Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor.

The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (Møllgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.

Oral S-adenosylmethionine in primary fibromyalgia. Double-blind clinical evaluation.

S-adenosylmethionine is a relatively new anti-inflammatory drug with analgesic and anti-depressant effects. Efficacy of 800 mg orally administered s-adenosylmethionine daily versus placebo for six weeks was investigated in 44 patients with primary fibromyalgia in double-blind settings. Tender point score, isokinetic muscle strength, disease activity, subjective symptoms (visual analog scale), mood parameters and side effects were evaluated. Improvements were seen for clinical disease activity (P = 0.04), pain experienced during the last week (P = 0.002), fatigue (P = 0.02), morning stiffness (P = 0.03) and mood evaluated by Face Scale (P = 0.006) in the actively treated group compared to placebo. The tender point score, isokinetic muscle strength, mood evaluated by Beck Depression Inventory and side effects did not differ in the two treatment groups. S-adenosylmethionine has some beneficial effects on primary fibromyalgia and could be an important option in the treatment hereof.

Timegadine: more than a non-steroidal for the treatment of rheumatoid arthritis. A controlled, double-blind study.

Timegadine is a tri-substituted guanidine derivative which inhibits both arachidonate cyclo-oxygenase and lipoxygenase activity. In a 24-week randomized double-blind controlled trial, timegadine 500 mg/day was compared with naproxen 750 mg/day in two groups of 20 patients with active rheumatoid arthritis. In the timegadine group, significant improvements were seen in both biochemical and clinical markers of disease activity, i.e. ESR, serum IgG and IgM, leukocyte and platelet counts, duration of morning stiffness, Ritchie index, number of swollen joints, pain, and general condition. In the naproxen group, only the Ritchie index improved. Differences between treatments, when present, were always in favour of timegadine. Serum alkaline phosphatase rose during the first 8 weeks of treatment in the timegadine group. A transient rise was also seen in the naproxen group. The side effects reported were mainly gastrointestinal and allergic, the latter being more frequently found in the timegadine group. Timegadine is superior to naproxen in controlling disease activity in rheumatoid arthritis, and appears to possess disease-modifying properties.

A long-term randomised trial on tenoxicam and piroxicam in osteoarthritis of the hip or knee: a 24-month interim report focusing on the 12-24 month interval.

The long-acting antiphlogistics tenoxicam (Ro 12-0068, Tilcotil) and piroxicam in single daily oral doses of 20 mg are compared in a double-blind, group-comparative, randomised trial planned to last for five years. Results of 12 months' treatment of 108 patients with osteoarthritis of the hip or knee have been reported. This interim analysis focuses mainly on the 12 to 24 month interval. The clinical improvements obtained within the first 12 months persisted during the second year in the 55 patients remaining on treatment. After 24 months, 53 patients had been withdrawn prematurely, three-quarters because of inefficacy or intolerance. Only six patients were withdrawn between 12 and 24 months, three for lack of efficacy, two for side-effects and one for reasons unrelated to therapy. There was no difference between the treatment groups with regard to incidence, time or reason for withdrawal, and only small, insignificant differences in efficacy and tolerability. This trial shows that long-term treatment of osteoarthritis with tenoxicam and with piroxicam is beneficial. Once efficacy and tolerability have been established, maintenance of therapy is feasible.

Fibrin dissolution in synovial fluid.

Fibrinogen, fibrinogen-fibrin breakdown products (b.d.p.), fibrin stabilizing factor (FSF), plasminogen, alpha-2 macroglobulin and alpha-1 antitrypsin concentrations were estimated immunologically in normal and in pathological synovial fluids from patients with RA, gout, monoarthritis, rheumatic fever, osteoarthritis and traumatic arthritis. The fibrinolytic activity was determined by the fibrinplate method. Normal synovial fluid contained no fibrinogen, b.d.p., FSF or alpha-2 macroglobulin, but traces of plasminogen and alpha-1 antitrypsin. By increasing inflammatory reactions increasing amounts of the various factors were demonstrable. No distinct, specific, patterns were found. Spontaneous fibrinolytic activity was demonstrable in the most exsudative cases, and in these high concentrations of fibrin breakdown products were present. These were also demonstrable in most pathological synovial fluids proving a recent proteolytic activity. It is suggested, that the fibrin breakdown products in pathological synovial fluids are partly resistent to proteolytic activity.

