Separate and overlapping specificities in rheumatoid arthritis antibodies binding to citrulline- and homocitrulline-containing peptides related to type I and II collagen telopeptides

IntroductionOur objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence.MethodsA total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA.ResultsOf the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence.ConclusionsAntibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0515-z) contains supplementary material, which is available to authorized users.     

Protein engineering: opportunities and challenges

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.     

Patterns of nitrogen and sulfur accumulation and retention in ombrotrophic bogs, eastern Canada

Nitrogen (N) and sulfur (S) play important roles in peatlands, through their influence on plant production and peat decomposition rates and on redox reactions, respectively, and peatlands contain substantial stores of these two elements. Using peat N and S concentrations and dry bulk density and ²¹⁰Pb dating, we determined the rates of N and S accumulation over the past 150 years in hummock and hollow profiles from 23 ombrotrophic bogs in eastern Canada. Concentrations of N and S averaged 0.80% and 0.18%, respectively, generally increased with depth in the profile and there was a weak but significant correlation between N and S concentrations. Rates of N and S accumulation over the past 50–150 years ranged from 0.5 to 4.8 g N m^?2 yr^?1 and from 0.1 to 0.9 g S m^?2 yr^?1. There were significant but weak correlations between C, N and S accumulation rates over 50‐, 100‐ and 150‐year periods. Over the last 50 years, rates of S accumulation showed little differentiation between hummocks and hollows, whereas the pattern for N accumulation was more variable (hummock minus hollow rate ranged from ?1 to +1.5 g N m^?2 yr^?1), with hummocks generally having a larger N accumulation rate, correlated with the rate of carbon (C) accumulation. There was a modest but significant positive correlation between 50‐year rates of N accumulation and wet atmospheric deposition of N measured between 1990 and 1996, with accumulation rates about four times that of wet deposition. The difference between deposition and accumulation of N is attributed to organic N deposition, dry deposition and N₂ fixation. A weaker, but still significant, correlation was observed between 50‐year S accumulation and 1990–1996 wet atmospheric S deposition, with about 75% of the deposited S accumulating in the peat. A laboratory experiment with peat cores exposed to varying water table position and simulated N and S deposition, showed that on average 87% and 98% of the deposited NH₄⁺ and NO₃^?, respectively, and 58% of the deposited S were retained in the vegetation and unsaturated zone of the cores, supporting the results from the field study.     

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

Rôle of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris brassicae

The incorporation of labelled dietary palmitic, linoleic, and linolenic acids into neutral (NL) and phospholipids (PL) during the metamorphosis of Pieris brassicae was studied, and the ability of the fat body to incorporate acetate into PL determined. Thirty-three per cent of total lipid in early fifth instar larvae (minus haemolymph) is PL, while the corresponding value in female 4-day pupae is 13·0 per cent and in the fat body of 4-day pupae 6·3 per cent. Incorporation of label into PL was studied more closely and in all cases the label was recovered from phosphatidylcholine (PTC) and phosphatidylethanolamine (PTE). The label from palmitate was also found in sphingomyelin and possibly phosphatidylserine. Specific activity of PL in the case of palmitic and linolenic acids was greatest in late fifth instar larvae. In early fifth instar larvae on palmitic acid-1-¹⁴C 39·0 per cent of label was in PTC, 52·8 per cent in PTE, and 2·0 per cent in sphingomyelin. In late fifth instar 45·0 per cent was in PTC, 45·5 per cent in PTE, and 6·5 per cent in sphingomyelin, while in 4-day female pupae 45·2 per cent was in PTC, 41·3 per cent in PTE, and 13·5 per cent in sphingomyelin. The label from linolenic acid only varied a little from early fifth instar to 4-day pupae, 51·8 per cent being in PTC and 48·2 per cent in PTE in early fifth instar larvae. The label from linoleic acid is incorporated in fat body PL almost exclusively in PTC and PTE, 55·8 and 43·2 per cent respectively in 4-day female pupae. Injected acetate is distributed after 1 hr between PTC (58·6 per cent), PTE (24·4 per cent), and sphingomyelin (17·0 per cent). It was concluded that the polyunsaturated acids are proportionately more common in PTE than in other PL types, and that the fatty acids of sphingomyelin are mainly those that the insect is capable of synthesizing from acetate. Palmitic acid is desaturated by Pieris to palmitoleic acid and the latter possibly utilized in PTE to compensate for a deficiency of linolenic acid in the artificial diet. No saturation of linoleic or linolenic acid was found. The rates of PL and NL synthesis during development and the rôle of the investigated fatty acids in the biosynthesis of PL are discussed.     

