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R J Reece

Publications and source records attributed to R J Reece.

At least 19 recordsLinked to original sources
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Treatment of poor-prognosis early rheumatoid arthritis. A randomized study of treatment with methotrexate, cyclosporin A, and intraarticular corticosteroids compared with sulfasalazine alone.

OBJECTIVE: To determine whether a regimen of methotrexate, cyclosporin A, and corticosteroids introduced at onset in poor-prognosis rheumatoid arthritis (RA) can produce a significant improvement in outcome compared with standard monotherapy with sulfasalazine (SSZ). METHODS: Eighty-two consecutive patients presenting with new, untreated RA of less than 12 months' duration who fulfilled criteria for poor long-term outcome were randomized to receive either combination therapy (n = 40) or SSZ alone (n = 42). The primary outcome measures were remission and American College of Rheumatology (ACR) criteria for 20% improvement at 48 weeks. RESULTS: After 48 weeks, the numbers of patients who met the ACR criteria for 20% improvement were not significantly different between the two groups (combination 58% versus SSZ 45%), and similar numbers of patients had persisting clinical remission (approximately 10% both groups). During the first 3 months, there were significantly greater reductions in parameters of disease activity in the combination group. By 24 weeks, the swollen and tender joint counts, C-reactive protein levels, and erythrocyte sedimentation rates had fallen significantly in both groups, with a greater improvement in the swollen and tender joint count in the combination group. At 48 weeks, the radiographic damage score had increased by a median of 1 (range 0-42.5) in the combination group and 1.25 (range 0-72.5) in the SSZ group (P = 0.28; although there were significant differences in the scores for the right hand). There were significantly fewer withdrawals due to lack of efficacy in the combination group than in the SSZ group (1 of 40 versus 10 of 42; P = 0.007). In the combination group, dose reduction was needed in 22.5% because of hypertension and in 22.5% because of elevated creatinine levels. Over 48 weeks, serum creatinine increased in both groups, but particularly in the combination arm. CONCLUSION: In poor-prognosis RA patients, "aggressive" combination therapy led to more rapid disease suppression but did not result in significantly better ACR response or remission rates. This suggests that in poor-prognosis disease, an approach based on identifying patients with poor treatment responses before extra therapy is added ("step-up" approach) may be more appropriate than the use of combination therapy in all patients from the outset.

Adrenal Cortex Hormones↗
PubMed2000

Modulation of inflammation and metalloproteinase expression in synovial tissue by leflunomide and methotrexate in patients with active rheumatoid arthritis. Findings in a prospective, randomized, double-blind, parallel-design clinical trial in thirty-nine patients at two centers.

OBJECTIVE: Leflunomide and methotrexate have proven to be efficacious in reducing joint inflammation and slowing destruction in clinical trials of patients with rheumatoid arthritis (RA). This study was conducted to provide more insight into the mechanism of action of these agents in synovial tissue. METHODS: In a 2-center, prospective, randomized, double-blind clinical trial, we compared leflunomide (20 mg/day, after a 3-day 100 mg/day loading dose) and methotrexate (increased stepwise to 15 mg/week) treatment in patients with active RA. Paired synovial tissue biopsy samples were obtained by knee arthroscopy at baseline and after 4 months of treatment. Frozen synovial tissue sections were stained for macrophages (CD68), T cells (CD3), adhesion molecules (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1]), cytokines (tumor necrosis factor alpha, interleukin-1beta [IL-1beta]), matrix metalloproteinase 1 (MMP-1), and tissue inhibitor of metalloproteinases 1 (TIMP-1). RESULTS: Paired synovial tissue sections were available in 35 patients (16 taking leflunomide, 19 taking methotrexate). Both drugs displayed equal clinical efficacy, with 8 leflunomide-treated patients (50%) and 10 methotrexate-treated patients (53%) fulfilling the American College of Rheumatology 20% response criteria. Both compounds showed similar effects on synovial tissue: reduced numbers of macrophages and reduced ICAM-1 and VCAM-1 expression were noted after 4 months of treatment. Both leflunomide- and methotrexate-treated patients exhibited a decreased MMP-1:TIMP-1 ratio in the synovial tissue. In the subset of patients fulfilling the 20% response criteria of the American College of Rheumatology, a more pronounced reduction in the expression of ICAM-1, VCAM-1, IL-1beta, and MMP-1 was found compared with the nonresponders. CONCLUSION: Leflunomide and methotrexate are clinically efficacious drugs that interfere with mechanisms involved in joint inflammation and destruction of joint integrity.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

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.

Culture Media↗
PubMed1999

Distinct vascular patterns of early synovitis in psoriatic, reactive, and rheumatoid arthritis.

OBJECTIVE: To examine the macroscopic vascular pattern of early synovitis in psoriatic arthritis (PsA), reactive arthritis (ReA), and rheumatoid arthritis (RA) and to assess the reliability of the grading features for synovitis. METHODS: Forty-four patients (14 PsA, 12 ReA, and 18 RA) with knee synovitis who were undergoing arthroscopy were assessed. Video recordings of the examination were scored independently by 3 arthroscopists who were blinded to the patient's identity and clinical details. Features of vascularity, villous formation, pannus, granularity, and capillary hyperemia were recorded and kappa values (-1 or =0.8) for features of vascularity, villous hypertrophy, and pannus. Seventy-three percent of the PsA and ReA patients had predominantly tortuous, bushy vessels; 89% of the RA patients had mainly straight, branching vessels. CONCLUSION: The distinct vascular patterns in PsA and ReA compared with those in RA may reflect different specific vascular factors in the pathogenesis of these arthritides. Vascularity and villous hypertrophy are the most reliable features of synovitis grading.