Pharmacokinetic profile of indoprofen in elderly subjects.

Multiple dosing four times daily for 7 days of indoprofen 200 mg, a non-steroidal anti-inflammatory drug with a short half-life (t1/2), revealed drug accumulation in eight elderly subjects. This indicates a substantially longer t1/2 (11 h) than that reported in young persons (3 h). A reduction in dose compared to younger subjects is recommended in elderly patients with osteoarthritis.

Bone mineral loss, bone histomorphometry and vitamin D metabolism in patients with rheumatoid arthritis on long-term glucocorticoid treatment.

Long-term glucocorticoid treatment might interfere with the vitamin D metabolism. The serum concentrations of 25-OHD were significantly reduced whereas the circulating levels of 1,25-(OH)2D were normal in 50 patients with rheumatoid arthritis on long-term treatment with small doses of prednisone. The bone mineral content of the forearm was significantly reduced, but the degree of bone loss did not correlate with duration of treatment or dose of prednisone given. Quantitative bone histomorphometry was performed in 18 patients. Apart from a significant correlation between serum 25-OHD and the fractional trabecular bone volume, no relationships were observed between bone histomorphometry and vitamin D metabolites or serum iPTH. The results indicate that the bone loss was due to a decreased osteoblastic activity rather than to an impaired vitamin D metabolism.

Fibrin and fibronectin in rheumatoid synovial membrane and rheumatoid synovial fluid.

Normal synovial membranes and synovial membranes from patients with classic rheumatoid arthritis were investigated for the presence of fibrin and fibronectin by an indirect immunoperoxidase technique. In normal synovial membranes, fibronectin was found around the monolayer of the synovial lining cells. Staining was most intense on the surface and beneath the lining cells, but not detectable in the cytoplasm. Fibronectin was also found in the cytoplasm of the endothelial cells. No staining for fibrin was found in the normal synovial membrane. In synovial membranes from patients with rheumatoid arthritis, large amounts of fibronectin were found around the multilayer of synovial lining cells, in the cytoplasm of the endothelial cells, and in argyrophilic fiber-rich connective tissue. In superficial areas denuded of synovial lining cells, high amounts of fibronectin were found incorporated in fibrin. In some areas with noninjured synovial lining cells, fibrin was also found, but in this case no fibronectin was incorporated. No fibronectin was found in connective tissue in areas with infiltration of inflammatory cells. After treatment of normal and rheumatoid synovial membranes with hyaluronidase, fibronectin was still present around the lining cells but the staining was found to be more distinct. This study relates the presence of fibrin and fibronectin in the rheumatoid synovial membrane to the high amount of these proteins, recently described, in rheumatoid synovial fluid. It also suggests that fibronectin present in the synovial membrane is produced and secreted by the endothelial cells.

Regional muscle tension and pain ("fibrositis"). Effect of massage on myoglobin in plasma.

In thirteen patients, all women, with regional muscle tension and pain ("fibrositis") plasma myoglobin concentration was measured before and after massage. A significant increase was observed in the plasma myoglobin concentration reaching a maximum three hours after the start of massage treatment (median 133 micrograms/l). A positive correlation was found between the degree of muscle tension and the increase in plasma myoglobin concentration. After repeated massage treatment a gradual decline could be demonstrated in the increase of the plasma myoglobin concentration in coincidence with the efficacy of the treatment. Opposed to this only normal levels of myoglobin were found in plasma when muscles without tenderness and pain were treated with massage. The observed increase of myoglobin in plasma after massage indicates a leak of myoglobin from the muscle fibres, which suggests that regional muscle tension and pain is associated with disorders in the muscle fibres.

Long-term treatment of rheumatoid arthritis with fenclofenac or gold: a controlled, single blind trial comparing clinical and biochemical parameters.

A randomized parallel group study to compare the effects of fenclofenac and gold was carried out in 35 patients with active rheumatoid arthritis. Patients were treated with either 1200 mg fenclofenac per day or gold in standard doses in addition to their non-steroidal anti-inflammatory therapy and monitored for up to twelve months. The two treatments produced similar improvements in erythrocyte sedimentation rate, orosomucoid and haptoglobin. In addition, gold produced improvements in haemoglobin, IgG and alpha 1-antitrypsin, but there were no statistically significant differences between the clinical responses. Fenclofenac caused significantly fewer and less serious unwanted effects.

