Chitinase activity was measured as the liberation of the fluorescent molecule 4-methylumbelliferyl (MUF) from three chitinase substrates (MUF-N-acetyl-β-d -glucosaminide, MUF-β-d -N,N’-diacetylchitobioside and MUF-β-d -N,N’,N’’-triacetylchitotrioside; Sigma Aldrich, UK), following the previously described method [42 (link), 60 (link), 61 (link)] (Additional file 1 : Supplementary information). Standards curves were obtained using chitinase from Streptomyces griseus (Sigma Aldrich, UK) dissolved in sterile phosphate-buffered saline solution (pH 7.4; 0.137 M) with a highest concentration of 0.1 UmL-1 (activity equivalent to 144 μM day-1). Samples were diluted prior to measurement if they were expected to be above this range.
Streptomyces griseus
Manufactured by Merck Group
Sourced in United States
Streptomyces griseus is a species of actinobacteria that is commonly used in the production of various antibiotics. It is a Gram-positive, aerobic, and filamentous bacterium. Streptomyces griseus can be utilized in laboratory settings for the identification and cultivation of this microorganism.
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Lab products found in correlation
Pronase, by Merck Group (2 mentions)
Remazol brilliant violet 5r dye, by Merck Group (2 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Claudin 1, by Thermo Fisher Scientific (1 mentions)
Brightvision polyhrp anti rabbit igg, by Immunologic (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Chitin powder, by Merck Group (1 mentions)
D glucono δ lactone, by Merck Group (1 mentions)
Collagenase b, by Roche (1 mentions)
Ms 222, by Merck Group (1 mentions)
Collagenase d, by Merck Group (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Mab flag, by Merck Group (1 mentions)
L 15 medium, by Merck Group (1 mentions)
17 protocols using streptomyces griseus
1
Chitinase Activity Quantification Protocol
2
Exploring Tight Junction Proteins in Plexuses
3
Assessing Chitinase Activity of Lysozyme Msp1
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To determine the potential chitinase activity of lysozyme Msp1, we used the purified protein for activity assay using a Chitinase Assay Kit (Macklin). The chitinase from Streptomyces griseus (Sigma-Aldrich, C6137) was used as a positive control [41 (link)]. In addition, we tested whether Msp1 could hydrolyze colloidal chitin to form the hydrolytic zone in agar plates. Thus, chitin powder (5 g; Sigma-Aldrich) was first added to the concentrated HCl (37%; 60 ml) and stirred with a magnetic rod at 4 °C overnight. The samples were then washed with 95% ethanol twice followed by washing with sterile water before being embedded in water agars (5%, w/v). The plates were punched with holes (7.5 mm in diameter), and 20 μl PBS or Msp1 protein solution (100 μg/ml) were individually inoculated for 12 h.
4
Primary Culture of Mouse CPE Cells
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Primary culture of mouse CPE cells was done as described (Menheniott et al, 2010 ). In brief, pups 2–9 days old were decapitated and the brains were isolated. Choroid plexus from all four ventricles were isolated under a dissection microscope. The cells were dissociated enzymatically by incubating them for 5–7 min with pronase (isolated from Streptomyces griseus, Sigma). Digestion was stopped by adding an excess of HBSS buffer, and the cells were washed twice with HBSS. The cell pellet was resuspended in DMEM‐F12 culture medium and plated on laminin‐coated plates or transwell systems. Two days later, they were shifted to DMEM‐F12 containing cytosine arabinoside (Ara‐C) to eliminate growth of fibroblasts. Cultures were maintained at 37°C in 5% CO2. Purity of the cultures was confirmed by checking the levels of transthyretin (TTR) both by qPCR and by immunostaining. Primary CPE cells retained the epithelial and the barrier properties which were confirmed by staining for ECDH, ZO1, and occludin and also by measuring the transepithelial electrical resistance (TEER, Millipore). To mimic the in vivo situation in the transwell system, LPS stimulation was always done from the basolateral side.
5
JMJD7-Mediated Peptide Hydroxylation
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Peptides (50 µM; DRG121-40, DRG116-40, and DRG215-39) were incubated with 100 µM Fe(II), 200 µM 2OG, and 500 µM ascorbate in the presence or absence of 10 µM recombinant His6-JMJD7 for 1 hour at 37°C in 50 mM HEPES, pH 7.5 (final volume 500 µL). The reaction was quenched with 2 drops of neat formic acid; hydroxylation (~40-80%) of the peptide was confirmed by MALDI-MS. Recombinant JMJD7 was removed using a Sep Pak C18 column according to the Kessler Lab Proteomics Protocol (University of Oxford, http://www.ccmp.ox.ac.uk/_asset/file/sep-pak-c18.pdf ), eluting with a 15% acetonitrile, 0.1% formic acid solution in double distilled water. Samples were then dried by vacuum centrifugation and subsequently re-suspended in 60 µL of 20 mM HEPES, pH 7.5. Enzymatic hydrolysis was carried out at 37°C with a non-specific protease from Streptomyces griseus (9 µL, 1 mg mL-1, Sigma-Aldrich) overnight.
