Cilia, Part A (Methods in Enzymology)

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In: Cilia, Part A. Wallace F. Marshall Methods in Enzymology ; Pigino, G. Axonemal radial spokes: 3D structure, function and assembly. Comparative structural analysis of eukaryotic flagella and cilia from Chlamydomonas, Tetrahymena, and sea urchins. J Structural Biol. Cryo-electron tomography of radial spokes in cilia and flagella.

Cell Biol. Bui KH. J Synchrotron Radiat. Electron-tomographic analysis of intraflagellar transport particle trains in situ.

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Simultaneous alignment of dual-axes tilt series. J Struct Biol — Salvi, E. J Struct Biol Caruso, T. The Berger Parker index as an effective tool for monitoring the biodiversity of disturbed soils: a case study on Mediterranean oribatid Acari: Oribatida assemblages. Biodiversity and Conservation 16 12 : Get Citation Citation. Get Permissions. The activity of MDP is widely distributed to various organs and has been detected in the lung, 12 kidney, 13 14 pancreas, 15 and small intestine.

In the experiment of the MDP knock-out mouse, Habib et al. Although the metabolisms of cysteinyl leukotrienes and glutathione have been reported in the eye, the physiological roles remain unclarified. The localization and activity of GGTP were investigated in the ocular tissues, 17 18 19 20 but the presence of MDP in the eye has not been studied to date.

A bioactive peptide amidating enzyme is required for ciliogenesis | eLife

As the result of screening of antibodies raised against bovine ciliary epithelium proteins, we established the monoclonal antibody mAb for MDP. In this study, we demonstrated for the first time the presence of MDP in bovine eye and the potential activities of MDP in bovine ciliary cells.

Bovine eyes were obtained from a local slaughterhouse. A razor blade was used to cut around the entire globe just posterior to the ora serrata. The lens, lens capsule, and zonulae were removed exposing the ciliary process. Bovine kidney and pancreas were obtained from the same slaughterhouse. The ciliary processes were dissected from the excised eyeball and minced with scissors.

The ciliary processes included mainly the epithelium and partially the ciliary stroma. After the undissociated materials were discarded, the cell suspension was collected and centrifuged for 5 minutes at g. Some of the cells were washed twice and suspended in phosphate-buffered saline and immediately used for immunization, and the rest were stored in liquid nitrogen for further immunization.

The spleen cells of an immunized mouse were fused with X63Ag8.

Gaia Pigino

An indirect immunofluorescence method with frozen sections of bovine tissue, as described later, was used for screening the supernatants of growing hybridomas. The positive hybridomas were cloned twice and injected intraperitoneally into mice previously treated with 2,6,10,tetramethylpentadecane Pristane; Tokyo Kasei Co.

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Ig isotype was determined using an isotype-specific antibody for mouse mAbs Serotec, Ltd. After a wash in phosphate-buffered saline, the slides were incubated with fluorescein isothiocyanate-conjugated second antibody diluted ; Dako Japan, Kyoto, Japan for 40 minutes at room temperature in the dark. The slides were then washed, mounted with aqueous mounting medium Perma Fluor; Immunon, Pittsburgh, PA and examined under a fluorescence microscope Nikon, Tokyo, Japan. The ciliary processes 0. The effluent was then incubated with the activated gel 0.

After sufficient washing with the buffer, the antigen was eluted with 0. The eluate was evaporated under reduced pressure at room temperature. The samples were dissolved in lysis buffer with 0. After the gel was washed, the antigenic molecules were eluted as just described. After electrophoresis, the proteins in the polyacrylamide gel were transblotted onto a polyvinylidene difluoride PVDF membrane Millipore Corp. In some experiments, the proteins in the polyacrylamide gel were stained with Coomassie blue and the After treatment with lysylendopeptidase, the fragments released from the Bovine ciliary process, neural retina NR , or retinal pigment epithelium RPE partially containing choroid 0.

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After a brief centrifugation, the supernatant was used for the fluorometric assay. All samples were assayed in duplicate from three experiments. D-Phe Nacalai Tesque was used as a standard. After 60 minutes, the fluorescence of each sample was measured in a fluorescence spectrophotometer model F; Hitachi, Tokyo, Japan at an excitation of nm and an emission of nm. Protein concentrations were measured using a detergent-compatible protein assay kit Bio-Rad.

Preincubation, addition of Gly-D-Phe, and the termination were performed as described earlier. After brief centrifugation, the supernatant was used for the fluorometric assay. Preincubation, the addition of Gly-D-Phe, and termination were performed as just described. All samples were assayed by enzyme-linked immunosorbent assay ELISA in duplicate from three experiments. Preincubation, the addition of LTD4, and the termination were as described earlier. After a brief centrifugation, the supernatant was removed.

The pelleted resin was washed with 1 mL buffer by centrifugation and resuspended in 0. The methanol extraction procedure was performed twice.

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  7. The methanol in the supernatant was collected and evaporated under reduced pressure. These were incubated for 18 hours at room temperature in the enzyme immunoassay plate. The plate was washed with buffer, and Ellman reagent, which contains the substrate to acetylcholinesterase, was added to each well. The reagent was developed for 90 minutes. Optical densities ODs were determined for each well by using a spectrophotometer set to nm.

    Protein concentrations were measured with detergent-compatible protein assay kit. One hybridoma, 49C, which belongs to the IgG1 isotype, was selected by immunohistochemistry. In the bovine eye, 49C antigen was detected in the cytoplasm of the ciliary pigmented epithelial PE cells and nonpigmented epithelial NPE cells Fig. The antigen was highly expressed in the pars plicata and moderately in the pars plana. At the ora serrata, the expression of antigen in the PE and the NPE cells became weaker and was nonexistent in the retina.

    The weak expression of 49C antigen in the PE and NPE cells of the iris extended approximately 1 mm from the root of the iris. Beyond this point, iridial cells showed no expression. No other intraocular structures demonstrated the expression. The 49C antigen was highly expressed in the renal proximal tubular cells of the kidney and in the acinar cells of the pancreas data not shown. The purification was independently performed four times with high reproducibility. The Partial amino acid sequencing of the A homology search showed that five peptide sequences were highly homologous to sheep MDP 23 Fig.

    Activity was evaluated from the fluorometric detection of D-Phe released from the substrate Gly-D-Phe. The amount of D-Phe in the supernatant of the homogenate of the ciliary process increased during the incubation period, and the release rate of D-Phe between 5 and 30 minutes was 3. The hydrolysis of Gly-D-Phe in the supernatant of the homogenate of ciliary process was slightly inhibited by 49C mAb.