Effects of a novel tyrosine kinase inhibitor in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Biologicals have revolutionised the treatment of rheumatoid arthritis (RA). However, progressive joint destruction can still be observed in many patients and the search for novel molecular therapies targeting specific signalling pathways is ongoing. In the present study, we investigated the effects of GW282974, a novel compound directed against tyrosine kinase activity with respect to the potential suppression of inflammation and destruction. METHODS: Synovial tissue specimens were obtained from RA patients undergoing surgical joint replacement. Rheumatoid arthritis synovial fibroblasts (RASFs) were stimulated with cytokines and GW282974 was added in different concentrations. Gene expression was checked by TaqMan PCR, using 18S as housekeeping gene. Protein analysis was quantified by ELISA. Cell growth and proliferation was measured using the "ViaLight" proliferation assay. RESULTS: EGF had no effect on the gene expression profile of RASFs when used as single stimulatory agent. In combination with pro-inflammatory mediators however, EGF showed a synergistic effect. The expression of matrix metalloproteinases, inflammatory cytokines and cyclooxygenase-2 on mRNA levels was strongly increased, whereas the addition of GW282974 abrogated these effects in a dose-dependent manner. These data could be confirmed on protein/lipid levels analysing the supernatants of RASFs by ELISA. Similarly, cell growth and proliferation of RASFs were inhibited by GW282974 in a dose- and time-dependent manner. By contrast, no cytotoxic effects were seen within the concentrations used. DISCUSSION: GW282974 appears to interfere with the inflammatory and the destructive pathways in RASFs and might therefore be used as novel therapeutic strategy for the treatment of RA.

Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis.

OBJECTIVE: Leukotrienes are a family of arachidonic acid derivatives with potent proinflammatory and profibrotic properties, and 5-lipoxygenase (5-LOX) catalyzes two key steps in the leukotriene biosynthetic pathway. Since inflammatory cell infiltrates and excessive fibrosis are hallmarks of systemic sclerosis (SSc) skin lesions, we undertook the present study to investigate the expression of 5-LOX in skin biopsy specimens from patients with SSc. METHODS: Expression of 5-LOX in skin sections from 10 SSc patients and 8 healthy controls was examined by in situ hybridization with specific riboprobes and by immunohistochemistry analysis with 5-LOX monoclonal antibodies. Synthesis of 5-LOX by cultured dermal fibroblasts from 7 patients with SSc and 4 controls was measured by fluorescence-activated cell sorter analysis. In addition, concentrations of leukotriene B4 (LTB4) and LTE4 in fibroblast supernatants after stimulation were determined using enzyme immunoassays. RESULTS: Expression of 5-LOX was found in all skin sections from SSc patients as well as from controls. However, the number and percentage of 5-LOX-positive cells were significantly higher in SSc skin sections compared with control sections. Expression of 5-LOX was seen in cells within perivascular inflammatory infiltrates as well as in fibroblasts throughout the skin. The experiments with cultured skin fibroblasts revealed that 5-LOX was constitutively expressed in these cells, which resulted in the production of leukotrienes after cell stimulation. Whereas no difference was found for LTE4, SSc fibroblasts produced significantly higher amounts of LTB4 after stimulation, compared with healthy control fibroblasts. CONCLUSION: The results of this study suggest that the 5-LOX pathway may be of significance in the pathogenesis of SSc and may represent a target for new treatment strategies.

Expression of osteoclast differentiation factor in rheumatoid arthritis.

OBJECTIVE: To analyze the expression pattern of osteoclast differentiation factor (ODF) and its contribution to osteoclastogenesis in rheumatoid arthritis (RA). METHODS: The expression of ODF was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) in RA synovial fibroblasts (RASF) isolated from 7 RA patients and in normal skin fibroblasts. Using RNA probes specific for ODF, in situ hybridization was performed. Immunohistochemical double labeling for CD68 was applied to characterize the ODF-expressing cells. ODF protein and messenger RNA (mRNA) expression by RASF with or without 1,25(OH)2D3 was studied by Western blot analysis and quantitative real-time PCR. In addition, we performed coculture experiments with RASF and normal peripheral blood mononuclear cells with or without 1,25(OH)2D3. RESULTS: By RT-PCR, ODF mRNA expression was found in all RASF investigated, but not in normal skin fibroblasts. In situ hybridization revealed that in RA synovial tissues, ODF mRNA was expressed mainly in the lining layer and at sites where synovium was attached to bone. Immunohistochemical double labeling demonstrated ODF mRNA expression mainly in CD68-fibroblast-like synoviocytes and CD68+ multinucleated osteoclast-like cells. By Western blotting, all RASF expressed ODF protein. However, different levels of ODF expression were found in the RASF from different patients. Interestingly, RASF expressing higher levels of ODF induced a larger number of osteoclast-like cells than did RASF expressing only low levels of ODF. Although 1,25(OH)2D3 did not alter the levels of ODF expression in RASF on either Western blot or quantitative real-time PCR, osteoclastogenesis required the presence of 1,25(OH)2D3. CONCLUSION: The present results suggest that activated RASF, by expressing ODF, play an important role in rheumatoid bone destruction. Moreover, the data provide evidence that RASF not only activate osteoclasts, but also contribute directly to osteoclastogenesis.

