Helen H. Hess

A linear Lowry--Folin assay for both water-soluble and sodium dodecyl sulfate-solubilized proteins.

Abstract The protein method of Lowry, Rosebrough, Farr and Randall was modified to give a linear standard curve of absorbance versus μg of bovine serum albumin at 650 and 750 nm wavelengths (1 cm optical path) over the range up to 50 μg protein per ml and an absorbance of 1.1. This was achieved mainly by using a high concentration of Folin-phenol reagent, added rapidly in a volume that was large relative to the final volume. Sodium dodecyl sulfate (SDS) incorporated in the test did not change the albumin standard curve, and linear results were obtained with water-insoluble membrane proteins (myelin proteolipid and rhodopsin) solubilized by SDS.

Cataracts in the Royal College of Surgeons rat: evidence for initiation by lipid peroxidation products.

The Royal College of Surgeons (RCS) rat has been extensively studied as a model system for inherited retinal degeneration. As in a number of human retinal degenerative diseases, posterior subcapsular cataracts (PSC) are associated with the retinal changes. It has been hypothesized recently that such cataracts may be initiated by toxic products generated by the peroxidation of polyunsaturated lipid components from degenerating photoreceptor outer segments. In the present study, the possibility that such a mechanism might be responsible for cataract initiation in the RCS rat has been investigated. The degeneration of the rod outer segments (ROS) occurs rapidly in these animals, beginning a few weeks after birth. Due to the failure of the retinal pigmented epithelium to phagocytize normally, ROS degeneration is accompanied by an accumulation of debris in the eye. During the brief period of maximal debris accumulation there is a marked increase in lipid peroxidation products in the vitreous. Cataract formation is correlated temporally with these events, becoming evident immediately following the time during which peroxidation products are present in the vitreous. In addition, the primary damage detected in the RCS lenses is an increase in the passive permeability of the lens membranes. Similar lens damage has been found in studies in which normal rat lenses were exposed to degenerating ROS in vitro. These findings are consistent with the hypothesis that cataracts in the RCS rat may be initiated by toxic lipid peroxidation products.

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.

The high calcium content of retinal pigmented epithelium

Abstract The concentration of calcium was measured in isolated retinal pigmented epithelial cells, erythrocytes, whole retina, and rod outer segments of bullfrogs. The tissues were solubilized in an organic base and calcium was determined by flameless atomic absorption spectrophotometry. The buffer used in preparation of tissues contained 0·2 m -sucrose, sodium phosphates to give a pH of 6·5, and the calcium concentration was 2·5 μ m by analysis. The calcium concentration was found to be 15 mmol/kg wet weight in pigmented epithelial cells, 0·05 in the nucleated red cells, 0·7 in dark-adapted whole retina, and 0·6 in dark-adapted rod outer segments (equivalent to 0·25 mol Ca/mol rhodopsin). The calcium in the pigmented epithelium exceeds that in muscle and is as great as that in platelets, which have been considered to be high calcium cells in which calcium plays a role in several cell functions. Possible functions of calcium in pigmented epithelium were discussed.

Ultrastructural demonstration of calcium in retina, retinal pigment epithelium and choroid.

Abstract Calcium has been demonstrated ultrastructurally in the retina, pigment epithelium and choroid of the frog using the potassium pyroantimonate (KPA) technique. Optimum tissue preservation was obtained with 2% potassium pyroantimonate in 1% osmium tetroxide at pH 9·2. With this fixative, electron-dense precipitate was found within the discs of the rod outer segments, as a dense band of granules along Bruchs membrane, in the extracellular space of the choroid, and to some extent within the nuclei and mitochondria of all cells. The precipitate has been identified as calcium antimonate by the use of EGTA pre-chelation and with the EMMA-4 electron microprobe. The possible significance of calcium in the various locations demonstrated by the KPA technique is discussed. This study establishes the specificity of the technique in demonstrating calcium in these tissues and suggests future applications in evaluating changes in the amount or location of this cation with both physiologic and pathologic processes.

Prevention of cataracts in pink-eyed RCS rats by dark rearing

Royal College of Surgeons rats have hereditary retinal degeneration and associated posterior subcapsular opacities (PSO) of the lens, detectable by slitlamp at 7-8 postnatal weeks in both pink- and black-eyed rats. The retinal degeneration is intensified by light, especially in pink-eyed rats. A fourth of pink-eyed rats developed mature cataracts by 9-12 months of age, but black-eyed rats whose retinas are protected from light by pigmented irises and pigment epithelium rarely have mature cataracts (3% or less), indicating light may be a factor in cataractogenesis. Prior work had shown that dark rearing reduced the rate of retinal degeneration in pink- but not black-eyed rats, but cataracts were not studied. In the present work, pregnant pink-eyed females were placed in a darkroom 1 week before parturition. Pups were removed over intervals at 20-85 postnatal days for: (a) microscopic study of fresh lenses and of fixed, stained retina and lens, and (b) counts of cells mm-2 of the web-like vitreous cortex after it had been dissected free. The macrophage-like cells are a quantitative index of immune reaction to retinal damage. At 50-53 postnatal days, in pink-eyed cyclic light reared RCS, the mean number of macrophages was 4.6-fold that in congenic controls, but in those that were dark reared it was only 1.4-fold. This was less than the increase in cyclic light reared black-eyed RCS (2.3-fold that in congenic black-eyed controls). Total absence of light reduced retinal degeneration and the number of macrophages, and prevented PSO detectable microscopically.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of liposomes with a phospholipid ratio similar to that of retinal rod outer segment membranes: Interaction with opsin and other proteins

Abstract Liposomes have been prepared with a phospholipid ratio and a cholesterol content similar to that of the outer segments of rod photoreceptor cells. Studies were made of the appearance, electrophoretic mobility and sodium leakage rates of the liposomes, with and without the inclusion of protein (albumin, myoglobin, ribonuclease, lysozyme, proteolipid or opsin). The findings indicate that although the appearance and properties of these liposomes closely resemble those of other liposomes reported in the literature, the liposomes become markedly changed in appearance, electrophoretic mobility and sodium permeability by the addition of opsin. These data emphasize the hydrophobic nature of the opsin-lipid interaction and suggest an ability of opsin to alter the ion leakage properties of a rod outer segment membrane.