Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.

Efficacy and safety of intraarticular hylan or hyaluronic acids for osteoarthritis of the knee: a randomized controlled trial

OBJECTIVE: To compare the efficacy and safety of intraarticular hylan and 2 hyaluronic acids (HAs) in osteoarthritis (OA) of the knee. METHODS: This was a multicenter, patient-blind, randomized controlled trial in 660 patients with symptomatic knee OA. Patients were randomly assigned to receive 1 cycle of 3 intraarticular injections per knee of 1 of 3 preparations: a high molecular weight cross-linked hylan, a non-cross-linked medium molecular weight HA of avian origin, or a non-cross-linked low molecular weight HA of bacterial origin. The primary outcome measure was the change in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain score at 6 months. Secondary outcome measures included local adverse events (effusions or flares) in injected knees. During months 7-12, patients were offered a second cycle of viscosupplementation. RESULTS: Pain relief was similar in all 3 groups. The difference in changes between baseline and 6 months between hylan and the combined HAs was 0.1 on the WOMAC pain score (95% confidence interval [95% CI] -0.2, 0.3). No relevant differences were observed in any of the secondary efficacy outcomes, and stratified analyses provided no evidence for differences in effects across different patient groups. There was a trend toward more local adverse events in the hylan group than in the HA groups during the first cycle (difference 2.2% [95% CI -2.4, 6.7]), and this trend became more pronounced during the second cycle (difference 6.4% [95% CI 0.6, 12.2]). CONCLUSION: We found no evidence for a difference in efficacy between hylan and HAs. In view of its higher costs and potential for more local adverse events, we see no rationale for the continued use of hylan in patients with knee OA.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.