The development of interneurons in the chick embryo spinal cord following in vivo treatment with retinoic acid.

To investigate the role of retinoic acid (RA) in the development of interneurons in the spinal cord, we examined the expression of cellular retinoic acid binding protein type I (CRABP I). The earliest developing interneurons in the chick spinal cord can be divided into two major groups: circumferential (C) neurons and primitive longitudinal (PL) neurons. In brachial segments, both types of interneurons began to express CRABP I at stage (st.) 13+ of the V. Hamburger and H.L. Hamilton (1951, J. Morphol. 88:49-92) stage series, which is before the onset of axonogenesis. Subsequently, with the onset of axonal outgrowth, C neurons and PL neurons expressed CRABP I in their cell bodies, axons, and growth cones. The expression of CRABP I was developmentally regulated. CRABP I immunoreactivity gradually decreased after st. 36 (embryonic day [E] 10) such that no interneurons expressed this protein by E21. The transient expression of CRABP I during a period of intensive axonal growth suggested that RA may be involved in the development of interneurons. To test this idea, we implanted an all-trans RA-containing ion exchange bead into either rostral segments of the spinal cord at st. 12-13 or into caudal segments at st. 15-16, all stages that are well before the appearance of CRABP-I-positive neurons in these segments. In the RA-treated spinal cord, increased numbers of pyknotic cells were found predominantly in dorsal regions, presumably reflecting the death of neuroepithelial cells, C neurons and premigratory neural crest cells. Surviving C neurons in the RA-treated spinal cord extended their axons ventrally toward the floor plate as in control embryos. PL neurons also projected their axons rostrally or caudally in the RA-treated spinal cord, similarly to control embryos. However, the proportion of caudally projecting PL neurons was significantly increased in segments rostral to the RA-containing bead. These results suggest that RA may regulate the survival and axonal orientation (directionality) of subpopulations of spinal interneurons.

Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a).

Lipoprotein(a) [Lp(a)] has been measured in numerous clinical and epidemiological studies by a variety of immunochemical methods. However, little, if any, consideration has been given to the confounding effect of the size heterogeneity of apolipoprotein(a) [apo(a)] on the measurement of Lp(a). We developed three direct-binding enzyme-linked immunosorbent assays (ELISAs) with detecting antibodies of different specificities to evaluate the effect of apo(a) size on Lp(a) measurement. The three assays used the same monoclonal antibody to capture the apo(a)-containing particles and were calibrated (in nanomoles per liter) with a serum containing apo(a) with 21 kringle 4 domains. Using all three ELISAs, we measured Lp(a) in a group of 723 subjects selected to have a single apo(a) band, as determined by a high-resolution phenotyping system. Essentially identical results were obtained by the two methods that measured Lp(a) by use of either a polyclonal antibody against apo B or a monoclonal antibody against apo(a) that does not recognize the kringle 4 type 2 repeats. In contrast, the ELISA using a monoclonal antibody specific for apo(a) kringle 4 type 2 repeats overestimated Lp(a) concentration in samples containing apo(a) with more than 21 kringle 4 domains and underestimated Lp(a) samples containing apo(a) with fewer than 21 kringle 4 domains. Thus, these differences in Lp(a) values varied as a function of apo(a) size. We conclude that antibody specificity and apo(a) size heterogeneity can significantly affect Lp(a) measurements.

Biological variability of cholesterol, triglyceride, low- and high-density lipoprotein cholesterol, lipoprotein(a), and apolipoproteins A-I and B.

Biological variability is a major contributor to the inaccuracy of cardiovascular risk assessments based on measurement of lipids, lipoproteins, or apolipoproteins. We obtained estimates of biological variation (CVb) for 20 healthy adults and calculated the percentiles of CVb as an expression of the variability of CVb among individuals for cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein (apo) A-I, apo B, and lipoprotein(a) [Lp(a)] by four biweekly measurements of these analytes. The CVb for the group was approximately 6-7% for cholesterol, HDL cholesterol, apo A-I, and apo B; approximately 9% for LDL cholesterol; and 28% for triglyceride. However, for each analyte, there was a considerable variation of CVb among individuals. For all analytes except Lp(a), there was no relation between the individual's CVb and the analyte concentration. Lp(a) was inversely related to CVb, and there was a very wide variation in the CVb for Lp(a) among the participants, ranging from 1% to 51%. The number of independent analyses to perform to accurately assess an individual's risk for coronary artery disease should be determined on the basis of the individual CVb for a given analyte rather than the average CVb.

Identification of 34 apolipoprotein(a) isoforms: differential expression of apolipoprotein(a) alleles between American blacks and whites.

A total of 34 different apo(a) isoforms was identified in a population sample of 806 American Whites and 701 American Blacks by a high resolution SDS-agarose gel electrophoretic method followed by immunoblotting. Among the 1507 individuals tested, 79% revealed double-banded phenotypes and 21% single-banded phenotypes. The frequencies of the apo(a) isoforms differed between American Blacks and Whites (p < 0.001).

Retinal pigment epithelial cell transplantation into aging retina: a possible approach to delay age-related cell death.