Retrotransposable L1 elements expressed in rheumatoid arthritis synovial tissue: association with genomic DNA hypomethylation and influence on gene expression.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by a progressive destruction of joints by invasive synovial fibroblasts (SF). We searched for retroviral sequences in RA synovial fluid pellets, identified a sequence similar to that of open reading frame 2 (ORF2)/L1 retrotransposable elements, explored the expression of L1 in RA synovial tissues and cultured RA SF, and investigated the link to genomic DNA hypomethylation and the influence of functional L1 on gene expression. METHODS: RA synovial fluid pellets were screened by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerated pol primers. The sequences were identified by GenBank search. Riboprobes to ORF2/L1 and galectin-3 and antibodies to the ORF1/L1-related p40 protein were used for in situ hybridization and immunohistochemistry of synovial tissues and cultured RA SF. Real-time quantitative RT-PCR was used for detecting ORF1 messenger RNA (mRNA). Since DNA hypomethylation occurs in inflammatory diseases, we incubated cells with the methylation inhibitor 5-aza-2'-deoxycytidine (5-azaC) and compared RA SF and osteoarthritis (OA) SF. L1-negative RA SF were transfected with the functional L1.2 construct, and differential gene expression was analyzed by subtractive hybridization combined with nested PCR. RESULTS: RNA sequences similar to those of ORF2/L1 retrotransposable elements, THE1 transposon, human endogenous retrovirus (ERV)-E, human ERV-HC2, and gibbon ape leukemia virus pol genes were isolated from different RA synovial fluid pellets. In RA synovial tissues, ORF2/L1 transcripts were detected in the sublining layer and at sites of cartilage and bone destruction. Galectin-3 mRNA and L1-related ORF1/ p40 protein showed similar expression patterns. In contrast, OA synovial tissues in situ and cultures in vitro were negative. Real-time quantitative RT-PCR confirmed the presence of ORF1 mRNA in cultured RA SF (30-300-fold the amount in normal SF), demonstrating the existence of a nondegenerated and functional L1 element. In vitro, the majority of RA SF expressed ORF2/L1 mRNA. After incubation of SF with 5-azaC, L1 mRNA appeared in a time- and dose-dependent manner. Compared with OA SF, RA SF were more sensitive to 5-azaC. After transfection of RA SF with a functional L1.2 element, human stress-activated protein kinase 2 delta (SAPK2delta [or SAPK4]), met protooncogene, and galectin-3 binding protein genes were differentially expressed. The transcription of the SAPK2delta gene, favored also by DNA hypomethylation in vitro, was confirmed in RA synovial tissues. CONCLUSION: Taken together, these data suggest that L1 elements and SAPK2delta pathways play a role in the activation of RA SF.

New nerve root holders during spinal decompression.

New nerve root holders have been developed to protect nerve roots and dural sac during spinal bony decompression, especially if it takes place in the region of the lateral recess by means of a high speed air drill. Their design derives from the adaptation of common brain spatulas to the anatomy of the lumbar nerve roots. Their use, together with the contralateral approach (the operative microscopical visualization of the lesion from the side of the patient opposite to it), represents an available method to protect nervous structures from operative injuries without superfluous retraction of them. The contralateral approach permits the visualization of the deepest angles of the lateral recess without withdrawal of lateral parts of the articular facets, important for vertebral stability. In order to achieve satisfactory results without vertebral instrumental stabilization, the holder assists in the drilling procedure to enlarge the periradicular space covering the nerve root and helps avoid undue dural tearing or radicular damage.

Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli.

