Potential role of arthroscopy in the management of inflammatory arthritis.

Despite its advantages in diagnosis, treatment and research, the role of arthroscopy in the management of rheumatic diseases has diminished due to the development of other less invasive means of joint assessment including advances in imaging techniques, e.g. ultrasound and magnetic resonance imaging. However, arthroscopy still provides invaluable information. By direct and precise internal inspection of a joint, arthroscopy allows the collection of synovial membrane samples (biopsies) of excellent quality, notably from the most representative pathological areas. Arthroscopy may also play a therapeutic role in the management of inflammatory arthritis (IA) by providing pain relief (lavage). Here we describe the procedure of knee arthroscopy under local anaesthesia, as well as an in situ visual assessment of synovial inflammation and its correlation with degree of histological and immunological abnormalities. With the emphasis being placed on early diagnosis and treatment initiation in patients with IA and as earlier initiation of targeted biologic therapies becomes more commonplace, the ability to predict which patients will respond to the different therapies available would be invaluable. Assessment of arthroscopic derived synovial biopsies has potential to play an important role in management of early IA in the future.

Mode of action of abatacept in rheumatoid arthritis patients having failed tumour necrosis factor blockade: a histological, gene expression and dynamic magnetic resonance imaging pilot study.

OBJECTIVES: Abatacept is the only agent currently approved to treat rheumatoid arthritis (RA) that targets the co-stimulatory signal required for full T-cell activation. No studies have been conducted on its effect on the synovium, the primary site of pathology. The aim of this study was to determine the synovial effect of abatacept in patients with RA and an inadequate response to tumour necrosis factor alpha (TNFalpha) blocking therapy. METHODS: This first mechanistic study incorporated both dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) and arthroscopy-acquired synovial biopsies before and 16 weeks after therapy, providing tissue for immunohistochemistry and quantitative real-time PCR analyses. RESULTS: Sixteen patients (13 women) were studied; all had previously failed TNFalpha-blocking therapy. Fifteen patients completed the study. Synovial biopsies showed a small reduction in cellular content, which was significant only for B cells. The quantitative PCR showed a reduction in expression for most inflammatory genes (Wald statistic of p<0.01 indicating a significant treatment effect), with particular reduction in IFNgamma of -52% (95% CI -73 to -15, p<0.05); this correlated well with MRI improvements. In addition, favourable changes in the osteoprotegerin and receptor activator of nuclear factor kappa B levels were noted. DCE-MRI showed a reduction of 15-40% in MRI parameters. CONCLUSION: These results indicate that abatacept reduces the inflammatory status of the synovium without disrupting cellular homeostasis. The reductions in gene expression influence bone positively and suggest a basis for the recently demonstrated radiological improvements that have been seen with abatacept treatment in patients with RA.

Determinants of binding-site specificity among yeast C6 zinc cluster proteins.

Related DNA binding proteins often recognize similar DNA sites but can distinguish among them with the use of different protein-DNA contacts. Here, it is shown that members of the C6 zinc cluster family of yeast transcriptional activators distinguish related DNA sites by a different mechanism. The DNA binding site for each of these proteins contains identical nucleotide triplets (CGG ... CCG) but differs in the spacings between the triplets. It is shown that zinc clusters of these proteins work interchangeably to recognize the conserved triplets and that the region 19 amino acids to the carboxyl-terminal side of the zinc cluster, comprising the linker and the beginning of a dimerization element as inferred from the GAL4 crystal structure, directs the protein to its preferred site.

The value of synovial cytokine expression in predicting the clinical response to TNF antagonist therapy (infliximab).

OBJECTIVES: Clinical response to TNF-alpha blockade in the treatment of RA is heterogeneous. The study aims were to determine whether pre-treatment synovial cytokine expression predicted infliximab response and whether synovial changes after therapy correlated with response. METHODS: Fifty-one patients had arthroscopic biopsies of the knee joint prior to infliximab (3 mg/kg) treatment. Synovial tissue cell numbers (CD68 and CD3 positive) and cytokine expression (TNF-alpha, lymphotoxin-alpha, IL-1alpha, -beta and receptor antagonist, and IL-6) pre-treatment was assessed using semi-quantitative immunohistochemistry. Changes in these parameters were assessed 16 weeks after infliximab in 32 patients who underwent repeat arthroscopic biopsy. RESULTS: Of the total patients, 47% (n = 24) achieved an ACR20 response; 53% (n = 27) did not. Baseline synovial TNF-alpha, IL-1alpha and -beta expression did not differ between the two groups. No differences in baseline TNF-alpha levels were observed with ACR levels of response (ACR20 and ACR50/70 groups). Post-treatment biopsies (17 ACR responders, 15 ACR non-responders) revealed significant reductions in sub-lining layer TNF-alpha expression in both response and non-response groups with significant reduction in vascularity and membrane proliferation scores. The worst ACR non-responders (<20% CRP suppression) demonstrated no reduction in any of the parameters. CONCLUSION: Pre-treatment synovial TNF-alpha or IL-1 expression does not predict TNF blockade response. Both ACR response and non-response was associated with reduction in synovial TNF-alpha-level expression. Suppression in TNF-alpha levels was not observed in the worst non-responders. The improvements (including in vascularity), independent of ACR clinical response, are compatible with the reduced structural damage documented in all groups of patients independent of response.

