is thought to have hindered disease control. Cattle herd incidents, termed breakdowns, that are either ‘prolonged’ (lasting ≥240 days) or ‘recurrent’ (with another breakdown within a specified time period) may be important foci for onward spread of infection. They drain veterinary resources and can be demoralising for farmers. Randomised Badger Culling Trial (RBCT) data were re-analysed to examine the effects of two culling strategies on breakdown prolongation and recurrence, during and after culling, using a Bayesian hierarchical model. Separate effect estimates were obtained for the ‘core’ trial areas (where culling occurred) and the ‘buffer’ zones (up to 2 km outside of the core areas). For breakdowns that started during the culling period, ‘reactive’ (localised) culling was associated with marginally increased odds of prolongation, with an odds ratio (OR) of 1.7 (95% credible interval [CI] 1.1–2.4) within the core areas. This effect was not present after the culling ceased. There was no notable effect of ‘proactive’ culling on prolongation. In contrast, reactive culling had no effect on breakdown recurrence, though there was evidence of a reduced risk of recurrence in proactive core areas during the culling period (ORs and 95% CIs: 0.82 (0.64–1.0) and 0.69 (0.54–0.86) for 24- and 36-month recurrence respectively). Again these effects were not present after the culling ceased. There seemed to be no effect of culling on breakdown prolongation or recurrence in the buffer zones. These results suggest that the RBCT badger culling strategies are unlikely to reduce either the prolongation or recurrence of breakdowns in the long term, and that reactive strategies (such as employed during the RBCT) are, if anything, likely to impact detrimentally on breakdown persistence.