We report on a system to improve expression of mature c-type cytochromes in Escherichia coli. It is based on the use of plasmid pEC86 that expresses the E. coli cytochrome c maturation genes ccmABCDEFGH constitutively, whereby the production of both endogenous and foreign c-type cytochromes was increased substantially. The periplasmic soluble domains of the c-type cytochrome subunits FixO and FixP of the Bradyrhizobium japonicum cbb3 oxidase could be expressed in E. coli only when pEC86 was provided in a degP-deficient strain. This shows that a stimulation of heme attachment by the Ccm maturase system combined with the diminished proteolytic activity in the periplasm can increase c-type cytochrome yields.

Translocation to the periplasm and signal sequence cleavage of preapocytochrome c depend on sec and lep, but not on the ccm gene products.

Post-translational maturation of soluble cytochrome c includes translocation of the precursor polypeptide and heme through the cytoplasmic membrane, proteolytic cleavage of the signal sequence, and covalent attachment of heme. Specific genes for cytochrome c maturation (ccmABCDEFGH in Escherichia coli) are required for holocytochrome c formation, among them genes encoding an ABC transporter (ccmABC). We investigated the requirements of apocytochrome translocation to the periplasm and characterized specific intermediates of the cytochrome c maturation pathway. Apocytochrome precursor was present in the membrane fraction. Translocation of the polypeptide was independent of ccm gene products, but dependent on a functional secretion machinery, as shown by accumulation of preapocytochrome c in the membranes of secA and secY mutants. After translocation, cleavage of the signal sequence allowed the release of apocytochrome into the periplasm, where heme was bound in a ccm-dependent manner. By contrast, non-cleaved holocytochrome c containing covalently bound heme accumulated in the membranes of a lepB mutant, which indicated that signal sequence cleavage and heme attachment are independent steps in the cytochrome c maturation pathway.

Characterization of the Bradyrhizobium japonicum CycY protein, a membrane-anchored periplasmic thioredoxin that may play a role as a reductant in the biogenesis of c-type cytochromes.

A new member of membrane-anchored periplasmic thioredoxin-like proteins was identified in Bradyrhizobium japonicum. It is the product of cycY, the last gene in a cluster of cytochrome c biogenesis genes. Mutational analysis revealed that cycY is essential for the biosynthesis of all c-type cytochromes in this bacterium. The CycY protein was shown to be exported to the periplasm by its N-terminal signal sequence-like domain. Results from Western blot analyses of membrane and soluble fractions indicated that the CycY protein remains bound to the membrane. A soluble version of the protein devoid of its N-terminal membrane anchor (CycY*) was expressed in Escherichia coli and purified to homogeneity from the periplasmic fraction. The protein showed redox reactivity and properties similar to other thioredoxins such as fluorescence quenching in the oxidized form. Its equilibrium constant with glutathione was determined to be 168 mM, from which a standard redox potential of -0.217 V was calculated, suggesting that CycY might act as a reductant in the otherwise oxidative environment of the periplasm. This is in agreement with our hypothesis that CycY is required, directly or indirectly, for the reduction of the heme-binding site cysteines in the CXXCH motif of c-type apocytochromes before heme attachment occurs.

The Bradyrhizobium japonicum aconitase gene (acnA) is important for free-living growth but not for an effective root nodule symbiosis.

The Bradyrhizobium japonicum acnA gene encoding the tricarboxylic acid cycle enzyme aconitase was cloned and characterized. The gene was mapped immediately upstream of the cytochrome c biogenesis gene cycV and found to be transcribed in the opposite direction. The nucleotide sequence of acnA was determined; the derived amino acid sequence shared a significant similarity with bacterial aconitases and with the human iron-responsive-element-binding protein. The level of expression of the acnA gene under aerobic growth conditions was 10-fold higher than that under anaerobic conditions. The start of transcription was mapped by primer extension experiments, and the putative promoter was found to contain a typical -10 but no -35 consensus sequence for a sigma70-type RNA polymerase. A 5' deletion removing all but 19 nucleotides upstream of the start of transcription completely abolished gene expression. An acnA mutant was constructed by gene disruption, and the mutant phenotype was characterized. Growth of the mutant was severely affected and could not be corrected by the addition of glutamate as a supplement. Although aconitase activity in free-living cells was decreased by more than 70%, the ability of the mutant to establish an effective root nodule symbiosis with soybean plants was not affected. This suggested either the existence of a second aconitase or the compensation for the mutant defect by symbiosis-specific metabolites synthesized in the root nodules.

Requirements for maturation of Bradyrhizobium japonicum cytochrome c550 in Escherichia coli.

