Isolation and characterization of the crystallins of the normal and cataractous canine lens.

The lens crystallins were analyzed in normal dogs and Miniature offSchnauzer dogs with congenital cataract formation. There was an increase in the relative proportions of alpha and beta L-crystallin and a decrease in the beta H and gamma-crystallin with increasing age in the noncataractous lens. These trends were advanced in the age-matched cataractous lenses. "Advanced aging" trends were also noted in various polypeptide components of beta-crystallin. Specifically, the appearance of a 29K band as well as a reversal of the 26K to 27.6K ratio occurred at an earlier age in the cataractous lens than in the clear lens. Three subunits of approximately 19K, 20K, and 21.5K were present on SDS-PAGE for alpha-crystallin from the cataractous lens as opposed to only two of 19K and 21.5K from the clear lens. However, if the protein was not heated following resolubilization in buffer containing 2% SDS and 5% 2-mercaptoethanol, only two subunits of 20K and 21.5K were evident in both clear and cataractous lenses. The electrophoretic behavior observed for both alpha and gamma-crystallins did not appear to be age related.

Reduced, oxidized, and protein-bound glutathione concentrations in normal and cataractous lenses in the dog.

Amounts of reduced, oxidized, and protein-bound glutathione (GSH) were measured in normal and cataractous lenses of pups and adult dogs. Lenses from pups included normal lenses from clinically normal pups, clear lenses from Beagle pups bred for glaucoma, and congenital cataractous lenses from Miniature Schnauzer pups. Lenses from adults included normal lenses from normal mixed-breed dogs, congenital cataractous lenses from Miniature Schnauzers, and complete mature cataractous lenses from clinical patients of different breeds. Glutathione in the normal lenses from pups and adult dogs is predominantly reduced GSH; oxidized GSH is about 2.1% to 2.6% of the reduced GSH values. The reduced GSH values are lower in normal pups [7.08 mumoles/g (wet wt) of lens] than in adults [7.83 mumoles/g (wet wt) of lens]; reduced GSH values decrease further in cataract formation. The decrease in oxidized GSH values parallel those of reduced GSH, except in the advanced cataracts of clinical patients in which oxidized GSH [0.045 mumoles/g (wet wt) of lens] was 9% of the GSH values. The GSH bound to soluble and insoluble lens proteins of congenital cataractous Miniature Schnauzer pups was significantly (P less than 0.01 and P less than 0.02, respectively) lower per gram of protein than that in pups with normal lenses. However, the soluble and insoluble protein-bound GSH of congenital cataractous lenses of adult Miniature Schnauzers and lenses in clinical patients with mature cataracts [based on mumole of GSH/g (wet wt) of lens] were not significantly different (P greater than 0.05) from that in adult dogs with normal lenses.

Effect of fibronectin on the adhesion of an established cell line to a surface reactive biomaterial.

We have demonstrated that an established hamster cell line (NIL 8 M-2) will adhere to the bioceramic bioglass. The rate at which the NIL 8 M-2 cells assume a spread morphology on bioglass is density dependent and the morphology displayed by NIL 8 M-2 cells attached to bioglass is much more elongated than that displayed by NIL 8 M-2 cells attached to nonreactive glass. Precoating the bioglass with the plasma form of human fibronectin significantly reduces the density dependent nature of cell spreading. Coating the bioglass with fibronectin also reduces the time required for cell spreading and changes the morphology of the attached cells from an elongated to an extremely flattened shape. Our work raises the possibility that bone-implant adhesion might be improved by introducing molecules relevant to cell-substrate attachment into the biomaterial prior to implantation.

Variations of plasma enzymes in the pony and the dog after carbon tetrachloride administration.

Adult female dogs or pony mares were subjected to a nonlethal dose of CCl4 (0.5 ml/kg of body weight). Amounts of several plasma enzymes thought to be indicative of hepatic disease were monitored. Plasma enzymes alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase (ALP), arginase, gamma-glutamyltransferase (GGT), and iditol dehydrogenase (ID), as well as total plasma bilirubin, were determined in these animals before and after the administration of the CCl4. In the dog, GGT was not significantly increased, whereas ALP values were increased during days 1 to 6. In the pony, GGT was significantly increased during the entire course of the study, whereas ALP exhibited only small, transient (though significant) increases. Responses of ID, AST, and ALP were unremarkable when compared between the pony and the dog. Total bilirubin was significantly (P less than or equal to 0.05) increased from days 1 to 4 (pony) or days 5 to 8 (dog) after the CCl4 dose, but subsequently returned to or decreased below base-line values. Animals did not have evidence of icterus at any time. Seemingly, the dog and the pony are distinct clinical entities, and only the appropriate laboratory tests for each species should be used to provide information for the clinicopathologic evaluation of hepatic disease.

Nuclear envelope of Chinese hamster ovary cells. Re-formation of the nuclear envelope following mitosis.

We have developed a technique for isolating nuclei and nuclear envelope(s) (NE) from Chinese hamster ovary (CHO) cells which does not depend on the use of detergents to solubilize contaminating cytoplasm. In our procedure NE are prepared from purified nuclei by nuclease digestion and subsequent high salt-sucrose gradient centrifugation. The nuclei and NE fractions are free of significant contamination by other subcellular organelles as judged by electron microscopy and enzyme analysis. Examination of the peptide and glycopeptide composition of the NE fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a very complex coomassie blue staining profile with prominent bands in the 55 000-75 000 molecular weight range. Using this NE isolation technique, we have examined the breakdown and re-formation of the NE during a limited stage (late G2, M, and early G1) of the replicative cycle in synchronized populations of CHO cells. Our data demonstrate that a minimum of 60% of the early G1 NE protein and a minimum of 50% of the early G1 NE phospholipid were present in the cell during the preceding G2 phase of the cell cycle and were reutilized in the re-formation of the NE occurring during late M and early G1. Our evidence suggests that the vast majority of the newly synthesized peptides and glycopeptides of the NE which appear in the daughter NE are synthesized during the early G1 phase of the replicative cycle. Examination of the NE peptides by one-dimensional gel electrophoresis suggests that no reproducible changes in NE peptide composition can be correlated with specific phases of the cell cycle.

A new animal model with hyperbilirubinemia: the indigo snake.

An unconjugated hyperbilirubinemia has been observed in all species of normal indigo snakes. The plasma clearance of large organic anions such as sulfobromophthalein and unconjugated bilirubin was markedly delayed when compared to other snake species. Endogenous bile flow and biliverdin and bilirubin excretory rates and the excretion of bile pigments after a bilirubin load were measured in various snakes. The indigo snake represents a new animal model in which to study mechanisms important to hepatic anion uptake and biliary transport.

The effect of TLCK on transcription and its role in modifying cell growth.

The synthetic protease inhibitor N-tosyl-L-lysine-chloromethyl ketone (TLCK) acts to inhibit transcription when added to cell lines growing in vitro. This inhibition of transcription is most pronounced in transformed cells where TLCK is very toxic at concentrations as low as 25 mug/ml of culture medium. Non-transformed cells are more resistant to the effect of TLCK, requiring ten times more TLCK to produce a comparable inhibition of transcription. The effect of this protease inhibitor on transcription can be prevented by preincubation of the cells in reduced glutathione or cysteine; however, the cells can not be rescued from the effect of TLCK even if glutathione or cysteine are added to the culture medium within five minutes of the addition of TLCK.