6
Preparation of Chitosan Oligomers
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Fully de-N-acetylated chitosan (FA = 0.001) was prepared by a further heterogeneous de-N-acetylation of a commercially available chitosan (Kimica, Japan). Fully de-N-acetylated chitosan oligomers were prepared by enzymatic hydrolysis of the fully de-N-acetylated chitosan using a commercial chitosanase from Streptomyces griseus (Sigma C9830) and a subsequent separation using size exclusion chromatography.39 (link)In addition a chitosan oligomer mixture (DPn = 3.96, FA = 0.045) provided by Koyo Ltd Co (Japan), lot number 121017WG was used in comparison.37 (link)d -Glucono δ-lactone (GDL) was purchased from Sigma-Aldrich (U.S.A.). All other chemicals were of analytical grade and used without any further purification.
7
Isolation and Culture of Rabbit Chondrocytes
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Normal articular cartilage was collected from five white New Zealand buck rabbits, which were anesthetized with overdose of propofol and euthanized using Potassium chlorate 60 mg/Kg. After knee joint surgery, the pieces of articular cartilage were dissected and separated from the underlying bone and connective tissues. The pieces were washed three times with PBS 1× and 5% penicillin/streptomicin. The extracted cartilage was digested in a solution of 2 mg/mL Protease Type XIV. Bacterial from (Streptomyces griseus) (Sigma Aldrich, St Louis, MO, USA) in PBS 1× for 1.5 h and 1.5 mg/mL collagenase B (Roche, Meylan, France) in basic medium DMEM at 37 °C overnight. This suspension was centrifuged at 1200 rpm for 8 min. to collect the chondrocytes and was washed with basic medium DMEM. The chondrocytes were cultured in DMEM/F12 (1:1 with 15% FBS plus 1% antibiotic mixture of penicillin/streptomycin) at a density of 1 × 105 cells/mL and incubated in a humidified atmosphere of 5% CO2 at 37 °C. Culture medium was changed every two days and each passage was made when the confluence reached between 80–90%. We only used the second passage of cells in all experiments in order to avoid loss of chondrocyte phenotype [92 ].
8
JMJD7-Mediated Peptide Hydroxylation
9
Zebrafish Angiogenesis Assay Protocol
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At about 24 h post-fertilization (hpf), the chorion was removed with the protease (1.5 mg/mL for 5 min) from Streptomyces griseus (Sigma-Aldrich Inc., St. Louis, MO, USA). The de-chorion embryos were distributed into 24-well culture plates with eight embryos per well containing E3/PTU (1-phenyl-2-thiourea, 0.003%) buffer. The Tg(fli1:EGFP) embryos were treated with drugs at various concentrations and resuspended in 1% DMSO. The embryos were anesthetized with tricaine (ethyl 3-aminobenzoate methanesulfonate, MS-222 (Sigma-Aldrich In., St. Louis, MO, USA) final concentration 0.016%) for about 50 hpf to prevent movement and their images were captured. The images were analyzed by measurement of the length of inter-segmental vessels to determine the anti-angiogenesis effect of individual compounds.
10
Isolation of Hepatic Stellate Cells and Peritoneal Macrophages
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After anesthesia, the mouse liver tissues were digested with Streptomyces griseus (Sigma, Saint Louis, MO, USA) and collagenase D (Sigma, USA). The digested liver was cut into pieces and then filtered through a sieve. The filtered cell fluid was centrifuged for 5 min to obtain hepatocyte sediment. Then, we centrifuged the supernatant obtained in the previous step at 600× g for 10 min, and then took the sediment. We added 5 mL of 20% Nycodenz into the centrifuge tube, mixed it with the sediment, and carefully added 5 mL of 12% Nycodenz and 3mL DMEM on the suspension surface. Then it underwent 1400× g centrifugation for 17 min. After centrifugation, the liquid in the centrifuge tube was in a stratified state. One layer of cells in the middle layer was hepatic stellate cells [29 (link)].
Other mice were selected to isolate macrophages. The mice were killed and then soaked in 75% alcohol for 5 min. The abdomen was lifted with tweezers, and a small wound was inflicted. To this end, 8 mL PBS was injected, and the peritoneal fluid was removed using a pasteurized straw. Finally, centrifugation was performed to obtain peritoneal macrophage precipitation.
Other mice were selected to isolate macrophages. The mice were killed and then soaked in 75% alcohol for 5 min. The abdomen was lifted with tweezers, and a small wound was inflicted. To this end, 8 mL PBS was injected, and the peritoneal fluid was removed using a pasteurized straw. Finally, centrifugation was performed to obtain peritoneal macrophage precipitation.
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