Dietary restriction retards age-related loss of gamma crystallins in the mouse lens.

The soluble crystallins in lenses from diet-restricted and control mice of diverse ages (2, 11, or 30 months) were studied by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained with both methods suggest that dietary restriction decelerates age-related loss of soluble gamma crystallins.

Assignment of the rhodopsin gene to human chromosome 3.

The human rhodopsin gene has been assigned to human chromosome 3 through the use of a mouse DNA probe and human/mouse somatic cell hybrids.

Immunocytochemical localization of glycerol-3-phosphate dehydrogenase in rat oligodendrocytes.

In this study, two indirect immunoperoxidase staining procedures were used to investigate the cellular localization of rat brain glycerol-3-phospate dehydrogenase (EC 1.1.1.8;GPDH). At the light and electron microscopic level, we found that the use of monospecific rabbit antibodies to GPDH consistently resulted in the specific staining of only one glial cell population. GPDH-positive cells in perineuronal, interfascicular and perivascular positions were identified as oligodendrocytes by classical morphological criteria. The specificity of GPDH antigen-antibody reaction was determined by qualitative and quantitative immunochemical methods and by imunocytochemical controls for immunologic and methodologic sources of nonspecific reaction product. The illustrative data from this study serve to qualitatively define GPDH as a biochemical marker for oligodendrocytes in rat central nervous tissue. In view of the fact that the synthesis of rat brain GPDH is specifically regulated by glucocorticoids, the positive results obtained in this study further warrant the interpretation that rat oligodendrocytes are target cells for glucocorticoids.

Glycerol phosphate dehydrogenase in developing retina of normal and mutant mice.

The specific activity of glycerol phosphate dehydrogenase (GPDH; E.C. 1.1.1.8) was determined in the retinas of C57BL/6J mice from 2 to 45 days after birth. Comparisons were made with age-matched mice congenic to C57BL/6J and homozygous for the autosomal recessive mutations for retinal degeneration (rd) and light ear (le). The total activity of GPDH in the developing retina was similar in each strain throughout development. However, following the loss of photoreceptor cells in the rdle mice, the specific activity of GPDH increased to approximately twice the level of that in the controls. These data suggest that GPDH is absent from the photoreceptor cells and that its expression in the remaining retinal cells is unaffected by the loss of the photoreceptor cells. Retinal GPDH does not exhibit the eight-to ten-fold increase seen during development of the brain, but instead remains essentially constant during this time period, and in adult animals the specific activity of GPDH in the retina is about 20-fold less than it is in the brain. Neither mouse retinal GPDH nor brain GPDH is affected by adrenalectomy, suggesting that they are not regulated by glucocorticoids. The major form of GPDH in the retina is distinct from the major form in mouse brain since immunotitration experiments demonstrated that 70-80% of retinal GPDH does not react with anti-GPDH IgG, whereas the reverse was true for the brain enzyme.

Soluble retinal proteins associated with photoreceptor cell death in the rd mouse.

In the mouse, the homozygous presence of the rd gene results in the genetically programmed death of the photoreceptor cells of the retina. Using congenic strains of mice and a novel, sensitive, immunological approach for visualizing unique retinal proteins, we identified four bands of protein whose concentrations are regulated by the homozygous presence of the gene for retinal degeneration. Since these proteins (with apparent molecular weights of 23, 33, 55, and 69 kD) are present in normal adult mouse retinas and absent from rodless retinas, and from other mouse non-retinal tissues including brain, heart, kidney and liver, the data support the identification of these proteins as being retina specific. These proteins are not peculiar to the normal mouse retina; but rather, all four (23, 33, 55 and 69 kD) are common to rat retina; three (23, 33, and 55 kD) are common to bovine retina; and presently at least two, 23 and 69 kD, are clearly detectable in normal, adult human retina. The temporal appearance and disappearance of the four retinal specific protein bands coincide with the morphological maturation and degeneration of the photoreceptor cell population. Collectively, the present data suggest that one or more may be photoreceptor specific. These observations present the first step in the identification and characterization of specific soluble proteins correlated with the biochemical phenotype of the rd gene and the death of photoreceptor cells of the retina.

