Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

Status and population trends of Starling Sturnus vulgaris in Great Britain

Capsule Starling populations have declined markedly since 1964, with the greatest declines in pastoral areas in the south and west of Britain.

Aims To establish the size of the Starling population and its recent decline in different habitats and regions.

Methods We use distance-based transect sampling to establish, for the first time, robust estimates of population size in different habitats and regions. We then analyse long-term trend data from two extensive monitoring schemes using generalized additive models to find correlates of the population decline.

Results The mean national breeding population of Starling over the period 1994–2000 was estimated at about 8.5 million birds, with a 95% confidence interval of 8.1–10.8 million. Most Starlings (36%) occur in southern Britain and densities are greatest in suburban habitats. Populations in both suburban areas and the wider countryside declined by over 50% between 1964 and 2000, being greatest in the south and west of Britain and in areas of livestock farming.

Conclusions Changes in pastoral farming practices are likely to account for at least some of the decline in the wider countryside, probably related to changes in food resources, though these are largely unquantified.     

Latitudinal and seasonal patterns in clutch size of some single‐brooded British birds

Capsule Daylength, rather than latitude, was found to be an important determinant of variation in clutch size.

Aims To describe the nature of spatial and temporal variation in clutch size, and explore the ecological correlates of these patterns.

Methods We tested the prediction that seasonal declines in clutch size will be greater at higher latitudes. The environmental variables focused on were the influence of daylength, plant productivity, seasonality (i.e. Ashmole's hypothesis) and physiological mechanisms that relate clutch size to ambient temperature. We used data from 1980 to 2003 on spatial variation in clutch size across Britain for single‐brooded species, in which clutch size can be taken as a measure of annual reproductive investment. We included all seven species, from five families, with sufficient data in the British Trust for Ornithology's Nest Record Scheme.

Results There are strong seasonal declines in clutch size but little evidence for latitudinal gradients in clutch size or in latitudinal gradients in the rate of seasonal clutch size decline. Of the environmental variables investigated, daylength had the most marked effect on clutch size; this was positive in diurnal species and negative in the one nocturnal species.

Conclusions Although this study was confined to a relatively small latitudinal range of 8°, we found marked latitudinal gradients in a number of factors thought to drive spatial patterns in clutch size. Moreover, such variation is of sufficient magnitude to generate spatial patterns in other ecological variables in Britain. There is thus no simple explanation for the lack of a latitudinal gradient in clutch size. The results concerning daylength indicate that the time available for foraging is an important determinant of variation in clutch size.     

New insights into the biosynthesis of mycobacterial lipomannan arising from deletion of a conserved gene

Genetic construction of a mutant strain (designated MSMEG4245) of Mycobacterium smegmatis, defective in a broadly conserved gene for a putative glycosyltransferase of the glycosyltransferase-C superfamily, results in a phenotype marked by the virtual absence of the phosphatidylinositol-containing lipomannan and lipoarabinomannan, replaced instead by a novel truncated form of lipomannan. The normal spectrum of phosphatidylinositol mannosides, long presumed precursors of these lipoglycans, was retained. Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry of the mutated form of lipomannan shows a family of phosphatidylinositol-anchored lipomannans with from only 5 to 20 Manp residues as compared with lipomannan from the wild type strain consisting of 21-34 Manp residues but with few changes in the branching pattern. Thus, MSMEG4245 is apparently a key mannosyltransferase, required for the proper elongation of lipomannan to its normal state and subsequent synthesis of lipoarabinomannan. The corresponding ortholog in Mycobacterium tuberculosis H37Rv has been identified as Rv2174. This previously unrecognized feature of the biosynthesis of lipomannan/lipoarabinomannan allows a significant revision of structural and biosynthetic schemata and provides a molecular basis of selectivity in biosynthesis, as conferred by the MSMEG4245 gene.     

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

The Na⁺/K⁺-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na⁺ for extracellular K⁺ to the hydrolysis of ATP. The asymmetric distribution of Na⁺ and K⁺ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers–Post model. It involves the presence of gates alternatively exposing Na⁺/K⁺-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K⁺, information is lacking about Na⁺-occluded intermediates, as occluded Na⁺ was only detected in states incapable of performing a catalytic cycle, including two Na⁺-containing crystallographic structures. The current knowledge is that intracellular Na⁺ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na⁺-occluded intermediates, we isolated species with tightly bound Na⁺ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na⁺ becomes spontaneously occluded in the E1 dephosphorylated form of the Na⁺/K⁺-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na⁺ occlusion as it would have been expected; on the contrary, occluded Na⁺ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers–Post model for explaining the Na⁺ transport pathway.     

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.     

Biosynthesis of the arabinogalactan-peptidoglycan complex of Mycobacterium tuberculosis

The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes. Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M. tuberculosis are fairly well understood. However, the current impetus for studying M. tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs. Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M. tuberculosis. The publication of the complete M. tuberculosis genome in 1998 has greatly aided these studies. To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M. tuberculosis have been identified and at least partially characterized. Eleven of these enzymes were reported subsequent to the publication of the M. tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome. In this article we review the current understanding of M. tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.     

Identification,cloning, purification,and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase

The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).