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Biomedical subjects

J I Clark

Publications and source records attributed to J I Clark.

At least 73 records · Page 4Linked to original sources
PubMed1992

Periods of oscillatory growth in developing ocular lens correspond with cell cycle times.

Lens growth rates oscillated throughout neonatal rat lens development. Changes in weight, soluble protein content, and cell division were quantitatively analyzed between birth and 26 days of age. Fourier analysis of daily changes in total weight and soluble protein content identified reproducible periodicities of 50-57 hours. Shorter periodicities of 14-18 hours and 18-21 hours were found by analyzing changes in lens dry weight/6 hours and cell division/7 hours. Changes in dry weight and cell division also contained a second periodicity of 36 hours and 30-43 hours, respectively. The periods of observed oscillations corresponded to the length of stages of the lens epithelial cell cycle. Oscillating growth rates during development emphasize the importance of oscillatory patterns during lens epithelial cell proliferation, differentiation, and tissue development.

Animals↗
PubMed1991

A quantitative evaluation of Fourier components in transparent and opaque calf cornea.

Fourier transform methods were applied to STEM (scanning transmission electron microscopy) images to detect and quantify the subtle differences between the structure of normal transparent calf cornea and opaque calf cornea. In order for a tissue to be transparent, it can scatter or absorb only a small amount of light. Light scattering is minimized when the principal Fourier components of the spatial fluctuations in the index of refraction have wavelengths which are small relative to the wavelength of light (Benedek, 1971). Corneal opacity was produced as a result of high intraocular pressure (100-150 mmHg) when liquid was injected into calf eyes (0-2 weeks old). Pressurization created large structural defects and slight disruptions in the organization of the collagen fibers. Although the fiber organization appeared similar in the micrographs of both opaque and transparent corneas, Fourier analysis of STEM images collected at 50K magnification identified statistically significant differences. Far fewer Fourier components with wavelengths in the light scattering range (200-1100 nm) were observed in the transparent corneas than in the pressurized corneas as predicted by Benedek's theory. It was of interest that corneas treated with 100% glycerol prior to pressurization remained transparent at high intraocular pressures, possibly because glycerol stabilized the structure of the corneas and maintained a uniform index of refraction across the corneal stroma. The results demonstrate the effectiveness of Fourier analysis in detection and quantification of slight changes in structure at the electron microscopic level.

Animals↗
PubMed1991

A quantitative analysis of transparency in the human sclera and cornea using Fourier methods.

Cellular microstructure observed by scanning transmission electron microscopy (STEM) was analysed using Fourier methods. Fourier components of the density fluctuations in the sclera and the cornea of the human eye were quantified. The results show that the Fourier components responsible for the opacity of the sclera have sizes of the order of the wavelength of visible light. In the sclera, approximately 54% of the spectral energy of the density fluctuation falls in the range of 200-1100 nm (scattering range). In the cornea, approximately 24% of the total spectral energy falls in this range. The predominant Fourier components of the density fluctuations in the opaque sclera are approximately 300 nm in wavelength, whereas those of the transparent cornea are approximately 80 nm in wavelength. This method will be useful in quantitative analysis of microstructural changes accompanying biological phenomena such as normal development of transparency in the human lens, and abnormal loss of transparency during cataract formation.

Cornea↗
PubMed1990

A new method for study of normal lens development in vitro using pulsatile delivery of PDGF or EGF in HL-1 serum-free medium.

A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens transparency. In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing 15 micrograms/ml insulin plus 1-2 ng/ml BB platelet-derived growth factor (PDGF), or 5-7 ng/ml epidermal growth factor (EGF) allowed similar growth in vitro, during the same time period. Normal lens growth occurred in culture when fresh medium was delivered to lenses as a pulse every 4-6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF stimulates lens growth during development in vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation, differentiation, and receptor regulation in a developing tissue.

Animals↗
PubMed1990

31P nuclear magnetic resonance and laser spectroscopic analyses of lens transparency during calcium-induced opacification.

