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Biomedical subjects

J I Clark

Publications and source records attributed to J I Clark.

At least 91 records · Page 5Linked to original sources
PubMed1986

Inhibition of lens opacification in x-irradiated rats treated with WR-77913.

Radiation induced cataracts are models for studying mechanisms of lens opacification. WR-77913, S-3-(amino-2-hydroxypropyl) phosphorothioate (NCS-318809), has been identified as a radioprotective agent. Injection of WR-77913 (1160 mg/kg, i.p.) 15 to 30 min before exposure to 15.3 gray of x-irradiation inhibited rat lenses from developing radiation cataracts. Irradiated rats which did not receive the drug developed dense cataracts. Lenses from control rats which received no radiation remained transparent. Individual lenses were weighed, homogenized, and assayed for protein content using the Lowry method. The molecular weight distribution of soluble protein was determined by HPLC. Mean lens weights were: controls 48.2 mg; irradiated, drug-treated 45.9 mg; and irradiated, nontreated 45.5 mg. Protein accounted for over 40% of the lens weight in control and drug-treated rats and less than 20% for the nontreated cataractous lenses. Water was less than 60% of the lens weight in control and drug-treated rats and over 80% in cataractous lenses. Insoluble protein ranged from 12 to 17% of the total lens weight for each group. The ratio of insoluble to soluble lens protein was 0.40 for control, 0.65 for drug-treated, and 11.28 for cataractous rat lenses. HPLC confirmed a dramatic loss of soluble protein and a complete absence of protein below 25K daltons in cataractous lenses. Proteins below 25K daltons accounted for over 25% of the soluble protein in control and drug-treated rat lenses. WR-77913 stabilizes protein composition and appears to be an effective inhibitor of radiation cataractogenesis.

Amifostine↗
PubMed1985

A quantitative microprobe analysis of elements in cortical and nuclear cells of the calf lens.

The outer cortical cells in the calf lens remain transparent under conditions that produce opacity in central nuclear cells. The nuclear cells opacify by a mechanism of cellular restructuring that is associated with a cytoplasmic phase separation while cortical cells do not opacify by this mechanism. In this study the differences in elemental composition of nuclear and cortical cells were analyzed using X-ray emission spectroscopy (XES) of tissue that was prepared for scanning electron microscopy. It was necessary to develop special methods of fixation and dehydration to prevent significant distortion of lens tissue and minimize solubilization and redistribution of elements during the histological processing of the tissue. We calibrated the microprobe for the quantitative analysis using gelatin standards which contained known concentrations of sulfur, potassium, phosphorus, chlorine, and cesium. The standard curves were used to determine proportionality constants, which related the intensity of X-ray emission to the molar concentration of each element, and to determine the minimum detectable levels of each element. An important finding is that the intensity of the X-ray emission is dependent on sample density only at low protein concentration. At the high protein concentrations that exist in lens, the intensity is not affected by sample density.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Low temperature and acrylamide inhibit lens opacification caused by calcium.

We studied lens opacification of rabbit lenses in culture. Opacification was produced by maintaining rabbit lenses in medium containing calcium concentrations as high as 10 mmol/l and the opacity was measured using LASER spectroscopy. Increasing calcium caused significant opacification in lens cortex while the transparency in lens nucleus was unchanged by these concentrations. The cortical opacity was inhibited by low temperature and acrylamide. The maximum inhibition of opacity occurred at a concentration of 1% acrylamide and a temperature of 4 degrees C. The opacification due to a phase separation in nuclear cells was unaffected by these concentrations of calcium.

Acrylamide↗
PubMed1984

In vivo observation of lens protein diffusivity in normal and X-irradiated rabbit lenses.

Using the techniques of quasielastic light scattering spectroscopy we have studied in vivo the effect of X-irradiation on the diffusivity of lens proteins as a function of position in the lens and time after irradiation. The findings were compared with those for normal lenses in vivo. The experimentally observed correlation functions show marked changes in the protein diffusivity well before substantial biochemical changes take place.

Animals↗
PubMed1983

Phase separation and lens cell age.

Using laser light scattering spectroscopy, we are studying age-related changes in the microstructure of lens cytoplasm. We have established in animal models that one of the earliest identifiable stages in cataract development is the presence of a phase transition in the lens cytoplasm. As a result of the phase transition, the cytoplasm separates into microvolumes that differ in their protein concentration. These microvolumes scatter light and cause the lens to become opaque. This phase separation occurs in normal lens cells at a characteristic temperature, Tcat, which varies across the lens with the cell age. As the animal becomes older, the Tcat for the nuclear cells decreases to a value well below body temperature. In X-irradiated eyes, however, Tcat increases with animal age until the phase separation occurs at or near body temperature. At this point, a well-developed nuclear cataract appears. We are now attempting to understand the biochemical basis for the differences between Tcat of normal and Tcat of X-irradiated lens cells during the aging process.

Aging↗
PubMed1982

Phase separation of X-irradiated lenses of rabbit.

The phase separation temperature (Tcat) was studied as a function of time (age) after the administration of a single dose of radiation (2000 rad), which induces cataract in the rabbit lens. In the normal unirradiated lens, Tcat decreases linearly with age at a rate (DTcat/dt) approximately 2.2 degrees/week. In the irradiated lens, Tcat initially decreases with age much less than the normal lens, then rises sharply with age at the time of the appearance of opacity in the living rabbit eye. We suggest that the phase separation temperature may serve as a sensitive and early indicator of cataractogenic processes in the lens.

Animals↗
PubMed1982

Identification of the scattering elements responsible for lens opacification in cold cataracts.

