Microglia extracellular traps in Oreochromis niloticus infected with Weissella cibaria.

The mechanism of extracellular traps (ETs) is important in the cellular response against bacteria. Thus, in the present study, we describe for the first time the capacity of the Nile tilapia (Oreochromis niloticus) microglia in the formation of ETs in Weissella cibaria in vitro infection. Thus, we evaluated the ultrastructure of the microglia culture and observed the formation of ETs 6 h after stimulation with lipopolysaccharide (LPS) and during the course of infection. Our results shed light on the mechanism of formation of ETs in the microglia of teleost fish and the ability of W. cibaria to infect these cells.

Microglia extracellular traps in Oreochromis niloticus infected with Weissella cibaria

The mechanism of extracellular traps (ETs) is important in the cellular response against bacteria. Thus, in the present study, we describe for the first time the capacity of the Nile tilapia (Oreochromis niloticus) microglia in the formation of ETs in Weissella cibaria in vitro infection. Thus, we evaluated the ultrastructure of the microglia culture and observed the formation of ETs 6 hours after stimulation with lipopolysaccharide (LPS) and during the course of infection. Our results shed light on the mechanism of formation of ETs in the microglia of teleost fish and the ability of W. cibaria to infect these cells.

Phagolysosomal activity of macrophages in Nile tilapia (Oreochromis niloticus) infected in vitro by Aeromonas hydrophila: Infection and immunotherapy.

The biochemical mechanisms involved in phagocytosis and the intracellular survival of Aeromonas hydrophila (Ah) in host macrophages (MΦs) are complex processes that affect infection success or failure. Thus, in the present study, we described the in vitro infection of Nile tilapia MΦs by a homologous bacterium and tested the effects of anti-A. hydrophila immunoglobulin Y (IgY) on the phagolysosomal activity and intracellular survival of the pathogen. The anti-Ah IgY modulated lysosomal acid phosphatase (LAP) activity as well as the production of reactive oxygen intermediates (ROIs) and nitric oxide (NO), thereby potentiating phagocytosis and the elimination of Ah. Thus, we assume that the specific IgY had a beneficial effect on infection control and postulated the use of the Nile tilapia MΦs as an important in vitro experimental model for the functional and therapeutic study of Ah infection.

Some coagulase negative Staphylococcus spp. isolated from buffalo can be misidentified as Staphylococcus aureus by phenotypic and Sa442 PCR methods.

OBJECTIVE: Staphylococcus aureus is a commonly reported cause of buffalo mastitis. However, its prevalence may be overestimated. The aim of this study was to compare S. aureus identification by conventional phenotypic and genotypic assays versus Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and novel real-time quantitative PCR tests for the cytochrome oxidase subunit D II (cydB) and staphylocoagulase (coa) genes. RESULTS: From 408 samples obtained from buffalo milk/milking environment, 32 putative S. aureus strains were identified based on characteristic growth on Baird Parker agar, positive catalase reaction, ability to clot rabbit plasma, and positive Sa442 PCR assay. However, in further testing, only 10 of these strains were positive in latex agglutination tests and by MALDI-TOF MS, only eight of the 32 strains were S. aureus while the rest were S. chromogenes (19), S. agnetis (3), S. cohnii (1), or S. xylosus (1). All eight strains identified as S. aureus by MALDI-TOF analysis and confirmed by 16S RNA gene sequencing were positive in a S. aureus-specific cydB PCR test. As well, 7/8 S. aureus strains were PCR positive in a real-time coa PCR test as were 2/69 S. chromogenes and the lone S. xylosus strain tested.

Validation of IgY for the diagnosis of Streptococcus agalactiae-caused endocarditis and bacterial meningitis in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae (Sta), which belongs to Lancefield group B, causes sepsis, endocarditis and bacterial meningitis in human neonates and Nile tilapia. Because the pathophysiology of Sta infection is partially similar in both species, the identification of biomarkers for the diagnosis and study of this disease is of importance for human and animal health. Therefore, in the present study, we produced an immunoglobulin Y (IgY) by immunizing laying hens with Sta proteins and evaluated its ability to detect Sta in paraffinized tilapia brain and cardiac tissue by direct immunofluorescence (IMF) and indirect immunohistochemistry (IHC). The IgY produced was effective in the diagnosis of Sta infection in Nile tilapia, justifying the use of this species as a biomodel for the study of this disease.

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase.

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.

The zymogen-enteropeptidase system: A practical approach to study the regulation of enzyme activity by proteolytic cleavage*.

The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to prepare a laboratory class that would stimulate student interest in enzyme regulation, exploring the fact that the catalytic activity of some enzymes is regulated by different mechanisms. The regulation of proteolytic enzymes requires the synthesis of an inactive zymogen and its being irreversibly "switched on" by specific proteolytic cleavage.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study.

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper, we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4h of incubation at 25 degrees C. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degrees C. Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate. The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle.