Low-dose prednisone chronotherapy for rheumatoid arthritis: a randomised clinical trial (CAPRA-2).

OBJECTIVE: To assess the efficacy and safety of low-dose prednisone chronotherapy using a new modified-release (MR) formulation for the treatment of rheumatoid arthritis (RA). METHODS: In this 12-week, double-blind, placebo-controlled study, patients with active RA (n=350) were randomised 2:1 to receive MR prednisone 5 mg or placebo once daily in the evening in addition to their existing RA disease-modifying antirheumatic drug (DMARD) treatment. The primary end point was the percentage of patients achieving a 20% improvement in RA signs and symptoms according to American College of Rheumatology criteria (ie, an ACR20 response) at week 12. Changes in morning pain, duration of morning stiffness, 28-joint Disease Activity Score and health-related quality of life were also assessed. RESULTS: MR prednisone plus DMARD treatment produced higher response rates for ACR20 (48% vs 29%, p<0.001) and ACR50 (22% vs 10%, p<0.006) and a greater median relative reduction from baseline in morning stiffness (55% vs 35%, p<0.002) at week 12 than placebo plus DMARD treatment. Significantly greater reductions in severity of RA (Disease Activity Score 28) (p<0.001) and fatigue (Functional Assessment of Chronic Illness Therapy-Fatigue score) (p=0.003) as well as a greater improvement in physical function (36-item Short-Form Health Survey score) (p<0.001) were seen at week 12 for MR prednisone versus placebo. The incidence of adverse events was similar for MR prednisone (43%) and placebo (49%). CONCLUSION: Low-dose MR prednisone added to existing DMARD treatment produced rapid and relevant improvements in RA signs and symptoms. CLINICALTRIALS.GOV, NUMBER: NCT00650078.

An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose.

A flexible enzyme module system is presented that allows preparative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-beta-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, +/-RmlB) yielded the precursor dTDP-alpha-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-alpha-D-glucose (3) on a 0.2-0.6 g scale with overall yields of 62 % and 72 %, respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmlC and RmlD) resulted in the synthesis of dTDP-beta-L-rhamnose (4; 24.1 micromol, overall yield: 35.9 %). Substitution of RmlC by DnmU from the dTDP-beta-L-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerase with 3 as substrate to yield 4 (32.2 mumol, overall yield: 44.7 %). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-alpha-D-quinovose (6) and dTDP-alpha-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.

Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate engineering.

The gene sus1 from Solanum tuberosum L. encoding for sucrose synthase 1 was cloned into the plasmid pDR195 under the control of the PMA1 promotor. After transformation of Saccharomyces cerevisiae strain 22574d sus1 was constitutively expressed giving a specific activity of 0.3Umg(-1) protein in the crude extract. A one-step purification by Q-Sepharose resulted in an 14-fold purified enzyme preparation in 74% yield. SuSy1 was subsequently purified by immobilized metal ion affinity chromatography and characterized for its utilization in synthesizing different nucleotide sugars and sucrose analogues. The kinetic constants for the cleavage and synthesis reaction were determined: K(m) (UDP) 4microM; K(iS) (UDP) 0.11mM; K(m) (sucrose) 91.6mM; K(m) (UDP-Glc) 0.5mM; K(iS) (UDP-Glc) 2.3mM; K(m) (D-fructose) 2.1mM; K(iS) (D-fructose) 35.9mM. Different nucleoside diphosphates as well as different donor substrate were accepted as follows: UDP>dTDP>ADP>CDP>GDP in the cleavage reaction and UDP-Glc>dTDP-Glc>ADP-Glc>CDP-Glc in the synthesis reaction. SuSy1 shows also a broad acceptance of D- and L-ketoses and D- and L-aldoses. The acceptance of aldoses was deduced from the binding of the inhibitor 5-deoxy-D-fructose (K(i) 0.3mM), an analogue of the natural substrate D-fructopyranoside. The broad substrate spectrum renders SuSy1 from potato a versatile biocatalyst for carbohydrate engineering.

Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato.

To gain insight into the regulatory mechanisms of sugar signaling in plants, the effect of derivatives of the transport sugar sucrose (Suc), the Suc isomers palatinose and turanose, and the Suc analog fluoro-Suc were tested. Photo-autotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used to study their effect on the regulation of marker genes of source and sink metabolism, photosynthesis, and the activation of mitogen-activated protein kinases (MAPKs). Suc and glucose (Glc) resulted in reverse regulation of source and sink metabolism. Whereas the mRNA level of extracellular invertase (Lin6) was induced, the transcript level of small subunit of ribulose bisphosphate carboxylase (RbcS) was repressed. In contrast, turanose, palatinose, and fluoro-Suc only rapidly induced Lin6 mRNA level, whereas the transcript level of RbcS was not affected. The differential effect of the metabolizable and non-metabolizable sugars on RbcS mRNA regulation was reflected by the fact that only Suc and Glc inhibited photosynthesis and chlorophyll fluorescence. The activation of different signal transduction pathways by sugars was further supported by the analysis of the activation of MAPKs. MAPK activity was found to be strongly activated by turanose, palatinose, and fluoro-Suc, but not by Suc and Glc. To analyze the role of sugars in relation to pathogen perception, an elicitor preparation of Fusarium oxysporum lycopersici was used. The strong activation of MAPKs and the fast and transient induction of Lin6 expresssion by the fungal elicitor resembles the effect of turanose, palatinose, and fluoro-Suc and indicates that non-metabolizable sugars are sensed as stress-related stimuli.