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique.

Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2 x 10(6) units/l) for 60 min at 37 degrees C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to "lamina splendens", the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.

Different molecular forms of fibronectin in rheumatoid synovial fluid.

The concentration of fibronectin in rheumatoid synovial fluid was found to be 2-3 times higher than in the corresponding plasma. Normal plasma revealed a homogeneous precipitate by cross- immunoelectrophoresis using antifibronectin, while rheumatoid plasma and rheumatoid synovial fluid exhibited a heterogeneous precipitate. The heterogeneous precipitate in rheumatoid plasma was found to be a complex between fibronectin and fibrinogen as evidenced by cross-immunoelectrophoresis. Synovial fluid fibronectin demonstrated a lower molecular weight by gelfiltration on Sepharose CL6B than did normal plasma fibronectin. We suggest that the presence of degraded fibronectin in rheumatoid synovial fluid may be the result of either the degradation of fibrin-fibronectin complexes or the destruction of matrix fibronectin from the synovial tissue.

Myopathy in bone loss of ageing: improvement by treatment with 1 alpha-hydroxycholecalciferol and calcium.

1. Eleven patients with the bone loss of ageing were treated with the vitamin D analogue 1 alpha-hydroxycholecalciferol and calcium for 3--6 months. 2. Muscle biopsies were taken from the vastus lateralis before and after the treatment and the activity of several enzymes was measured. Succinate dehydrogenase and total phosphorylase activities, which are a measure of the oxidative capacity, were low and increased significantly with the treatment. The lactate dehydrogenase activity, which can be taken as a measure of the anaerobic metabolism, was normal and did not change with treatment. The phosphagen stores, ATP and creatine phosphate were low and increased to normal with treatment. 3. Histochemical classification of the fibre composition revealed that the treatment induced an increase in the relative number of fast-twitch a (FTa or type II A) fibres accompanied by a reduction of the fast-twitch b (FTb or type II B) fibres. The cross-sectional area of the FTa fibres also increased with the treatment. 4. The present findings indicate that treatment with the active vitamin D analogue, 1 alpha-hydroxycholecalciferol, and calcium improves the myopathy associated with the bone loss of ageing.

The extra-articular deposition of fibrinogen antigenic material in patients with rheumatoid arthritis.

Experimental suction blisters were elicited on extensor and flexor sites or forearm skin of patients both with rheumatoid arthritis and without rheumatoid diseases. The blister fluid obtained was investigated, together with rheumatoid bursa fluid and rheumatoid noduli material by crossed and quantitative immunoelectrophoresis for fibrinogen antigenic material, plasminogen and inhibitors of fibrinolysis. In rheumatoid noduli and rheumatoid bursa fluid, fibrinogen degradation products identical with those previously described in rheumatoid synovial fluid and in bullous dermatosis were found (3, 14). On the other hand, no fibrinogen antigenic material was found in the experimental suction blisters. These results indicate that the fibrinogen degradation products in rheumatoid inflammatory exudate are probably not specific for a rheumatoid process, but rather part of a general mechanism in inflammation.

Properties of fibrinogen-antigenic material on the rheumatoid synovial membrane and in the rheumatoid synovial fluid.

Fibrin deposits on rheumatoid synovial membranes and fibrinogen-antigenic material in rheumatoid synovial fluid were found to be identical by crossed immunoelectrophoresis into immunoglobulin against fibrinogen, by SDS-polyacrylamide gel electrophoresis, and by gel filtration on Sepharose CL 6B. The material was found to be neither fibrinogen nor fibrin, but degradation products. One of the fragments was purified by preparative agarose electrophoresis, and the physicochemical properties of this fragment were found to be different from those obtained by plasmin digestion of fibrinogen or fibrin. This indicates that other proteases than plasmin are responsible for the degradation products. The material was easily degraded by plasmin to D- and E-antigenic end products, identical to those obtained by plasmin digest of fibrinogen. The solubility of the material was poor in synovial fluid compared to serum and buffer. On the basis of these results, it is suggested that the fibrinlike material on the synovial membrane represents fibrinogen degradation products from the inflamed tissue. These products are likely released into the synovial fluid, and when their concentration here exceeds their solubility, they precipitate on the synovial membrane.