The authors studied the relationships between lens phosphate metabolites and transparency in (1) normal intact calf lenses, (2) whole-lens homogenates, (3) cortical homogenates, and (4) cortical homogenates that were opacified by the addition of calcium. Transparency was measured with the use of laser spectroscopy. Phosphate metabolites and pH were measured with the use of phosphorus nuclear magnetic resonance (31P NMR) spectroscopy. 31P NMR spectra of fresh whole-lens homogenates and fresh intact lenses were nearly identical. In fresh tissue, adenosine triphosphate (ATP) accounted for 59%, 55%, and 61% of the total phosphorus detected in intact lenses, whole-lens homogenates, and cortical homogenates, respectively. As ATP decreased over time to undetectable levels, no loss of transparency was measured, indicating no relationship between ATP levels and lens opacification. In contrast, the authors found that the loss of transparency in cortical homogenates produced by additions of 5, 10, and 20 mM calcium chloride was associated with increased levels of sugar phosphates and glycerol phosphorylcholine and with decreased levels of inorganic phosphate. Loss of cortical transparency was associated with both increases or decreases in pH from the normal value.

Adenosine Triphosphate↗
PubMed1989

Relaxometry of lens homogenates. II. Temperature dependence and comparison with other proteins.

We have extended our earlier work (C.F. Beaulieu, J.I. Clark, R.D. Brown III, M. Spiller, and S.H. Koenig, Magn. Reson. Med. 8, 45 (1988] on the magnetic field dependence of 1/T1 (NMRD profiles) of calf lens nuclear homogenates, at 25 degrees C, to 5 degrees C, and to other protein systems as well. These include concentrated solutions of myoglobin and bovine serum albumin, both globular proteins, the first compact and roughly spherical, the other extended, flexible, and with weak internal bonding; chicken lens homogenate, for which the dominant crystallins (lens proteins) are approximately 70% alpha-helical compared with calf crystallins, which are essentially all beta-sheet; and hen egg white, both native and heat-denatured. Our earlier conjectures regarding a reversible change in protein organization of the calf lens crystallins as a function of solute protein concentration is given added support. Our findings suggest that cytoplasmic homogenate can be characterized as a heterogeneous and polymorphic solution of crystallins. At high concentrations the NH moieties of the protein backbone become accessible to solvent with water (not NH proton) exchange rates greater than 10(4) s-1. This conclusion is based on two aspects of the observed NMRD profiles. At low crystallin concentration, the profiles of calf and chicken lens homogenates are similar in form to those of myoglobin and native hen egg white, a form that has been studied previously for a range of diamagnetic globular proteins and has been demonstrated to arise from the rotational thermal motion of the solute molecules. At high crystallin concentrations, the NMRD profiles of the lens homogenates develop a monotonic background (high rates at low fields), much like that of the heat-denatured egg-white sample and those of most tissues. In addition, there is a set of peaks in the central part of the profiles of the concentrated crystallins, seen also in the denatured egg white and some tissues but not in the myoglobin sample, which is known to arise from cross-relaxation interactions between the water protons and (through the intermediary of the NH proton) the 14N quadrupolar levels. The magnitude of these peaks, which is larger by an order of magnitude for native calf lens homogenates than for any tissue, requires that the majority of the NH moieties be accessible to water. Finally, going to 5 degrees C for the native calf lens homogenate takes the sample below the temperature of reversible phase separation, and it becomes opaque.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

From E.M. microprobe analysis to NMRD studies of the lens.

We describe how electron microscope X-ray emission spectroscopy (XES) was the basis for recent studies of lens cytoplasm using proton nuclear magnetic resonance, NMR, relaxation methods. Although electron microscopy and NMR are very different techniques, the phosphorus distributions that were observed in whole lens using XES led to studies of proton relaxation rates in lens cytoplasm. Proton NMR is sensitive to water-protein interactions that exist in transparent lens cytoplasm. The cytoplasm is transparent because the interactions produce Fourier components in the density fluctuations of lens cytoplasm that remain small relative to the wavelength of light (1). New information on the interactions that are involved in the development and maintenance of transparent cytoplasm may be obtained using methods of proton NMRD (2).

Animals↗
PubMed1989

Effect of radioprotective agents on X-ray cataracts.

The effect of some protective agents on cataract development is briefly reviewed and new evidence is presented on the efficacy of a phosphorothioate compound (Amifostine) in inhibiting the development of X-ray-induced cataract. Morphological studies showed that at the end of 4 months, lenses from X-irradiated rats which had not received any drugs showed liquefaction in the equatorial region and at the posterior pole, as well as a marked swelling of the fibers in the anterior cortex. Animals which received 1.16g/kg of WR77913 showed considerable protection against the development of radiation induced cataracts with morphological changes in the lens being less severe than in animals receiving no drugs. When animals were treated with 0.5g/kg of Amifostine (WR2721) the lenses showed much greater protection against cataract development than with WR77913. Amifostine appears to be more effective than WR77913 in inhibiting X-ray-induced cataract development.