Various forms of Bradyrhizobium japonicum cytochrome c550 (the cycA gene product) were overexpressed in Escherichia coli cells grown under different conditions. Antibodies directed against a synthetic cytochrome c550 peptide were used as tools to detect both, apoprotein and holoprotein. Complete maturation of the apoprotein into its holo form with haem covalently bound to the polypeptide was observed only under anaerobic growth conditions and in E. coli K12 derivatives, whereas haem binding did not occur in the E. coli BL21 host. When maturation was complete, holocytochrome c550 was found exclusively in the periplasmic fraction. A cycA-expressing plasmid construct lacking the genetic information for the signal sequence produced apoprotein that was rapidly degraded without further maturation. Mutations in the haem-binding site resulted in products that were translocated through the cytoplasmic membrane, but apparently became degraded. Our results support the view that attachment of haem to the apoprotein is not a prerequisite for cleavage of the signal sequence and occurs on the periplasmic side of the membrane, subsequent to translocation of the apoprotein precursor.

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.

Pathological, physiological, and evolutionary aspects of short unstable DNA repeats in the human genome.

One of the salient features of the mammalian genome is the vast excess of DNA without obvious function, such as repetitive DNAs, spacers, and introns. In recent years, microsatellites, which include short triplet repeats (mostly CAGn and CGGn) and dinucleotide repeats (notably CAn) have gained widespread attention, along with minisatellites which consist of somewhat longer repeat units. Micro- and minisatellites, collectively called variable number tandem repeats (VNTRs), can be highly unstable and display an amazing degree of polymorphism. This property is exploited for gene mapping, for tumor diagnosis, and in forensic medicine. Undue expansion of gene-associated microsatellites is also responsible for some severe genetic diseases, such as fragile X syndrome. Most or all of these diseases are caused by expansion of CAG and CGG triplets. Within protein-coding regions these triplets usually code for polymers of glutamine, serine, alanine or proline. Physiologically, such amino acid repeats are often found in transcription factors and can increase or decrease their activity, depending on the repeat number. Alone or in conjunction with DNA methylation, such repeats may offer a unique opportunity for subtle, semi-stable modulation of gene activity. Also, at least in some plants and perhaps other organisms, a quasi-Lamarckian inheritance is mediated by repetitive DNA. Generally, repetitive DNA sequences, whether represented by short or by long DNA segments, may be beneficial for the evolution of a species.

Short introns interrupting the Oct-2 POU domain may prevent recombination between POU family genes without interfering with potential POU domain 'shuffling' in evolution.

Transcription factors are often encoded by gene families that share the same type of DNA binding domain. The POU domain genes are one such paradigm. We compared the genomic DNA encoding the POU domain of the Oct-2 genes in human and mouse. In both species this domain is split into a cluster of four exons by short, highly diverged introns. We postulate that the main role of these introns is to prevent ectopic homologous recombination with other members of the POU gene family, with its potentially deleterious effects in somatic and germline cells. Such rapidly diverging introns may generally promote evolution by facilitating the maintenance of duplicated genes. The use of different codons for the same protein domain among members of a gene family may be a slower process that serves a similar purpose. Introns that split conserved domains such as the POU domain do not conform to the exon shuffling hypothesis originally put forward by W. Gilbert (1978). However, we note that the introns flanking the POU domain are in the same phase, i.e. interrupt codons in the same reading frame. Thus we propose that the entire POU domain, which is encoded by a tight cluster of exons, has been shuffled together during evolution as a functional unit, or 'shufflon'.

The linear extrachromosomal DNA of Physarum polycephalum replicates and is maintained under non-selective conditions in two different lower eukaryotes.

The slime mould Physarum polycephalum contains 100 to 200 molecules of extrachromosomal linear DNA (PeDNA). Two sets of the 19S and 26S ribosomal genes are located on each molecule of PeDNA. In the nonmitotic phase of the cell cycle PeDNA is localised in the nucleolus. The molecules are maintained throughout vegetative growth. In order to study the signals responsible for its maintenance, PeDNA was purified and introduced into the two distantly related yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Surprisingly, intact PeDNA transforms both yeasts with high frequency and PeDNA sequences are maintained in the absence of selective pressure.

Regions in the ribosomal minichromosome of Physarum polycephalum are protected from restriction nucleases; protection is insensitive to high salt in the G phase and sensitive in the M phase of the cell cycle.