A biomolecular approach to the study of the expression of specific genes in the retina.

The central nervous system contains a number of populations of neurons that are morphologically and functionally distinct. To study the genes responsible for the development and maintenance of proteins with unique structural and biochemical properties, polyspecific antisera were produced against normal mouse retinal proteins and used in combination with the rd (retinal degeneration) neurological mutant of the mouse to immunologically identify specific retinal proteins. Western transfer analysis of the proteins present in normal and in mutant retinas identified three classes of neural proteins: those found only in normal retina; those found in normal and in photoreceptorless retinas but not in other tissues; and those found in both normal and mutant retinas, as well as in brain, but not elsewhere. Some of these class 1 proteins were shown to be present in the retinas of other species, including humans, suggesting their importance in the process of vision. The poly A+ RNA was isolated from the retinas of normal mice and used to generate a cDNA expression library in lambda gt-11. This library was screened with polyspecific antisera absorbed with the proteins present in mutant retina, and a number of immunologically positive plaques were cloned. Four of these were shown to code for rhodopsin, the major visual protein in mammalian retinas. The approach described is applicable to other systems in order to generate specific immunological and recombinant DNA probes for examining the expression of specific genes during development and differentiation.

Isolation of cells from rabbit renal proximal tubules by using a hyperosmolar intracellular-like solution.

A novel method of isolation of cells from rabbit kidney proximal tubules by using an intracellular-like solution (ICS) and gentle mechanical agitation in the absence of enzymes or chelators is described. Metabolic and functional characteristics of these cells were studied after washing and resuspension in modified Hanks medium, and the results were compared with those obtained in cells similarly prepared in extra-cellular-like solution (ECS). Trypan Blue exclusion and protein content were not different between the two preparations. However, oxygen consumption, ATP content and time- and concentration-dependent rates of uptake of phosphate, alpha-methyl glucoside and L-alanine were severalfold higher in cells prepared in ICS. Na+-dependent uptake of these solutes was 95% and 80% of total uptake in cells prepared in ICS and ECS respectively. Maximum transport rates (Tmax.) of phosphate, alpha-methyl glucoside and L-alanine were significantly higher in cells prepared in ICS. We propose that the use of ICS in the isolation procedure would yield a functionally more viable cell preparation, and therefore provides an ideal model for transport and metabolic studies at a cellular level.

Evidence for glucocorticoid target cells in the rat optic nerve. Hormone binding and glycerolphosphate dehydrogenase induction.

Biochemical evidence suggests that neuroglia are responsive to glucocorticoids, yet previous studies of glucocorticoid localization have typically failed to demonstrate significant uptake by neuroglial cells. To further investigate this problem, we measured glycerol-3-phosphate dehydrogenase (GPDH) activity and glucocorticoid receptor binding capacity in normal rat optic nerves and in those undergoing Wallerian (axonal) degeneration. Binding studies were also performed on hippocampus and anterior pituitary for comparison purposes. Normal optic nerve preparations possessed a high level of GPDH activity that was glucocorticoid-inducible and that increased further following axonal degeneration. Antibody inactivation experiments demonstrated the presence of more enzyme molecules in the degenerating nerve preparations. correlative immunocytochemical studies found GPDH-positive reaction product only in morphologically identified oligodendrocytes, a result that is consistent with the previously reported localization of this enzyme in rat brain. Optic nerve cytosol fractions displayed substantial high-affinity binding of both dexamethasone (DEX) and corticosterone (CORT) that, like GPDH, was elevated approximately two fold in degenerating nerves. Finally, in vivo accumulation of [3H]DEX and [3H]CORT by optic nerve and other myelinated tracts was examined using nuclear isolation and autoradiographic methods. Although neither steroid was found to be heavily concentrated by these tissues in vivo, a small preference for DEX was observed in the nuclear uptake experiments. These results are discussed in terms of the hypothesis that glial cells are targets for glucocorticoid hormones.