Adult↗
PubMed1999

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.

Animals↗
PubMed1999

Intra-articular primatised anti-CD4: efficacy in resistant rheumatoid knees. A study of combined arthroscopy, magnetic resonance imaging, and histology.

OBJECTIVES: CD4+ T cells sustain the chronic synovial inflammatory response in rheumatoid arthritis (RA). SB-210396/CE 9.1 is an anti-CD4 monoclonal antibody that has documented efficacy in RA when given intravenously. This study aimed to establish the safety and efficacy of the intra-articular administration of SB-210396/CE 9.1 compared with placebo, examining its mode of action using a combined imaging approach of arthroscopy, magnetic resonance imaging (MRI), and histology. METHODS: Thirteen RA patients with active, resistant knee synovitis, were randomised to intra-articular injection of placebo (n=3), 0.4 mg (n=3) or 40 mg (n=7) of anti-CD4 after sequential dynamic gadolinium enhanced MRI, followed by same day arthroscopy and synovial membrane biopsy. Imaging and arthroscopic synovial membrane sampling were repeated at six weeks. This study used a unique region of interest (ROI) analysis mapping the MRI area analysed to the specific biopsy site identified arthroscopically, thus providing data for all three modalities at the same synovial membrane site. RESULTS: 12 patients completed the study (one placebo treated patient refused further MRI). Arthroscopic improvement was observed in 0 of 2 placebo patients but in 10 of 10 patients receiving active drug (>20% in 6 of 10). Improvement in MRI was consistently observed in all patients of the 40 mg group but not in the other two groups. A reduction in SM CD4+ score was noted in the 40 mg group and in the 0.4 mg group. Strong correlations both before and after treatment, were identified between the three imaging modalities. Intra-articular delivery of SB-210396/CE 9.1 was well tolerated. CONCLUSIONS: SB-210396/CE 9.1 is safe when administered by intra-articular injection. A trend toward efficacy was found by coordinated MRI, arthroscopic, and histological imaging, not seen in the placebo group. The value of ROI analysis was demonstrated.

Adult↗
PubMed1998

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.

Amino Acid Substitution↗
PubMed1998

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.

Adenosine Triphosphate↗
PubMed1997

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.

DNA, Fungal↗
PubMed1997

Structure and mobility of the PUT3 dimer.

The solution structure and backbone dynamics of the transcriptional activator PUT3 (31-100) has been characterized using NMR spectroscopy. PUT3 (31-100) contains three distinct domains: a cysteine zinc cluster, linker, and dimerization domain. The cysteine zinc cluster of PUT3 closely resembles the solution structure of GAL4, while the dimerization domain forms a long coiled-coil similar to that observed in the crystal structures of GAL4 and PPR1. However, the residues at the N-terminal end of the coiled-coil behave very differently in each of these proteins. A comparison of the structural elements within this region provides a model for the DNA binding specificity of these proteins. Furthermore, we have characterized the dynamics of PUT3 to find that the zinc cluster and dimerization domains have very diverse dynamics in solution. The dimerization domain behaves as a large protein, while the peripheral cysteine zinc clusters have dynamic properties similar to small proteins.

Amino Acid Sequence↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

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.

Base Sequence↗
PubMed1994

Domain structure of Escherichia coli DNA gyrase as revealed by differential scanning calorimetry.

The domain structure of DNA gyrase from Escherichia coli has been examined using differential scanning microcalorimetry. The intact enzyme (an A2B2 tetramer) shows at least four transitions with apparent Tm's at 44.8, 53.3, 58.6, and 60.7 degrees C. Comparison with the thermal stabilities of the two separate subunits and genetically-engineered protein fragments has been used to assign these transitions to individual domains within the intact gyrase proteins. The thermal unfolding of DNA gyrase and all individual fragments are irreversible under the conditions of the calorimetric experiment. Further evidence for the assignment of transitions to particular domains has been obtained by studying the effects of tight-binding ligands such as novobiocin on the thermal stabilities of the various protein fragments.

Adenosine Triphosphate↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization.

In many vertebrates, UV-sensitive photoreceptors have been identified by microspectrophotometry and UV-visual sensitivity has been identified by behavioral studies, but as yet no vertebrate UV-sensitive pigment gene has been isolated. We have sequenced a cDNA clone that hybridizes to short single cone cells in the zebrafish (Brachydanio rerio). These cells, which make up 25% of the cone population in zebrafish retinae, are UV-sensitive (lambda max approximately 360 nm). The visual pigment encoded by this gene is unusual in that its amino acid sequence is more homologous to the rod pigment rhodopsin (up to 89%) than to other cone pigments (35-83%). Like all other vertebrate visual pigments, it contains a lysine residue at position 296, the presumptive retinal binding site, and a glutamate residue at position 113. However, it is unique in possessing a lysine residue at position 126, which may account for the UV-sensitivity of the pigment.

Amino Acid Sequence↗