Patricia N. Farnsworth

Calf lens neutral proteinase activity using calf lens crystallin substrates

Activity of the calf lens neutral proteinase was measured with the calf lens crystallin fractions and purified α-crystallin A and B chains. For all substrates, rates of hydrolysis were linear for at least 1 hr. With α-crystallin, the rate of hydrolysis began to level off after 1 hr, but continued for at least 48 hr at a slower rate. The β L -crystallin fraction was hydrolyzed at the greatest rate, followed in decreasing order by α and β H -crystallin. ψ-Crystallin was not hydrolyzed. The purified B chains of α-crystallin were hydrolyzed at a greater rate than the A chains or unfractionated α 2 -crystallin. In unfractionated α 2 -crystallin a higher rate of degradation of B chain compared to A chain was shown by SDS gel was not extensive, polypeptide products of hydrolysis were identified

Methylisocyanate and actin polymerization: the in vitro effects of carbamylation.

Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4 degrees C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.

An Apparent Small Cluster of Choroidal Melanoma Cases

Three choroidal melanomas were detected in a 2.5-year period in a small community of 3,592 persons. This small cluster represented an incidence about 20 times that expected (P = .0006). The community has an isolated water supply and very little industry. We determined the incidence of cancer in this and two adjacent communities and found no other unexpectedly high incidence. The three patients had no common exposures. Analyses of air and water from the involved community by mass spectroscopy, chromatography, and Ames (mutagenicity) tests were noncontributory. Nine of 60 mice given community water after weaning developed lens opacities eight to 16 months later; electron microscopy showed an abnormal monolayer of cells on the outer surface of the anterior lens capsule. The genesis of this monolayer was not clear. None of the 30 controls showed such lesions.

Morphological studies of an ion-dependent perinuclear cataract model

The perinuclear region of the rabbit lens is susceptible to alterations in the ionic composition of incubation medium. Rabbit lenses and a comparable cell type, red blood cells, were stressed during ex vivo incubations in isotonic modified Earles medium with 131 mM NaCl replaced by either 232 mM sucrose or 131 mM choline chloride at pH 7.2 (normal) or 9.2. Our parallel NMR study revealed that these experimental media maintain normal intracellular pH and phosphorus metabolite levels. The present study demonstrates that lens transparency, normal fiber cell ultrastructure and volume were maintained in either sodium chloride or choline chloride containing media at normal or elevated pH. Similarly, normal morphology, mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC), 86.8 +/- 0.03 micron 3 and 33.2 +/- 1.0 g dl-1, respectively, were maintained in red blood cells in either sodium chloride or choline chloride containing media. In sodium chloride deficient media at both normal and elevated pH the lens developed a nuclear cataract based on slit-lamp examination; however, SEM examination showed that fiber cell morphological abnormalities were confined to a narrow band, 50 micron wide, in the perinuclear region of the transition zone. Damage consisted of ruptured cell membranes and an absence of identifiable interdigitations with the combination of sodium chloride deficiency and elevated pH. The major abnormality produced during incubation in sodium chloride deficient medium at normal pH was the presence of numerous smooth-surfaced cellular protrusions along the vertices of the perinuclear fiber cells. In addition, the sodium chloride deficient medium, pH 9.2, produced a volume loss both in the lens and RBC (4.5 +/- 1.5% and 5.6 +/- 1.1%, respectively). The sodium chloride deficient medium, pH 7.4, produced no volume loss in the lens or red blood cells (MCV 86.0 +/- 0.05 micron 3). Further studies indicated that the cataract induced by sodium chloride deficiency (pH 9.2) is irreversible. The mechanism for perinuclear opacification due to ion deficiency remains to be elucidated.

Localization and quantification of intact, undamaged right-handed double-stranded B-DNA, and denatured single-stranded DNA in normal human epidermis and its effects on apoptosis and terminal differentiation (denucleation).

Quantification of two types of nucleic acids [double-stranded (ds-) and single-stranded (ss-) DNA] was performed to understand the distribution of DNA within the epidermal strata and to examine the effects of DNA structure on gene expression, viz., apoptosis and terminal differentiation. In addition, we examined the precise starting point of cell death within the epidermis (suprabasal layer); examined how DNA structure affects gene expression of melanocytes; and characterized the “transitional cells” located between the stratum granulosum and stratum corneum, viz., epidermal phase transition zone (EPTZ). Ultrasensitive anti-DNA antibody probes (ds-DNA, ss-DNA), the Feulgen reaction, histological stains (morphological characterization) and the terminal deoxyribonucleotidyl transferase (TUNEL) assay (apoptosis) were used to characterize cell death in normal human epidermis. This study characterized, for the first time, the deterioration of right-handed ds-B-DNA and the increase in denatured ss-DNA during epidermal maturation. For the first time, this approach also allowed for the quantitative and qualitative characterization of DNA content and structure in all epidermal strata, using anti-ds-B-DNA and anti-ss-DNA antibodies. In order to improve the retention and quality of DNA, a novel histotechnological processing procedure was used. The results indicate that the largest decline in DNA occurred within the stratum granulosum, followed by the EPTZ, and the stratum spinosum. Not all epidermal nuclei lost DNA, indicating two differentiating keratinocyte pathways, viz., apoptotic and non-apoptotic. Both pathways united in the stratum granulosum. These results suggest that keratinocyte terminal differentiation and apoptosis are distinct cellular events, cell death begins earlier than expected, and molecular epidermal events take place in a gradual and orderly manner within keratinocytes. During maturation, ds-B-DNA decreases as ss-DNA increases. Therefore, during differentiation of keratinocytes, both DNA content and DNA structure are altered.

Procion Yellow Fluorescent Microscopy: A New Method of Studying Arterial and Venous Pathology:

A new means of studying vessel wall pathology is presented. This technique utilizes Procion yellow, a vital fluorescent dye, to stain and delineate the connective elements, matrix, and elastic tissue within the arterial wall. In addition, penetration of the dye into cells provides evidence of membrane pathology. A canine model was used, and clamped segments of femoral artery were stained and examined. The appreciation of this approach for the examination of the vessel wall shows alterations in tissue elements that have not been reported previously. This technique makes possible a unique means of studying connective tissue elements as well as membrane integrity.