Activation of transcription by metabolic intermediates of the pyrimidine biosynthetic pathway.

Saccharomyces cerevisiae responds to pyrimidine starvation by increasing the expression of four URA genes, encoding the enzymes of de novo pyrimidine biosynthesis, three- to eightfold. The increase in gene expression is dependent on a transcriptional activator protein, Ppr1p. Here, we investigate the mechanism by which the transcriptional activity of Ppr1p responds to the level of pyrimidine biosynthetic intermediates. We find that purified Ppr1p is unable to promote activation of transcription in an in vitro system. Transcriptional activation by Ppr1p can be observed, however, if either dihydroorotic acid (DHO) or orotic acid (OA) is included in the transcription reactions. The transcriptional activation function and the DHO/OA-responsive element of Ppr1p localize to the carboxyl-terminal 134 amino acids of the protein. Thus, Ppr1p directly senses the level of early pyrimidine biosynthetic intermediates within the cell and activates the expression of genes encoding proteins required later in the pathway. These results are discussed in terms of (i) regulation of the pyrimidine biosynthetic pathway and (ii) a novel mechanism of regulating gene expression.

Preliminary crystallographic analysis of the breakage-reunion domain of the Escherichia coli DNA gyrase A protein.

The 64 x 10(3) Mr N-terminal breakage-reunion domain of the Escherichia coli DNA gyrase A protein was purified from an over-expressing strain. When complexed with the gyrase B protein, this truncated A protein has all of the enzymic properties of the full-length counterpart, although with reduced efficiency in some cases. The 64 x 10(3) Mr protein has been crystallized in several forms, a number of which were too small for crystallographic analysis. However, two forms grew to sufficient size for preliminary X-ray analysis. Both forms were tetragonal with a primitive lattice. One form (type I) had cell dimensions of a = b = 170 A, c = 145 A a space group of either P41212 (P43212) or P42212, and diffracted to 6 A resolution. The type II crystals had cell dimensions of a = b = 177 A, c = 175 A, a space group of P41212 (P43212) or P42212, and diffracted to at least 4.5 A resolution. Both crystal forms apparently contained four subunits (possibly a tetramer) in the asymmetric unit. We are attempting to increase the size and quality of these crystals.

Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.

Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.

Overproduction and single-step purification of GAL4 fusion proteins from Escherichia coli.

A DNA fragment encoding the yeast GAL4 transcriptional activator DNA-binding domain (amino acids 1-93) was cloned into an Escherichia coli expression vector such that the overproduced protein is tagged with six histidine residues and a factor Xa protease cleavage site. The vector also contains unique restriction sites at the 3' end of the gene to allow the construction of fusion proteins. These fusion proteins can easily be purified to homogeneity and their activity tested in vitro.

Phosphorylation of Zn(II)2Cys6 proteins: a cause or effect of transcriptional activation?

Many Zn(II)2Cys6 transcriptional regulators exhibit changes in phosphorylation that are coincident with their roles in transcriptional activation. It is, however, unclear whether these changes occur as a cause of, or as a result of, transcriptional activation. In this paper, we explore the relationship between these two events and collate data available on the phosphorylation state of those transcriptional regulators where the relationship has not been clearly identified.

Molecular basis of nutrient-controlled gene expression in Saccharomyces cerevisiae.

The ability of a unicellular organism to alter patterns of gene expression in response to nutrient availability is essential to its survival in a changing environment. How is the cell able to identify individual metabolites amongst a myriad of other similar molecules, and convert the information of its presence into a concerted change in the transcription of the genes required for the response to that metabolite? There is increasing evidence that the activity of transcription factors can be influenced directly by interaction with metabolites. A variety of mechanisms have been identified by which this type of gene regulation by small molecules can occur.

