The quaternary structure of RNase G from Escherichia coli

RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.     

Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K⁺ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

“An Impediment to Living Life”: Why and How Should We Measure Stiffness in Polymyalgia Rheumatica?

Objectives

To explore patients’ concepts of stiffness in polymyalgia rheumatica (PMR), and how they think stiffness should be measured.

Methods

Eight focus groups were held at three centres involving 50 patients with current/previous PMR. Each group had at least one facilitator and one rapporteur making field notes. An interview schedule was used to stimulate discussion. Interviews were recorded, transcribed and analysed using an inductive thematic approach.

Results

Major themes identified were: symptoms: pain, stiffness and fatigue; functional impact; impact on daily schedule; and approaches to measurement. The common subtheme for the experience of stiffness was “difficulty in moving”, and usually considered as distinct from the experience of pain, albeit with a variable overlap. Some participants felt stiffness was the “overwhelming” symptom, in that it prevented them carrying out “fundamental activities” and “generally living life”. Diurnal variation in stiffness was generally described in relation to the daily schedule but was not the same as stiffness severity. Some participants suggested measuring stiffness using a numeric rating scale or a Likert scale, while others felt that it was more relevant and straightforward to measure difficulty in performing everyday activities rather than about stiffness itself.

Conclusions

A conceptual model of stiffness in PMR is presented where stiffness is an important part of the patient experience and impacts on their ability to live their lives. Stiffness is closely related to function and often regarded as interchangeable with pain. From the patients’ perspective, visual analogue scales measuring pain and stiffness were not the most useful method for reporting stiffness; participants preferred numerical rating scales, or assessments of function to reflect how stiffness impacts on their daily lives. Assessing function may be a pragmatic solution to difficulties in quantifying stiffness.     

Implications of land‐use change to Short Rotation Forestry in Great Britain for soil and biomass carbon

Land‐use change can have significant impacts on soil and aboveground carbon (C) stocks and there is a clear need to identify sustainable land uses which maximize C mitigation potential. Land‐use transitions from agricultural to bioenergy crops are increasingly common in Europe with one option being Short Rotation Forestry (SRF). Research on the impact on C stocks of the establishment of SRF is limited, but given the potential for this bioenergy crop in temperate climates, there is an evident knowledge gap. Here, we examine changes in soil C stock following the establishment of SRF using combined short (30 cm depth) and deep (1 m depth) soil cores at 11 sites representing 29 transitions from agriculture to SRF. We compare the effects of tree species including 9 coniferous, 16 broadleaved and 4 Eucalyptus transitions. SRF aboveground and root biomass were also estimated in 15 of the transitions using tree mensuration data allowing assessments of changes in total ecosystem C stock. Planting coniferous SRF, compared to broadleaved and Eucalyptus SRF, resulted in greater accumulation of litter and overall increased soil C stock relative to agricultural controls. Though broadleaved SRF had no overall effect on soil C stock, it showed the most variable response suggesting species‐specific effects and interactions with soil types. While Eucalyptus transitions induced a reduction in soil C stocks, this was not significant unless considered on a soil mass basis. Given the relatively young age and limited number of Eucalyptus plantations, it is not possible to say whether this reduction will persist in older stands. Combining estimates of C stocks from different ecosystem components (e.g., soil, aboveground biomass) reinforced the accumulation of C under coniferous SRF, and indicates generally positive effects of SRF on whole‐ecosystem C. These results fill an important knowledge gap and provide data for modelling of future scenarios of LUC.     