Using both quasi-elastic light scattering spectroscopy and angular dissymmetry in the intensity of the scattered light, we examined the onset of turbidity for intact calf lenses and for isolated nuclear cytoplasm. In the case of the nuclear cytoplasm these measurements demonstrate the presence of two kinds of scatterers: small units of approximately 100-A radius and larger elements whose size is distributed around 1,500 A. As the temperature is decreased towards the cold cataract temperature, the intensity of light scattered by the small units stays almost constant while the intensity scattered by the large elements increase very strongly. The opacification of the lens cytoplasm produced by decreasing the temperature results principally from an increase in the concentration of the large scattering elements. For the intact nucleus the situation is qualitatively similar, but the mean size of the large scattering elements shows a more substantial increase than in the isolated cytoplasm as temperature is lowered towards the cold cataract temperature.

Animals↗
PubMed1981

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy.

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.

Animals↗
PubMed1980

The effects of glycols, aldehydes, and acrylamide on phase separation and opacification in the calf lens.

Glycols, aldehydes, and acrylamide inhibit the in vitro formation of cold cataracts in calf lenses. The inhibition is reversed when the glycols or the acrylamide are diffused out of the lenses. The inhibition by aldehydes or polacrylamide is irreversible, suggesting that the crosslinking reagents may permanently modify lens structure to prevent the development of lens opacities due to a phase transition.

Acrylamides↗
PubMed1978

Fluorescein colchicine. Synthesis, purification, and biological activity.

The synthesis of a fluorescent colchicine derivative permits the localization of colchicine-binding receptors in cells. Fluorescein colchicine (FC) was prepared by the addition of fluorescein isothiocyanate to deacetyl colchicine. The product, FC, was separated from the reactants by thin-layer chromatography (TLC). The purity of FC was demonstrated by TLC, UV spectral analysis, and analysis of the kinetics of photodecomposition. FC inhibited [3H] colchicine binding to purified brain tubulin. The biological activity of FC was compared to the activity of unlabeled colchicine on mitosis, motility, secretion, and myogenesis. The effects of FC were identical to those of unlabeled colchicine in all biological systems tested. The results demonstrate that FC may be substituted for colchicine in biological experiments without significant loss in specificity or effectiveness.

Colchicine↗
PubMed1975

Membrane-microtubule interactions: concanavalin A capping induced redistribution of cytoplasmic microtubules and colchicine binding proteins.

The relationship between microtubules and concanavalin A surface receptors during concanavalin A capping in primary cultures of rabbit ovarian granulosa cells was examined by electron microscopic and fluorescence labeling techniques. Cells treated with concanavalin A and hemocyanin at 4 degree or 37 degree and then incubated at 37 degree for 1 hr formed large juxtanuclear caps that were observed with shadow cast replicas of the cell surface. Thin section analysis of capped cells revealed an abundance of microtubules immediately beneath the cap which were arranged approximately perpendicular to the plane of the membrane. The capping process was unaffected by the antimicrotubule agents colchicine or vinblastine. Further, vinblastine treatment of capped calls resulted in the formation of numerous paracrystals that were confined to the cytoplasm underlying the capped region of the membrane; uncapped cells displayed paracrystals that were randomly dispersed in the cytoplasm. Exposure of fixed cells to fluorescein thiocarbamyl colchicine, which localizes colchicine binding proteins, revealed an intensely fluorescent region that corresponded to the cap; this staining pattern was absent in uncapped cells. These findings indicate that concanavalin A mediated capping modifies the cytoplasmic disposition of microtubules and colchicine binding proteins. Further, it is suggested that the capped region of the plasma membrane is a preferred site of microtubule polymerization.

Animals↗
PubMedDate not supplied

Characterization of the cellular microstructure of ocular lens using 2D power law analysis.

Power law analysis provides a quantitative method for characterization of spatial fluctuations in the cellular microstructure of the ocular lens. In the power law analysis, Fourier components of the spatial fluctuations are computed, and the relationship between the amplitude, A, and spatial frequency, f, of the components is defined by a power law function: [formula, see text]. The exponent of the function, beta, defines the scaling of the amplitude of the Fourier components as a function of spatial frequency. We performed two-dimensional power law analysis on electron micrographs of lens cells ranging from transparent to opaque. We identified two values of power law exponent, beta, for the spatial fluctuations of all lens cells, one for low- and a second for high-spatial frequencies. In the low-spatial frequency region, the value of beta was in the range of 0.53 to 1.33, for transparent and opaque cells. In the high-spatial frequency region, the value of beta increased from 2.78 for transparent lens cells to 3.60 for opaque lens cells. The power law analysis provides a new method for quantitative characterization of the spatial fluctuations in the microstructure of transparent and opaque lens cells.

Animals↗
PubMedDate not supplied

Digital image capture and quantification of subtle lens opacities in rodents.

A rapid, sensitive, and cost-effective method is reported for the subjective and objective documentation of subtle opacities in lenses of unanesthetized transgenic mice or selenite-injected rat pups as models for cataract formation. Animal eyes were dilated with eye drops and the animal was positioned in front of a Nikon FS2 photo slit lamp. Slit-lamp observations were recorded using a Canon Optura Pi digital video recorder. High-quality images of opacifying lenses were captured from the video and quantified using densitometry at progressive stages of opacification. In mice, targeted genomic deletion of the proteins CP49 (a lens-specific filament) or Six5 (a model for myotonic dystrophy) resulted in subtle cataracts that were easily recorded and quantified using this instrumentation. In rats, the early progressive changes leading to a dense nuclear opacity caused by selenite injection were easily documented using this instrumentation. Low-cost components combined with a conventional slit-lamp ophthalmoscope were used to capture high-quality images of selected stages of cataract formation for quantitative analysis using commercial software.

Animals↗