Purification and characterization of 50 kDa extracellular metalloprotease from serratia sp. ZF03

Proteolytic enzymes have an important role in variety of physiological and pathological functions. They have been used in therapeutic and pharmaceutical applications. Characterizations of extracellular proteases from various strains of S. marcescens indicate that most strains produce a very similar major metalloprotease. This metalloprotease [serrapeptidase, serrapeptase] is an important pharmaceutical agent. Serrapeptase has been used in Asian and European countries for the treatment of inflammatory diseases, cardiovascular disorders, and bacterial infections. In the present study, purification and characterization of extracellular metalloprotease from Serratia sp. ZF03 for therapeutic purposes were reported. In this study the protease gene encoding a zinc-metalloprotease was isolated from the previously isolated red-pigmented Serratia sp. ZF03. The gene was sequenced and submitted to the GenBank. Proteolytic activity was detected by skim milk agar plate method and zymography. This fragment was found to encode an extracellular zincmetalloendopeptidase with a molecular weight of approximately 50 kDa. The metalloprotease was purified by ammonium sulfate precipitation and dialysis, and then characterized. The effects of various inhibitors and reagents on protease activity and its kinetic parameters were also determined. The nucleotide sequence demonstrated that deduced amino acid sequence has a higher identity with those of metalloprotease from serralysin family. Production of metalloprotease was highest at 48[th] h of cultivation. Optimum protease activity occurred at a temperature range of 50-55[degree sign]C and a pH range of 8.0-10. EDTA as a metal chelator, significantly inhibited protease activity. Zymography and inhibition assays showed that metalloprotease is the major secreted protease of Serratia sp. ZF03. The kinetic parameters, K[m] and V[m], were 0.00105 mg/ml and 0.0531 mM/min, respectively. Since the metalloprotease of this strain has strong proteolytic properties and good stability, it would be a suitable candidate to be used as an effective drug in the medicine and pharmaceutical industries

Effect of tetracycline on the bond performance of etch-and-rinse adhesives to dentin

This study evaluated the effect of modified tetracycline on the resin-dentin bond strength (µTBS), silver nitrate uptake (SNU) and solution homogeneity (SH) of two adhesives. Dentin surfaces were treated with phosphoric acid, rinsed off and either rewetted with water (control group - CO), 2 percent minocycline (MI), 2 percent doxycyline (DO) or 2 percent chlorhexidine (CH). Adhesive systems (Adper Single Bond 2 and Prime Bond NT) and composite were applied and light-polymerized. Specimens were sectioned to obtain bonded sticks (0.8 mm²) to test under tension at 0.5 mm/min. For SNU, specimens were immersed in silver nitrate and analyzed by EDX-SEM. SH was qualitatively analyzed after mixing the adhesives with different solvent-based solutions containing MI, DO and CH. Lower µTBS values were observed in the DO group compared with MI and CH (p = 0.01). Lower SNU was observed for MI and CH. The lowest µTBS for both adhesives was observed for the DO group (p = 0.01). Signs of phase separation were observed for DO with both adhesives. MI or CH used as rewetting solutions after acid etching did not affect the µTBS and hybrid layer quality.

Structure and secretory activity of cultured chondrocytes from patients with osteoarthritis

Cartilage samples were taken from OA patients in order to describe and quantify pro-inflammatory mediators. Samples were cultured under aseptic conditions in Dulbecco's modified Eagle medium at 37°C for 10 days. Control samples, taken from non-inflammatory cartilage, were cultured under the same conditions. The levels of NO-2 and NO-3 were measured in the supernatant using a spectrophotometric assay. The activity of MMP-1 was quantified by ELISA.The concentration of NO-x was 47.3 ± 4.1 µM in the OA cartilague and 10.7 ± 1.8 µM in the controls. The average MMP-1 activity was 3,650 ± 387 ng/ml in the OA cartilage and 2,150 ± 190 ng/ml in the control samples. These increased values of MMP-1 and NO- x observed in the OA cartilage suggest a higher catabolic activity. A morphological analysis of OA chondral tissue using light microscopy shows that the surface of the tissue is characterized by the presence of aggregated chondrocytes or "clones" but in the deeper areas isolated cells are found. These results could be a significant contribution towards the identification of biological markers indicating the presence of OA activity

The metalloproteinase of Leishmania: leishmanolysin

Zinc metalloproteinases are a diverse group of endo- and exoproteinases related only by their common catalytic mechanism and similar primary structure defining the metal binding domain. They are involved in tissue remodelling, metastasis, peptide hormone processing and digestion. Outside of the zinc binding site, their primary structures are highly divergent, suggesting that this group of enzymes is the product of convergent evolution. The three dimensional structures of small soluble bacterial (thermolysin) and eukaryote (astacin) metalloproteinases has allowed the establishment of several families of metalloproteinases based upon the zinc binding ligands of the enzymes. Thus far, no high-molecular weight membrane bound metalloproteinase has been crystallised; unfortunately these are among the most interesting in terms of human physiology. Leishmanolysin, the abundant surface metalloproteinase of several genera of kinetoplastid protozoans, most notably Leishmania, provides an abundant source of glycophosphatidylinositol-anchored glycoprotein for biochemical and structural studies, which will not only lead to a better understanding of the role of the proteinase in the life cycle of the protozoan, but will also provide a framework upon which to model the structures of mammalian metalloproteinases.