Addition of Escherichia coli K-12 Growth Observation and Gene Essentiality Data to the EcoCyc Database

The sets of compounds that can support growth of an organism are defined by the presence of transporters and metabolic pathways that convert nutrient sources into cellular components and energy for growth. A collection of known nutrient sources can therefore serve both as an impetus for investigating new metabolic pathways and transporters and as a reference for computational modeling of known metabolic pathways. To establish such a collection for Escherichia coli K-12, we have integrated data on the growth or nongrowth of E. coli K-12 obtained from published observations using a variety of individual media and from high-throughput phenotype microarrays into the EcoCyc database. The assembled collection revealed a substantial number of discrepancies between the high-throughput data sets, which we investigated where possible using low-throughput growth assays on soft agar and in liquid culture. We also integrated six data sets describing 16,119 observations of the growth of single-gene knockout mutants of E. coli K-12 into EcoCyc, which are relevant to antimicrobial drug design, provide clues regarding the roles of genes of unknown function, and are useful for validating metabolic models. To make this information easily accessible to EcoCyc users, we developed software for capturing, querying, and visualizing cellular growth assays and gene essentiality data.     

FcγRIIIa expression on monocytes in rheumatoid arthritis: role in immune-complex stimulated TNF production and non-response to methotrexate therapy

Objective

The expression of FcγRIIIa/CD16 may render monocytes targets for activation by IgG-containing immune complexes (IC). We investigated whether FcγRIIIa/CD16 was upregulated in rheumatoid arthritis (RA), associated with TNF production in response to IC-stimulation, and if this predicted response to methotrexate therapy.

Methods

FcγRIIIa/CD16 expression on CD14^low and CD14⁺⁺ monocytes was measured by flow cytometry in healthy controls and RA patients (early and long-standing disease). Intracellular TNF-staining was carried out after in vitro LPS or heat-aggregated immunoglobulin (HAG) activation. FcγRIIIa/CD16 expression pre- and post-steroid/methotrexate treatment was examined.

Results

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in long-standing RA patients compared to controls was demonstrated (p = 0.002) with intermediate levels in early-RA patients. HAG-induced TNF-production in RA patients was correlated with the percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 (p<0.001). The percentage of CD14⁺⁺ monocytes expressing FcγRIIIa/CD16 at baseline in early DMARD-naïve RA patients was negatively correlated with DAS28-ESR improvement 14-weeks post-methotrexate therapy (p = 0.003) and was significantly increased in EULAR non-responders compared to moderate (p = 0.01) or good responders (p = 0.003). FcγRIIIa/CD16 expression was not correlated with age, presence of systemic inflammation or autoantibody titers.

Conclusion

Increased FcγRIIIa/CD16 expression on CD14⁺⁺ monocytes in RA may result in a cell that has increased responsiveness to IC-stimulation. This monocyte subset may contribute to non-response to methotrexate therapy.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

Liver fatty acid binding protein gene ablation potentiates hepatic cholesterol accumulation in cholesterol-fed female mice

Although liver fatty acid binding protein (L-FABP) is postulated to influence cholesterol homeostasis, the physiological significance of this hypothesis remains to be resolved. This issue was addressed by examining the response of young (7 wk) female mice to L-FABP gene ablation and a cholesterol-rich diet. In control-fed mice, L-FABP gene ablation alone induced hepatic cholesterol accumulation (2.6-fold), increased bile acid levels, and increased body weight gain (primarily as fat tissue mass). In cholesterol-fed mice, L-FABP gene ablation further enhanced the hepatic accumulation of cholesterol (especially cholesterol ester, 12-fold) and potentiated the effects of dietary cholesterol on increased body weight gain, again mainly as fat tissue mass. However, in contrast to the effects of L-FABP gene ablation in control-fed mice, biliary levels of bile acids (as well as cholesterol and phospholipids) were reduced. These phenotypic alterations were not associated with differences in food intake. In conclusion, it was shown for the first time that L-FABP altered cholesterol metabolism and the response of female mice to dietary cholesterol. While the biliary and lipid phenotype of female wild-type L-FABP+/+ mice was sensitive to dietary cholesterol, L-FABP gene ablation dramatically enhanced many of the effects of dietary cholesterol to greatly induce hepatic cholesterol (primarily cholesterol ester) and triacylglycerol accumulation as well as to potentiate body weight gain (primarily as fat tissue mass). Taken together, these data support the hypothesis that L-FABP is involved in the physiological regulation of cholesterol metabolism, body weight gain, and obesity.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.