All procedures in this study conformed to the tenets of the
Declaration of Helsinki, the National Institutes of Health guidelines
on the care and use of animals in research and the ARVO Statement on
the Use of Animals in Ophthalmic and Vision Research. Human AH was
obtained at the time of either glaucoma surgery or cataract removal. An
institutionally approved human protocol governed the acquiring of the
human samples, and informed consent was obtained from all patients. AH
was obtained by paracentesis of the anterior chamber at the beginning
of the operation. Two samples from patients with primary open-angle
glaucoma and three samples from patients with cataract, but no history
of glaucoma were obtained. The AH was frozen at −70°C until use. To
determine whether the AH was contaminated with intracellular proteins
as a result of cells that were damaged during the paracentesis, a
commercially available assay for lactic acid dehydrogenase (LDH; Sigma,
St. Louis, MO) was performed on each of the AH samples. The assay
followed the manufacturer’s protocol, but the volumes were reduced to
allow the assay to be conducted in a 96-well plate. For gel
electrophoresis, after the human AH (0.75 μg) was thawed, it was
dried (SpeedVac system; Savant, Farmingdale, NY). The AH was then
reconstituted in sample buffer (62.5 mM Tris [pH 6.8]), 2% sodium
dodecyl sulfide [SDS], and 5% β-mercaptoethanol) and placed in
boiling water. Samples were run on 12.5% gels (PhastGels; Pharmacia
Biotech, Piscataway, NJ) and blotted onto nitrocellulose (BioRad,
Hercules, CA) as directed by the manufacturer’s protocol. Protein
standards (BenchMark Protein Ladders) were obtained from Life
Technologies (Grand Island, NY). The western blot analyses were
developed with a kit (Chemiluminescence Reagent Plus; NEN Life
Sciences, Boston, MA) after incubation with primary antibody to
myocilin
12 overnight at 4°C and incubation with
secondary antibody (Kirkegaard and Perry, Gaithersburg, MD) for 1 hour.
Chemiluminescence was detected using film (XAR; Kodak, Rochester, NY)
or with an image analysis system (Image Station 400; NEN Life
Sciences).
Bovine eyes were transported from the abattoir, and the AH was removed
within 5 hours of death. For column chromatography, the AH was
concentrated 10-fold (Centricon-10 concentrator; Amicon, Danvers, MA),
and sample was diluted 10-fold with water and reconcentrated. Samples
of up to 200 μl were applied to a gel filtration column (Zorbax
GF250; DuPont, Wilmington, DE) with a running buffer of 50 mM HEPES (pH
7.5), 0.2M NaCl, and 0.02% NaAzide. The flow rate through the column
was 330 μl/min. Fractions were collected each minute. Fractions were
concentrated on the concentrators, gel sample buffer was added, and the
samples were placed in boiling water. Samples were run on 12.5% gels
and western blot analysis developed according to the manufacturer’s
protocol (Pharmacia Biotech). The blotted gel was silver stained
according to the protocol.
Monkey AH was obtained at the time of death. The monkey AH was treated
in a manner similar to the bovine AH, with the exception that it was
not diluted with water after concentration.
To study the obstruction of microporous filters, the bovine AH was
first centrifuged at 100,000
g for 45 minutes. Between 1 and
1.25 ml of the supernatant was passed through a polycarbonate 0.2-μm
filter that had not been treated with polyvinylpyrrolidone (PVP;
Nucleopore, Pleasanton, CA). These PVP-free filters are
hydrophobic.
15 The filters were washed with 1 ml of
Dulbecco’s phosphate-buffered saline (PBS) after the aqueous had been
perfused through and then rinsed briefly in this saline. Ten filters
were placed in 1 ml of 0.1% Nonidet P40 (American Bioanalytical,
Natick, MA) overnight in the refrigerator. Samples of all the fractions
at each step of the process (uncentrifuged, centrifuged supernatant,
material perfused through the filter, and proteins eluted from the
filters) were concentrated (Centricon-10; Amicon) and then used in the
biochemical analysis.