Food utilization and esterase activity in Pieris brassicae during chronic exposure to lindane-containing food

Pieris brassicae larvae were reared on a meridic diet containing 0.75 ppm lindane. The results show that overal food consumption was lower but the approximate digestibility of food and the efficiency of conversion of ingested material into tissue biomass were higher than in control larvae. Total haemolymph esterase activity of larvae fed the lindane diet was reduced. Disc electrophoresis of midgut soluble proteins revealed eight zones of carboxylesterase activity. Results suggest that the activity of two esterase zones was reduced and that of one enhanced in larvae fed the lindane diet.

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Zusammenfassung Fünfte Larvenstadien von Pieris brassicae wurden für 72 h an einer meridischen Diät gehalten, die 0,75 ppm Lindan enthielt. Wie die Ergebnisse zeigen, war die Gesamtnahrungsaufnahme ca 50% niedriger als die der Kontrollarven, dagegen erreichte das Larvengewicht über 70% der Kontrolle. Angenäherte Verdaulichkeit (AD) betrug 80,4% für die lindanhaltige Diät gegenuber 61,7% für die Kontrolldiät. Die Effizienz des Umsatzes von aufgenommener Nahrung (ECI) war ebenfalls größer für lindanhaltige Nahrung, dagegen war die Effizienz des Umsatzes von verdauter Nahrung (ECD) bei lindanhaltiger und Kontrolldiät vergleichbar. Disc-Elektrophorese von löslichen, larvalen Mitteldarmproteinen ergab acht Zonen von Esterase-Aktivität, die verschiedene Empfindlinchkeit gegenüber in-vitro Organophosphat-Hemmung zeigten. Die Aktivität von zwei Carboxylesterasen war vermindert und die Aktivität einer schnell wandernden Esterase erschien bei lindangefütterten Larven vergrößert. Die Ergebnisse lassen auch vermuten, daß die totale hämolymphesterolytische Aktivität bei lindangehaltenen Larven reduziert war. Der Effekt von reduzierter hydrolytischer Aktivität auf larvalen Lipid-Metabolismus wird diskutiert.

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This study was supported by funds from the Finnish Cultural Foundation.     

Crocidolite asbestos causes an induction of p53 and apoptosis in cultured A-549 lung carcinoma cells

A number of genotoxic chemicals and agents, such as benzo(a)pyrene and ultraviolet light, are able to induce nuclear accumulation of p53 protein. Usually, this response is transient and a consequence of stabilization of the wild-type p53 protein. After withdrawal of the exposure, the amount of p53 protein returns to a normal level within hours or a few days. We have studied the p53 response to the exposure of crocidolite asbestos in A-549 lung carcinoma cells using three different methods, i.e., p53 immunohistochemistry, Western blotting and metabolic labelling followed by p53 immunoprecipitation. With these techniques we demonstrate a dose-dependent p53 nuclear response to crocidolite exposure. The half-life of p53 protein in A-549 lung carcinoma cells cultured in serum-free media increased from 30 up to 80 min, and the protein reacted with a wild-type specific antibody suggesting that it was in a wild-type conformation. In situ 3′-end labelling of A-549 cells demonstrated a dose-dependent increase in apoptotic activity. Our data support the idea that increased apoptotic activity, induced by crocidolite, is mediated by p53. This revised version was published online in June 2006 with corrections to the Cover Date.     

Paclitaxel succinate analogs: Anionic and amide introduction as a strategy to impart blood-brain barrier permeability

A focused library of TX-67 (C10 hemi-succinate) analogs has been prepared, including C7 regioisomers, esters, amides, and one-carbon homologs. These were prepared to investigate whether the lack of TX-67 interaction with P-glycoprotein (Pgp) is due to the presence of the carboxylic acid moiety and whether this phenomenon was restricted to C10 analogs. Tubulin stabilization ability, cytotoxicity, and Pgp interactions were evaluated. All carboxylic acid analogs and several of the amides had no apparent interactions with Pgp at the concentrations used, whereas the ester variants displayed characteristics of Pgp substrates. Furthermore, it was demonstrated that hydrogen-bonding properties were significant with respect to Pgp interactions. Calculations of logD and cross-sectional areas revealed that these analogs are predicted to partition into the membrane and can compete for Pgp binding sites. The anionic and amide introduction strategy may allow for delivery of paclitaxel into the CNS and may be a potential approach for the delivery of other, structurally complex and lipophilic non-CNS permeable drugs.     

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) differ in their suitability as hosts for the endangered freshwater pearl mussel (Margaritifera margaritifera) in northern Fennoscandian rivers

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