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.     

High-Salinity-Induced Iron Limitation in Bacillus subtilis

Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.     

Exaptation at the molecular genetic level

The realization that body parts of animals and plants can be recruited or coopted for novel functions dates back to, or even predates the observations of Darwin. S.J. Gould and E.S. Vrba recognized a mode of evolution of characters that differs from adaptation. The umbrella term aptation was supplemented with the concept of exaptation. Unlike adaptations, which are restricted to features built by selection for their current role, exaptations are features that currently enhance fitness, even though their present role was not a result of natural selection. Exaptations can also arise from nonaptations; these are characters which had previously been evolving neutrally. All nonaptations are potential exaptations. The concept of exaptation was expanded to the molecular genetic level which aided greatly in understanding the enormous potential of neutrally evolving repetitive DNA—including transposed elements, formerly considered junk DNA—for the evolution of genes and genomes. The distinction between adaptations and exaptations is outlined in this review and examples are given. Also elaborated on is the fact that such distinctions are sometimes more difficult to determine; this is a widespread phenomenon in biology, where continua abound and clear borders between states and definitions are rare.     

Interaction of Nucleic Acids with Lipid Membranes

Abstract

The thermodynamics of nucleic acids which were enclosed in reverse-phase evaporation vesicles was studied by thermal denaturation with optical recording. The denaturation curves were recorded with a dual wavelength spectrophotometer. The sum of the hypochromicity of the nucleic acid and of the change in turbidity of the vesicles was measured at 260 nm and was corrected for the change in turbidity at 320 nm. Cloned fragments of double-stranded DNA containing 180 base pairs and poly A:poly U were enclosed in REV with a yield up to every vesicle containing five nucleic acid molecules. Vesicles were prepared from egg- lecithin, and the surface charge of the vesicles was varied by addition of stearic acid, phosphatidyl-glycerol and phosphatidyl-serine. The helix-coil transition of the nucleic acid enclosed in the vesicle could be resolved from that of the free nucleic acid. Due to the enclosure into the egg-lecithin REV the transition is stabilized from 70.5° to 74°C, the transition is broadened from 0.7°C to 2.7°C. Varying the phosphatidyl-serine-lecithin-ratio from 0–100%, an optimum in the yield of enclosure at 20% was obtained, a further broadening of the transition to 5.5°C and a decrease of the stabilization down to a small destabilization at 100% phosphatidyl serine was observed. Qualitatively, similar effects were observed with poly A:poly U. Variation of the ionic strength led to the conclusion that the replacement of the counterions of the phosphate backbone by the surface charge of the membrane, as well as a direct contact between the nucleic acid and the membrane have to be assumed. At present, the biological relevance of the results may be more in the drastic decrease in cooperativity than in the slight modulation of the stability. From nearly 180 base pairs opening up cooperatively in free nucleic acid this number is lowered to less than 50, a size in the range of promotor regions.     

Architecture and activity-mediated refinement of axonal projections from a mosaic of genetically identified retinal ganglion cells

Our understanding of how mammalian sensory circuits are organized and develop has long been hindered by the lack of genetic markers of neurons with discrete functions. Here, we report a transgenic mouse selectively expressing GFP in a complete mosaic of transient OFF-alpha retinal ganglion cells (tOFF-alphaRGCs). This enabled us to relate the mosaic spacing, dendritic anatomy, and electrophysiology of these RGCs to their complete map of projections in the brain. We find that tOFF-alphaRGCs project exclusively to the superior colliculus (SC) and dorsal lateral geniculate nucleus and are restricted to a specific laminar depth within each of these targets. The axons of tOFF-alphaRGC are also organized into columns in the SC. Both laminar and columnar specificity develop through axon refinement. Disruption of cholinergic retinal waves prevents the emergence of columnar- but not laminar-specific tOFF-alphaRGC connections. Our findings reveal that in a genetically identified sensory map, spontaneous activity promotes synaptic specificity by segregating axons arising from RGCs of the same subtype.     

‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage A or a large portion of the coding region of the Herpes simplex virus type l glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.     

A transcriptional network associated with natural variation in Drosophila aggressive behavior

Background  

Aggressive behavior is an important component of fitness in most animals. Aggressive behavior is genetically complex, with natural variation attributable to multiple segregating loci with allelic effects that are sensitive to the physical and social environment. However, we know little about the genes and genetic networks affecting natural variation in aggressive behavior. Populations of Drosophila melanogaster harbor quantitative genetic variation in aggressive behavior, providing an excellent model system for dissecting the genetic basis of naturally occurring variation in aggression.     

Aging in the post-genomic era: simple or complex?

A report on the Third Genetic Effects on Aging Meeting, The Jackson Laboratory, Bar Harbor, Maine, August 4-8, 2000.