James F. McGinnis

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.

The photoreceptor-specific 33 kDa phosphoprotein of mammalian retina: Generation of monospecific antibodies and localization by immunocytochemistry

The distribution in mouse retina of a 33,000 Da phosphoprotein (33 kDa) that complexes with the beta/gamma subunits of transducin (T beta gamma) and undergoes light-induced dephosphorylation was determined by immunocytochemistry. An antiserum containing antibodies for the 33 kDa protein and beta-transducin of mouse and bovine retinas was generated against the purified 33 kDa-T beta gamma complex from bovine retina. The antiserum reacts with beta-transducin derived from either 33 kDa-T beta gamma complex or transducin complex (T alpha beta gamma), but not with the alpha- or gamma-transducin. It also reacts with both the phosphorylated and unphosphorylated form of the 33 kDa-T beta gamma complex. Antibodies, monospecific for the 33 kDa and beta-transducin subunits respectively, were purified from the antiserum by immunoadsorption and used in immunocytochemical analysis of the respective antigens. The 33 kDa protein was found to be associated exclusively with the photoreceptor cells of the retinas, with the most intense staining in the inner and outer segments layers and lighter staining in the synaptic terminal layers. beta-Transducin also is found in the photoreceptors, but some T beta immunoreactivity exists within the inner plexiform layer. The specific localization of the 33 kDa protein together with its light-modulated phosphorylation suggest that the 33 kDa-T beta gamma complex is involved in light-regulated activities of the rod photoreceptor cells.

Isolation and analysis of the mouse opsin gene

We have identified three overlapping 5′‐truncated mouse opsin cDNA clones by immunologically screening a λgt11 retina expression library. Using one of the cDNA clones as a probe, we isolated a 5 kb genomic fragment that encompassed the complete coding sequence for mouse opsin. The coding region for opsin was interrupted by four introns positioned precisely as those previously described for other mammalian opsins. In contrast to the single major opsin mRNA in the bovine and human retina, Northern analysis of mouse retina RNA demonstrated the presence of at least five distinct species of polyadenylated opsin mRNAs. Their sizes ranged from 1.7 kb to 5.1 kb.

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S‐modulin, the CAR (cancer‐associated retinopathy) protein and recoverin, the Ca2+‐dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.

Chromosomal assignment of the recoverin gene and cancer-associated retinopathy

The deduced amino acid sequence of the recently cloned mouse 23kD photoreceptor cell-specific protein showed it to be identical to the recoverin protein and the CAR (cancer-associated retinopathy) protein. DNA sequence variants were found in the mouse recoverin gene (Rcvrn), and segregation analysis of restriction fragment length variants in recombinant inbred strains of mice assigned Rcvrn to mouse Chromosome (Chr) 11, between Sparc (3.7 map units) and Zfp-3 (2.3 map units). These results demonstrate a close linkage of recoverin to the tumor suppressor gene, Trp53. On the basis of these data, knowledge of the function of recoverin, and the characteristics of CAR, an experimentally testable model is presented to explain the molecular basis for CAR.

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.

HORMONAL EFFECTS ON DIFFERENTIATION IN NEURAL CULTURES

The data presented clearly demonstrate that the induction of glycerol phosphate dehydrogenase (GPDH) by cortisol in C6 cells is due to an increased rate of synthesis without alteration of the rate of degradation of the enzyme, thus resulting in a greater number of molecules. Similarly, we have shown by immunotitration, Ouchterlony double diffusion, gel permeation chromatography, pH optimun, heat lability and polyacrylamide gel electrophoresis that GPDH in the brains of normal (induced) and hypophysectomized (uninduced) rats is identical. Thus in vivo as in cell culture, the hormonal regulation of GPDH activity is brought about by a change in the number of molecules, not in their catalytic efficiency. Similar questions now arise about the mRNA for GPDH, i.e., the influence of hydrocortisone on its rate of synthesis and degradation and efficiency of its translation. Since brain GPDH is identical to the enzyme present in muscle and liver, the mechanism for the specificity of the induction is an intrinsic property of brain tissue rather than related to the enzyme itself such as the coding by different structural genes.

Structural analysis of mouse S-antigen

Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe. The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence. The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent. The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930. An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins. This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively. The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well. There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database. Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts. Peptides 3 and K, however, were more divergent. The short repeats within these peptides were conserved.

Purification and characterization of rat brain glycerol phosphatase dehydrogenase

Abstract Glycerol-3-phosphate dehydrogenase ( sn -glycerol-3-phosphate: NAD + 2-oxidoreductase, EC 1.1.1.8) was isolated from rat brain tissue and its physical and kinetic properties investigated. The purification method resulted in the separation of fractions enriched for six other brain proteins and this procedure is presented. The apparent Michaelis constant ( K m ) for dihydroxyacetone phosphate is 0.17 mM whereas the K m for l -glycerol-3-phosphate is 0.30 mM. Under the conditions of the assay, the pH optimum occurs at pH 7.15. The molecular weight determined by Sephadex G-100 chromatography is 72 000 whereas ultracentrifugation in a sucrose gradient yields a molecular weight of 75 000. Furthermore using an antiserum prepared against purified rat brain glycerol-3-phosphate dehydrogenase, it was shown that rat brain glycerol-3-phosphate dehydrogenase is lighter than rabbit muscle glycerol-3-phosphate dehydrogenase ( M r 78 000). The purified enzyme is extremely sensitive to p -mercuribenzoate, being demonstrably inhibited by 10 nM, although it is much less sensitive to N -ethylmaleimide and iodoacetate. The specific activity of the purified enzyme is 152 units/mg of protein, representing a 2800-fold increase in specific activity. Analytical acrylamide gel electrophoresis separates the purified enzyme into two bands of protein, each of which exhibits glycerol-3-phosphate dehydrogenase activity. The isozymes differ from each in charge and neither one represents an oligomeric form of the other.

Assignment of the rhodopsin gene to human chromosome three, region 3q21-3q24 by in situ hybridization studies.

By Southern analysis of restriction digests of genomic DNA from human-mouse somatic cell hybrids, we recently assigned the rhodopsin gene to human chromosome 3. Using in situ hybridization techniques and a mouse rhodopsin cDNA probe, we now show that the rhodopsin gene is on the long end of human chromosome 3 at region 3q21-3q24.