In the central spacer region of the extrachromosomal ribosomal DNA of the slime mold Physarum polycephalum, four small regions of related sequence are completely inaccessible to restriction endonucleases (HinfI and MboI). In addition, some sequences neighboring the inaccessible ones, are partially inaccessible to restriction enzymes and micrococcal nuclease. Taking advantage of the natural synchrony of Physarum plasmodia, we found that this protection is present throughout the cell cycle. Treatment with high salt (2.5 M-NaCl) of nuclei from the G2 phase of the cell cycle left the protection essentially unchanged. When nuclei from the M phase were treated with salt, the protection was abolished. The inaccessible sites are located close to the origins of replication of the rDNA.

Histone H5 can correctly align randomly arranged nucleosomes in a defined in vitro system.

In eukaryotic cells, DNA is packed into regularly spaced chromatin subunits called nucleosomes. The average distance between nucleosomes (the repeat length) varies in a tissue- and species-specific manner, with values ranging from about 160 to 240 DNA base pairs (bp). Thus, it has been recognized that the repeat length could be one of the factors underlying selective gene expression. In cells growing in culture, the characteristic repeat length for that type of cell seems to arise from an immature chromatin structure in which nucleosomes are initially irregularly spaced or are arranged in small closely packed clusters. At present no in vitro system has been described which is capable of reconstituting the mature physiological nucleosome spacing from purified chromatin components. Moreover, neither the factors necessary for spacing nor the reaction mechanism are known. We describe here an in vitro system that can restore the native subunit spacing in rearranged chromatin samples which have irregularly spaced nucleosomes similar to the situation apparent in newly replicated chromatin.

Histone H5 can increase the internucleosome spacing in dinucleosomes to nativelike values.

Chicken erythrocyte chromatin was assembled with inner histones at about 60% of the ratio found in vivo and subsequently incubated with histone H5 (or H1 + H5) in a solution containing 0.1 M NaCl and poly(glutamic acid). Micrococcal nuclease digestion produced dinucleosomes of 360-390 base pair (bp) DNA content, similar to those from native chromatin and contrasting with the 270-280 bp species found in material incubated without H5. On sucrose gradients a dinucleosome sedimenting at 16 S containing 360 bp DNA was isolated. Removal of H1 + H5 after reconstitution did not change these results; H5 thus can induce rearrangements of nucleosome cores with respect to their neighbors. The results are interpreted as an H5-induced "sliding apart" of histone octamers, complementary to the "sliding together" found in native chromatin after removal of H1 + H5.

Cromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence.

Chicken erythrocyte inner histones (H2A, H2B, H3 and H4) were associated with the two complementary homopolymeric polydeoxyribonucleotides and the two alternating copolymeric polydeoxyribonucleotides. No evidence for formation of chromatin-like structures was obtained for the complexes with poly(dG) . poly(dC) or poly(dA) . poly(dT). Both poly (dGdC) . poly(dGdC) and poly(dAdT) . poly(dAdT) could be folded by histones to yield material digested by DNAase I to multiples of about 10 and by staphylococcal nuclease to 146 bp core particles. Due to the lack of sequence heterogeniety in the complex of histones with poly(dAdT) . poly(dAdT), core particles with remarkable fine structural detail are obtained. The internal organization of DNA in the AT-containing and GC-containing core particles appears not to be identical.

Structure and synthesis of a lipid-containing bacteriophage. Dissociation of bacteriophage PM2 into its morphological subunits.

The lipid-containing bacteriophage PM2 was dissociated stepwise in 1 M NcCl (pH 7.2)with increasing urea concentrations. In 2 M urea the following substructures could be identified: (a) a nucleocapsid containing all of the viral lipid, the DNA, proteins III and IV, plus a fraction of protein II, and (b) a second substructure respresenting particles which contained all viral elements except protein I, the spike protein. In 4 M urea the viral nucleocapsid containing all of proteins III and IV, the DNA, plus a fraction of protein II, was isolated. Upon increasing the urea concentration further, this nucleocapsid is stable up to 8.5 M urea; in 9 M urea protein III was partly dissociated from the nucleocapsid. The nucleocapsid in 4--8.5 M urea is stabilized by the addition of 0.1--3 M NaCl but dissociates if the NaCl concentration is less than 0.1 M. The nucleocapsid was also dissociated in 4--8.5 M urea at pH 4.5. The nucleocapsid structures and some intermediate morphological subunits have been analysed by physical methods, enabling us to draw some conclusions about the structure and hydration of the virus.