Tryptic fragments of the Escherichia coli DNA gyrase A protein.

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.

The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein.

We have constructed a clone which over-produces a 33 kDa protein representing the C-terminal portion of the Escherichia coli DNA gyrase A subunit. This protein has no enzymic activity of its own, but will form a complex with a 64 kDa protein (representing the N-terminal part of the A subunit) and the gyrase B subunit, that will efficiently catalyse DNA supercoiling. We show that the 33 kDa protein can bind to DNA on its own in a manner which induces positive supercoiling of the DNA. We propose that the 33 kDa protein represents a domain of the gyrase A subunit which is involved in the wrapping of DNA around DNA gyrase.

Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein.

In a previous report (Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments. The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity. We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein. We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product. The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment. We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced. The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523). The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein. Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction. Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523). A model is proposed for the domain organization of the gyrase A protein.

DNA gyrase: structure and function.

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.

The yeast galactose genetic switch is mediated by the formation of a Gal4p-Gal80p-Gal3p complex.

Saccharomyces cerevisiae responds to galactose as the sole source of carbon by activating the GAL genes encoding the enzymes of the Leloir pathway. Here, we show in vitro that the switch from repressed to activated gene expression involves the interplay of three proteins [an activator (Gal4p), a repressor (Gal80p) and an inducer (Gal3p)] and two small molecules (galactose and ATP). We also show that the galactose- and ATP-dependent interaction between Gal3p and Gal80p occurs without disruption of the Gal80p-Gal4p interaction. Thus, Gal3p-mediated activation of transcription occurs via the formation of a tripartite protein complex.

Signaling activation and repression of RNA polymerase II transcription in yeast.

Activators of RNA polymerase II transcription possess distinct and separable DNA-binding and transcriptional activation domains. They are thought to function by binding to specific sites on DNA and interacting with proteins (transcription factors) binding near to the transcriptional start site of a gene. The ability of these proteins to activate transcription is a highly regulated process, with activation only occurring under specific conditions to ensure proper timing and levels of target gene expression. Such regulation modulates the ability of transcription factors either to bind DNA or to interact with the transcriptional machinery. Here we discuss recent advances in our understanding of these mechanisms of transcriptional regulation in yeast.

Quantitation of putative activator-target affinities predicts transcriptional activating potentials.

We quantitate the 'activating potentials' of deletion and point mutation variants of a 42 amino acid yeast transcriptional activating region excised from the yeast activator GAL4 and, using surface plasmon resonance, we measure the relative affinities of these molecules for a variety of proteins, including plausible target proteins as well as GAL80, a specific inhibitor of GAL4. We find a remarkable correlation between the relative activating potentials of the derivatives and their relative affinities for yeast TBP and for yeast TFIIB; other tested proteins interacted significantly more weakly, if at all. These results provide an especially strong argument that TBP and TFIIB are activating region targets. We also show, using one set of yeast activating region mutants, that activator-target interactions are strongly correlated with the length of the activating region, that the effect of point mutants is highly dependent on the length of the activating region mutated and that, unlike interactions with TBP and TFIIB, interaction with the specific inhibitor GAL80 is destroyed by deletion of certain critical residues in the C-terminal half of the 42 amino acid activating region.

A transcriptional activating region with two contrasting modes of protein interaction.

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

Crystal structure of a PUT3-DNA complex reveals a novel mechanism for DNA recognition by a protein containing a Zn2Cys6 binuclear cluster.

PUT3 is a member of a family of at least 79 fungal transcription factors that contain a six-cysteine, two-zinc domain called a 'Zn2Cys6 binuclear cluster'. We have determined the crystal structure of the DNA binding region from the PUT3 protein bound to its cognate DNA target. The structure reveals that the PUT3 homodimer is bound asymmetrically to the DNA site. This asymmetry orients a beta-strand from one protein subunit into the minor groove of the DNA resulting in a partial amino acid-base pair intercalation and extensive direct and water-mediated protein interactions with the minor groove of the DNA. These interactions facilitate a sequence dependent kink at the centre of the DNA site and specify the intervening base pairs separating two DNA half-sites that are contacted in the DNA major groove. A comparison with the GAL4-DNA and PPR1-DNA complexes shows how a family of related DNA binding proteins can use a diverse set of mechanisms to discriminate between the base pairs separating conserved DNA half-sites.

The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase.

The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p+SA reveals that the K(m) for galactose is similar to that of Gal1p, but the K(m) for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.