Amifostine↗
PubMed1988

Growth and transparency in the lens, an epithelial tissue, stimulated by pulses of PDGF.

The rat lens undergoes dramatic growth during early postnatal development. Lens weight increased by a factor of 23 in 26 days. Growth rate per day oscillated between 0 and 87 percent. A new culture system was designed to study the oscillations in growth during development. Lens growth and transparency in vitro required pulsatile delivery of platelet-derived growth factor (PDGF) in HL-1 serum-free medium. Continuous delivery of HL-1 medium with PDGF or pulsatile delivery of HL-1 medium without PDGF resulted in lens opacity and no growth. These results provide direct evidence that PDGF stimulates an epithelial tissue and that oscillations in growth occur during normal development of the rat lens.

Animals↗
PubMed1988

Structure of WF-3681, 3-(2,5-dihydro-4-hydroxy-5-oxo-3-phenyl-2-furyl)propionic acid.

C13H12O5, Mr = 248.24, monoclinic, C2/c, a = 18.757 (8), b = 7.282 (2), c = 17.511 (8) A, beta = 91.20 (3) degrees, V = 2391 (3) A3, Z = 8, Dx = 1.379 Mg m-3, lambda(Cu K alpha) = 1.54178 A, mu = 0.859 mm-1, F(000) = 1040, T = 293 K. Final R = 0.054 for 1409 unique observed reflections. The molecule contains two planar regions which differ in orientation by 5.7 degrees. Distances from the carbonyl carbons to the center of the phenyl ring are not in the range found in the crystal structures of other potent aldose reductase inhibitor molecules.

Aldehyde Reductase↗
PubMed1988

Relaxometry of calf lens homogenates, including cross-relaxation by crystallin NH groups.

We studied the magnetic field dependence of the longitudinal relaxation rates of water protons (1/T1 nuclear magnetic relaxation dispersion (NMRD) profiles) in transparent homogenates of calf lens. The samples included nuclear homogenates with total (heterogeneous) crystallin contents between 34% (v/v) (native) and 14% (diluted) as well as cortical homogenate, 21% (native) and 34% (concentrated). The NMRD profiles had two components: a monotonic dispersive component (analogous to that of both globular protein solutions and diamagnetic tissue) and "14N quadrupolar peaks." 14N peaks have never been reported for protein solutions, only for tissues and dehydrated proteins. These peaks occur between 0.5 and 5 MHz proton Larmor frequency and arise from interactions of solvent water protons with NH moieties of proteins. The 14N peaks in lens cytoplasm are very large and may correlate with the crystallin structure and interactions required to maintain short-range order and lens transparency. The monotonic and 14N quadrupolar components were largest in concentrated samples, but with different concentration dependencies. The dispersive components of samples above approximately 19% protein concentration had a fixed functional form, the amplitude of which varied with protein volume fraction, f, by the multiplicative factor f/(1 - f), suggesting spatial organization and dynamics of the solute proteins that are relatively independent of water content. In contrast, at concentrations less than 19%, the NMRD profiles are concentration dependent, indicating a dependence of the orientational relaxation time of the proteins on protein-protein interactions seen previously in other globular proteins at these concentrations. The 14N peaks are not resolved below approximately 19% protein and increase linearly with incremental volume fraction at protein concentrations above 19%. In addition, the 14N peaks in nuclear homogenates are 50-100% larger than those of cortical homogenates at the same concentrations. Partial substitution of solvent D2O for H2O decreases the peak heights, indicating that an exchangeable proton mediates the interaction between solvent protons and protein 14N nuclei.

Animals↗
PubMed1988

Calcium decreases transparency of homogenate from lens cortex and has no effect on nucleus.

We studied the effects of calcium on transparency in homogenates of cortical and nuclear cells from calf lenses. Calcium was mixed into samples of homogenate to final concentrations between 0 and 50 mM and the transparency of the calcium-treated homogenates was measured using laser transmittance. In the presence of 10 mM calcium, the transmittance of cortical homogenate decreased 50% while the nuclear homogenate lost less than 4% transmittance after 24 h at 37 degrees C. To better understand the contribution of cytoplasm and membranes to opacity, the nuclear and cortical homogenates were centrifuged to separate membranes from the cytoplasm. When 10 mM calcium was added to cortical homogenate which was then centrifuged, the transmittance of the membrane fraction decreased nearly 60%, while the fraction without membrane decreased only 10%. The strong effect of calcium on the membrane fraction was accompanied by an increase in specific gravity of membranes from 1.23 to 1.32. Ten- and 20 mM calcium had no effect on transparency of membranes or cytoplasm separated from nuclear homogenate, and 50 mM calcium produced only a slight opacity. The results indicate that an effect on membrane-protein interactions may be important in the loss of transparency produced by calcium in cells of lens cortex.

