Structural effects of intra-articular TGF-ß1 in moderate to advanced knee osteoarthritis: MRI-based assessment in a randomized controlled trial.

BACKGROUND: To determine effects of allogeneic human chondrocytes expressing TGF-ß1 (TG-C) on structural progression of MRI features of knee osteoarthritis over a 1 year period. METHODS: This phase II randomized controlled trial of TG-C included patients with moderate to advanced osteoarthritis. Patients were randomized to receive an intraarticular 3:1 mixture of non-transduced allogeneic human chondrocytes and TG-C or placebo. 3 T MRI was acquired for all patients at baseline and follow-up (3, 6 and 12 months). MRIs were assessed using the WORMS system including cartilage damage, bone marrow lesions (BMLs), meniscal damage/extrusion, Hoffa-, effusion-synovitis, and osteophytes. Analyses were performed on a whole knee level, compartmental level, and subregional level. Binary logistic regression with Generalized Estimating Equation was used to compare risks of progression, adjusting for baseline age and gender. Mann - Whitney - Wilcoxon tests were used to assess differences for continuous variables. RESULTS: Fifty-seven Patients were included in the TG-C group and 29 in the placebo group. At 12 months, knees in the TG-C group showed less progression of cartilage damage compared to placebo on a whole knee level (34.6% vs. 47.9%; adjusted RR 0.7, 95%CI [0.5-1.1], p = 0.077). Less progression of Hoffa-synovitis and effusion-synovitis was observed in the TG-C group compared to placebo (9.6% vs. 21.1%, adjusted RR 0.5, 95%CI [0.2,1.2], p = 0.115). No statistically significant differences were seen for BMLs, meniscal damage and osteophytes. CONCLUSIONS: Intraarticular treatment with TG-C showed fewer patients in the treated group with progression in structural OA features and other MRI-defined inflammatory markers such as Hoffa-synovitis and effusion-synovitis. However, no differences were observed in regard to progression of BMLs and meniscal damage, or hypertrophic osteophyte formation. TRIAL REGISTRATION: NCT01221441 .Registered 13th October, 2010.

Identification of the transcription termination site of the mouse nkx-1.2 gene: involvement of sequence-specific factors.

We have identified a transcription termination site in the 3' flanking region of the mouse nkx-1.2 gene. A downstream transcription regulatory element in the mouse nkx-1.2 gene was characterized by transferring its 3'-fragment into a chloramphenicol acetyl transferase (CAT) expression vector. Analysis of recombinant plasmids transfected into mouse NIH3T3 cells by CAT assay showed the possible region of regulation. There were two direct repeat structures containing poly(dG-dT) x poly(dC-dA) sequences (GT repeats) in this region. The precise location of transcription termination was mapped by nuclease S1 analysis of the transcripts from recombinant plasmids transfected into COSM6 cells. It was approximately 20 nucleotides upstream of the first GT repeat within the 5' sequences of the first element of the two direct repeats. Gel mobility shift assay and footprinting analysis demonstrated that nuclear DNA binding proteins bound specifically to the sequences where the termination occurred as well as the other sequences in the second element of the direct repeats. Southwestern analysis showed that 90-, 54-, 36- and 15-kDa nuclear proteins bound to the region of the termination. It is possible that one or more of those proteins are involved in blocking the elongation of the mouse nkx-1.2 gene transcript and then result in termination.

Effect of plant matrix and fluid ethanol concentration on supercritical fluid extraction efficiency of schisandrin derivatives.

An investigation of the effect of plant matrix on the supercritical fluid extraction efficiency of five schisandrin derivatives is reported, exhibiting a great difference with respect to extraction efficiency depending on the matrix. Pure supercritical CO2 at 60 degrees C and 34.0 MPa cannot fully recover schisandrin derivatives from the leaves as much as from the other matrices. Only 36.9% of these compounds are extracted from leaves of Schisandra chinensis by supercritical CO2 in comparison with organic solvent extraction. However, more than 80% of schisandrin derivatives are obtained from both stem and fruit parts. Ethanol addition also shows a different effect depending on plant matrix; that is, CO2 modified with 10% ethanol could enhance the yield of schisandrin derivatives from leaves by four times when compared with that of pure CO2, but it has little effect on both stems and fruits.

Sequences responsible for transcription termination of the mouse gastrin gene.

To identify the transcription termination elements in the mouse gastrin gene, we examined RNA transcripts after stable transfection of gastrin expression plasmids into the NIH3T3 cell line. The GT-repeat region at the 3'-flanking sequence of the mouse gastrin gene acted as a transcription terminator. When the GT-repeat unit was deleted from its site, the effect of termination disappeared. Further experiment, using serial deletion mutants, revealed that the 56-38 nucleotide upstream region from the GT-repeat unit also participated in transcription termination. We propose that the upstream region of the GT-repeat unit might be recognized as a pause site by the RNA polymerase II, and an abnormal DNA structure, derived from the GT-repeat unit, might function as a blockage of polymerase processivity.

Different effects of base analog substitutions in BamHI restriction site on recognition by BamHI endonuclease and BamHI methylase.

BamHI endonuclease and BamHI methylase were used to investigate their specific interaction with the common recognition sequence, GGATCC. Five derivatives of the oligonucleotide, GACGGATCCGTC, containing a variety of single-base analog substitutions within the hexameric recognition core were synthesized. Steady-state kinetics for the reaction of the endonuclease and the methylase showed that both enzymes recognize the sequences by contacting with functional groups exposed in both major and minor grooves of the site but in different ways. Removal or substitution of the 5-methyl group in thymidine blocked the endonuclease reaction completely but still allowed the methylase reaction with less efficiency. The data also showed that the methylase made a critical minor groove contact with the 2-amino group of the first G but the endonuclease did with that of the second G.

Lipase and its modulator from Pseudomonas sp. strain KFCC 10818: proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator.

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.