muramidase and thiazolyl-blue

In this study, a blend mixture of chitosan-ethylenediaminetetraacetic acid (CS 2 wt%-EDTA) at a weight ratio of 30/70 and polyvinyl alcohol (PVA) solution (10 wt%) was electrospun to produce fibrous mats with lysozyme (10, 20 and 30 wt%) used for wound healing. The morphology and diameter of the electrospun fiber mats with and without lysozyme were analyzed by scanning electron microscopy (SEM). The amount of lysozyme loaded in the nanofiber mats was measured by HPLC. The cell lysis activity of the lysozyme was investigated with Micrococcus lysodeikticus cells as a substrate. The wound healing activity was performed in vivo using male Wistar rats. The SEM images of all lysozyme-loaded fibers show a smooth fiber without beads with an average diameter of 143-209 nm. The amount of lysozyme loaded in the nanofiber mats was slightly decreased when the initial concentration of lysozyme was increased. The rapid lysozyme release from the nanofiber mats was obtained and is dependent on the lysozyme-loading amount. In animal wound healing, lysozyme loaded CS-EDTA nanofiber mats accelerated the rate of wound healing when compared to the controls (gauze). In conclusion, our experiments demonstrated that biomaterials composed of lysozyme loaded CS-EDTA nanofibers have a potential for wound healing.

Topics: Animals; Bandages; Calorimetry, Differential Scanning; Cell Survival; Chitosan; Coloring Agents; Edetic Acid; Fibroblasts; Humans; Male; Micrococcus; Microscopy, Electron, Scanning; Muramidase; Nanofibers; Polyvinyl Alcohol; Rats; Rats, Wistar; Solubility; Tetrazolium Salts; Thiazoles; Wound Healing

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

Topics: Amyloid; Cell Line; Cell Survival; Humans; Microscopy, Electron, Transmission; Muramidase; Neurons; Protein Stability; Spectrophotometry, Infrared; Tetrazolium Salts; Thiazoles

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.

Topics: alpha-Amylases; Animals; Biocompatible Materials; Bone and Bones; Cell Adhesion; Cell Proliferation; Chitosan; Compressive Strength; Humans; Mice; Microscopy, Electron, Scanning; Muramidase; Stress, Mechanical; Tetrazolium Salts; Thiazoles; Tissue Engineering

The genomic DNA of Escherichia coli, which contains the unmethylated CpG motif, was used to evaluate the immunostimulating effect of bacterial DNA on innate immune responses in the bivalve mussel Hyriopsis cumingii Lea. The results showed that the E. coli DNA had no significant effect on the production of superoxide anion (O(2-)) or acid phosphatase (AP) by haemocytes in vitro. However, the bactericidal activity of the haemocytes was significantly increased when the cells were incubated with 50 or 100mug/ml bacterial DNA for 12 and 24h. Antibacterial activity, lysozyme activity, and prophenoloxidase (proPO) production of haemolymph were also increased, when the bivalve molluscs were injected with 50 or 100mug/ml of bacterial DNA for 12 and 24h. These activities returned to the control level after 48h. This work showed the bacterial DNA with unmethylated CpG motif could activate some parameters of the immune system of bivalve molluscs in vivo and in vitro.

Topics: Acid Phosphatase; Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Catechol Oxidase; DNA, Bacterial; Enzyme Induction; Enzyme Precursors; Escherichia coli; Hemocytes; Hemolymph; Immunity, Innate; Lipopolysaccharides; Muramidase; Superoxides; Tetrazolium Salts; Thiazoles; Time Factors; Unionidae

The dimer of lysozyme was administered to carp (Cyprinus carpio L.) in an attempt to reduce the suppression of response in fish exposed to the cypermethrin. Experimental studies were carried out on cells isolated from blood and pronephros of carp. In the in vivo studies fish were intoxicated with cypermethrin (synthetic pesticide) at a dose of LD5. After 24 h fish were injected intraperitoneally with a modulator-the dimer of lysozyme (KLP-602) at a dose of 0.02 mg/kg of body weight. In the study we determined the metabolic activity of phagocyte cells and the proliferative ability of lymphocytes stimulated by ConA and LPS. The results indicate that this modulator is useful for stimulation of cellular and humoral immunity after experimentally induced suppression by selected pesticide.

Topics: Animals; B-Lymphocytes; Carps; Cell Proliferation; Cell Separation; Immunity, Cellular; Immunologic Factors; In Vitro Techniques; Injections, Intraperitoneal; Insecticides; Kidney; Lymphocytes; Mitogens; Muramidase; Phagocytes; Pyrethrins; Respiratory Burst; T-Lymphocytes; Tetrazolium Salts; Thiazoles

The present study examined the effect of ambroxol on free radical production, granule enzyme release, and cell death in silica-activated rat alveolar macrophages. The action of ambroxol was assayed by measuring changes in the activities of protein kinase C (PKC) and tyrosine kinase (PTK) and in the intracellular calcium level. Ambroxol attenuated the production of superoxide, hydrogen peroxide, and nitric oxide and the release of acid phosphatase and lysozyme in macrophages activated by silica. Staurosporine, genistein, EGTA, and trifluoperazine inhibited the silica-induced free radical production and granule enzyme release. Silica induced the increase in PKC and PTK activities and the elevation of intracellular calcium level in macrophages, which was decreased by ambroxol. Silica induced a cell death and increased the caspase-3 activity in macrophages in a concentration-dependent manner. Ambroxol decreased the silica-induced cell viability loss in macrophages. The results show that ambroxol decreases the stimulated responses and cell death in rat alveolar macrophages exposed to silica, which may be accomplished by inhibition of activation processes, protein kinases, and calcium transport. The inhibitory effect of ambroxol on silica-induced cell death appears to provide the protective effect on pulmonary tissues against the toxic action of silica.

Topics: Acid Phosphatase; Ambroxol; Animals; Calcium; Caspase 3; Caspases; Cell Death; Cytoplasmic Granules; Expectorants; Hydrogen Peroxide; In Vitro Techniques; Macrophage Activation; Macrophages, Alveolar; Muramidase; Nitrites; Oxidants; Protein Kinase C; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Respiratory Burst; Silicon Dioxide; Superoxides; Tetrazolium Salts; Thiazoles