Animals↗
PubMed1987

Inhibition of phase separation by reagents that prevent X-irradiation cataract in vivo.

Galactose, and the phosphorothioates, WR-77913 and WR-2721, which inhibit cataract produced by X-irradiation, were evaluated for their effects on the phase separation temperature, Tc, of calf lens homogenate. The reagents were added to nuclear homogenate and the change in Tc per mol, dTc/dC, was measured. Galactose decreased Tc, -65 degrees C mol-1, WR-77913 decreased Tc, -28 degrees C mol-1 and WR-2721 decreased Tc, -76 degrees C mol-1. These are the first phase separation inhibitors that can be used in vivo to study the inhibition of lens opacification.

Amifostine↗
PubMed1987

Effect of calcium on the calf lens cytoplasm.

Opacification was induced in calf lens cytoplasmic extracts by addition of calcium. The sample turbidity was shown to increase with calcium molarity, incubation time and temperature and to decrease with the protein cytoplasmic concentration. Although this turbidity was enhanced when membrane fragments were left over in the cytoplasmic extracts, it did show up in the absence of any detectable vesicular fragment. Scattering techniques (X-ray and light) showed that the calcium-induced opacification is linked to enhanced light scattering, which results from the formation of additional scatterers, a few tens of nm in diameter. Additional structures were indeed visualized by freeze-fracture electron microscopy (FFEM): they appear as molecular clusters with diameters ranging from 20 to 90 nm, made of densely packed particles, with heterogeneous sizes. The turbidity expected from these clusters, as well as the expected variation of turbidity with cytoplasmic concentration, was calculated to be in agreement with the measurements. When compared with cold cataract, these results illustrate that similar opacities may result from completely different biophysical mechanisms.

Animals↗
PubMed1987

Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.

Animals↗
PubMed1987

Effect of aspirin and vitamin E on phase separation in calf lens homogenate.

Vitamin E (d-alpha-tocopherol) and aspirin were evaluated for their effects on the phase separation temperature, Tc, of calf lens cytoplasm. Vitamin E and aspirin were added to nuclear homogenate and the change in Tc was measured. Aspirin had no measurable effect on Tc. Vitamin E lowered Tc at a rate of -22 degrees C M-1 l-1. The results suggest that the effects of aspirin on cataract are unrelated to phase separation and that vitamin E may act on the phase separation in precataractous lens cells.

Animals↗
PubMed1986

Radioprotection against cataract formation by WR-77913 in gamma-irradiated rats.

Protection by WR-77913 against radiation-induced cataract formation in rats was observed following intraperitoneal (i.p.) administration of drug (1160 mg/kg) 15-30 min before exposure to 15.3 Gy of Cs-137 whole head irradiation. Control groups included irradiated, non-protected animals, and sham-irradiated aging controls. Protection was documented photographically and by analysis of eye lens constituents. All non-protected irradiated animals developed dense cataracts throughout the lens between 90-120 days post-irradiation, while WR-77913 protected animals developed minimal lens opacification through 200 days post-irradiation. No opacification in aging controls was seen. Lens protein analysis by Lowry assay and size exclusion HPLC showed radioprotected and aging control animals were similar in protein content, distribution of total and soluble protein, and degree of lens hydration. This contrasted significantly with cataractous lenses of non-protected animals. In cataractous lenses, the soluble protein concentration in the 25-43 K dalton range was approximately 10% of that found in radioprotected or aging control lenses. Hydration was substantially higher in cataractous lens. These results indicate that WR-77913 protects against lens opacification, protein insolubilization, and hydration in lenses of irradiated animals. Biodistribution studies with [S-35]-WR-77913 showed ocular uptake of drug within 15 minutes after i.p. injection, which remained relatively constant through 60 min. The relative order of drug concentration for individual eye components was: globe greater than total eye approximately equal to humor greater than lens. Although the mechanism of radioprotection observed remains to be elucidated, WR-77913 clearly prevents radiation-induced cataracts in rats. The potentially significant clinical use for this radioprotective compound is being investigated further.

Amifostine↗