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Dimethylmethylene Blue (dimethylmethylene + blue)

Distribution by Scientific Domains

Medical Sciences	71%

Selected Abstracts

Quinacrine Enhances Vesicular Stomatitis Virus Inactivation and Diminishes Hemolysis of Dimethylmethylene Blue,phototreated Red Cells,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2002
Stephen J. Wagner
ABSTRACT Several photodynamic methods for virus inactivation in red blood cell (RBC) suspensions have resulted in unwanted hemolysis during extended 1,6°C storage. To explore the possibility that hemolysis may be mediated by a membrane-bound dye, a molecule similar in structure to yet different in light absorption properties from the photosensitizer was used as an inhibitor for RBC membrane binding in virus photoinactivation and photohemolysis studies. The addition of 500 ,M quinacrine to oxygenated RBC before treatment with 3.6 ,M dimethylmethylene blue (DMMB) and 219 mJ/cm2 red light resulted in an increased extracellular concentration of the sensitizer, increased extracelluar viral inactivation kinetics, and decreased hemolysis during 1,6°C storage without alteration of quinacrine absorption properties. These results collectively suggest that despite its recognized affinity for viral nucleic acid, DMMB also binds to RBC membranes and that the bound dye is, in part, responsible for photoinduced hemolysis. [source]

Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude mice

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 7 2009
J. T. Oliveira
Abstract Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. [source]

Quinacrine Enhances Vesicular Stomatitis Virus Inactivation and Diminishes Hemolysis of Dimethylmethylene Blue,phototreated Red Cells,

Parathyroid hormone 1,34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

ARTHRITIS & RHEUMATISM, Issue 10 2009
Je-Ken Chang
Objective Parathyroid hormone 1,34 (PTH[1,34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone,related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1,34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). Methods We studied the effect of PTH(1,34) on human articular chondrocytes with azacytidine (azaC),induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. Results AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3,10 days of treatment with 10 nM PTH(1,34). SOX9 expression was not changed by either azaC or PTH(1,34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1,34) treatment did not reverse this effect. Furthermore, PTH(1,34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. Conclusion Our findings indicate that PTH(1,34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA. [source]

ADAM-8 isolated from human osteoarthritic chondrocytes cleaves fibronectin at Ala271

ARTHRITIS & RHEUMATISM, Issue 9 2009
Marc D. Zack
Objective Fibronectin fragments are thought to play a critical role in the initiation and progression of cartilage degradation in arthritis. In a recent study, fibronectin neoepitopes resulting from cleavage of intact fibronectin at the Ala271/Val272 scissile bond, generating an ,30-kd fragment with the new C-terminus VRAA271 and an ,50,85-kd fragment with the new N-terminus 272VYQP, were identified in osteoarthritis (OA) cartilage. The present study was undertaken to isolate the enzymes responsible for this cleavage from human OA chondrocytes. Methods Fibronectin-degrading activity in human OA chondrocyte,conditioned medium (OACCM) was purified using conventional chromatography. A fluorescent peptide was developed based on the fibronectin scissile bond 269RAA,Val272, and this peptide was used to track fibronectinase activity during purification. Western blotting with antibodies that detect the fibronectin neoepitopes VRAA271 and 272VYQP was used to confirm cleavage of intact fibronectin by the enzymatically active fractions. Mass spectrometry was used to identify the proteins found in the fibronectinase-enriched fractions, with further confirmation by Western blotting. In addition, a recombinant enzyme identified by mass spectrometry was tested by Western blotting and dimethylmethylene blue assay for its ability to produce fibronectin neoepitopes in OA cartilage. Results Purification of OACCM by chromatography resulted in isolation of a fibronectin-degrading enzyme, and mass spectrometry identified ADAM-8 as the fibronectinase present in these preparations. Furthermore, treatment of OA cartilage with recombinant human ADAM-8 promoted cartilage catabolism. Conclusion The results of this study identify ADAM-8 as a fibronectinase in human OA chondrocytes. Because ADAM-8 is capable of producing the fibronectin neoepitopes VRAA271 and 272VYQP in human OA cartilage, this enzyme may be an important mediator of cartilage catabolism. [source]

Neutrophil gelatinase,associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

ARTHRITIS & RHEUMATISM, Issue 10 2007
Kalpana Gupta
Objective Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ,125,130,kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF. Methods MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase,associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay. Results Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL,MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL,MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9,mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05). Conclusion Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA. [source]

Calcium signaling leads to mitochondrial depolarization in impact-induced chondrocyte death in equine articular cartilage explants

ARTHRITIS & RHEUMATISM, Issue 7 2007
C. A. M. Huser
Objective Chondrocyte apoptosis is an important factor in the progression of osteoarthritis. This study aimed to elucidate the mechanisms involved upstream of caspase 9 activation and, in particular, calcium signaling and mitochondrial depolarization. Methods Articular cartilage explants obtained from healthy horses were subjected to a single impact load (500-gm weight dropped from a height of 50 mm) and cultured in vitro for up to 48 hours. Chondrocyte death was quantified by the TUNEL method. Release of proteoglycans was determined by the dimethylmethylene blue assay. Weight change was measured, and mitochondrial depolarization was determined using JC-1 staining. To assess the role of calcium signaling in impact-induced chondrocyte death, explants were preincubated in culture medium containing various concentrations of calcium. Inhibitors were used to assess the role of individual signaling components in impact-induced chondrocyte death. Results Calcium quenching, inhibitors of calpains, calcium/calmodulin-regulated kinase II (CaMKII), and mitochondrial depolarization reduced impact-induced chondrocyte death after 48 hours in culture. Transient mitochondrial depolarization was observed 3,6 hours following a single impact load. Mitochondrial depolarization was prevented by calcium quenching, inhibitors of calpain, CaMKII, permeability transition pore formation, ryanodine receptor, and the mitochondrial uniport transporter. Cathepsin B did not appear to be involved in impact-induced chondrocyte death. The calpain inhibitor prevented proteoglycan loss, but the percentage weight gain and proteoglycan loss were unaffected by all treatments used. Conclusion Following a single impact load, calcium is released from the endoplasmic reticulum via the ryanodine receptor and is taken up by the mitochondria via the uniport transporter, causing mitochondrial depolarization and caspase 9 activation. In addition, calpains and CaMKII play important roles in causing mitochondrial depolarization. [source]