Retinal pigment epithelial cells (RPE) from young, healthy rats were transplanted into older rat eyes to test whether aging changes could be delayed or arrested in an appropriate model. The Fischer-344 rat, a model for studying age-related photo-receptor cell death, was used as the recipient, and the Long Evans rat (6- to 8-day-old) was used as the donor of young RPE. Morphometric analyses at 3 and 6 months after RPE transplantation demonstrated a significant delay in age-related cell death in the outer and inner nuclear layers and in the outer and inner plexiform layers, as compared with the findings in controls. These results indicated the beneficial effects of RPE transplantation into the aging eye.

[Effect of neonatal retinal pigment cell transplantation on aged retinas].

The authors performed neonatal healthy retinal pigment epithelium (RPE) transplantation into older eyes to test whether aging changes could be deterred or arrested in an appropriate model. Fischer-344 rats, a model for studying age-related photoreceptor cell death, were used as the recipient and 6- to 8-day-old Long Evans rats were used as the donor of neonatal RPE. Isolated RPE cells were transplanted into the subretinal space in 24, 3-month-old Fischer-344 rats. The presence of healthy neonatal RPE cells significantly maintained the thickness of each layer as compared to the findings in nongrafted and sham operated controls at 3 and 6 months after the transplantation. The transplanted RPE cells saved the reduction of cell population caused by aging from 40% to 17% in the outer nuclear layer and from 33% to 8% in the inner nuclear layer at 6 months after transplantation.

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro.

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.

[A comparative study of distribution of 70 kD stress protein in the retina between the normal and retinal dystrophic rat].

The immunolocalization of 70 kD stress protein (SP70) was investigated in the retinal tissues of normal Sprague-Dawley (SD) rat and that of the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. From postnatal day 2 to 15, SP70 was present in the maturing retinal tissues of both rat strains. In the RCS rat retina of postnatal day 22, at the onset of retinal degeneration, SP70 was expressed in the retinal pigment epithelium (RPE). At postnatal day 40, immunostaining for SP70 was considerably reduced in the degenerating RCS retina. In the RCS retina at postnatal day 90, immunostaining for SP70 was completely lost except for the ganglion cells and the inner plexiform layers. These results suggested that, at the onset of retinal degeneration, the RCS retina may have a state of metabolic stress, which induced SP70 expression in the RPE. At the end stage of retinal degeneration, the immunostaining for SP70 was lost, suggesting the lack of production of SP70 in the degenerated retinal tissue.

[Distribution of 70 kD stress protein in the optic nerve. A comparative study between the normal and retinal dystrophic rat].

The immunolocalization of the 70 kD stress protein (SP70) was investigated and compared in the optic nerve of normal Sprague-Dawley (SD) rats and that of the Royal College of Surgeons (RCS) rat with inherited retinal dystrophy. At postnatal day 8, SP70 was present in the maturing glial cell bodies of both rat strains. At postnatal day 22, SP 70 was observed in the glial cell bodies and optic nerve fibers of both rat strains. At postnatal day 40 RCS rat, SP70 was diminished in the optic nerve fibers and glial cell bodies but was increased in the glial cell nuclei. This suggests that axonal transportation of SP70 from the retina may be reduced following retinal degeneration. In the SD optic nerve, the normal distribution of immunostaining for SP70 was preserved. Stress proteins are thought to play an important role in cellular development and survival mechanisms. It was suggested that optic nerve was damaged by the retinal degeneration in the RCS rat.

[Localization of somatostatin mRNA in the rat retina detected by in situ hybridization histochemistry].

In order to determine the localization of the mRNA encoding somatostatin in the rat retina, we studied Sprague-Dawley rats by in situ hybridization histochemistry. Radiolabelled oligodeoxyribonucleotides complementary for rat somatostatin mRNA were used in this study. Among the layers of the retina, we found specific labelling in the soma of some cells in the innermost and outermost laminae of the inner-nuclear layer and in the ganglion cell layer. These results indicate the major site of somatostatin synthesis within the rat retina.

Vitreoretinal surgical technique for transplanting retinal pigment epithelium in rabbit retina.

Transplantation of retinal pigment epithelial (RPE) cells has been proposed as a potential remedial procedure for previously untreatable retinal diseases. In this study, a vitreoretinal surgical technique was used to transplant pigmented RPE cells obtained from pigmented rabbits into the subretinal space of New Zealand White rabbits. At the time the animals were sacrificed, the retina was re-attached in all but 4 of the 24 experimental eyes. Histologically, by one week the transplanted RPE cells had formed a monolayer in patchy areas beneath the attached retina. By electron microscopy, RPE cells with prominent melanin granules were found attached to Bruch's membrane. Three weeks after transplantation, grafted RPE cells had formed apical microvilli and tight junctions with adjacent cells. The nucleus of the cells containing pigment had become oval, and their contact with Bruch's membrane appeared to be composed of bsal infoldings that were well formed. Our findings demonstrated the functional appearance of the transplanted RPE cells.

Cellular retinoic acid-binding protein in rat lacrimal gland.

We employed a monoclonal antibody to cellular retinoic acid-binding protein (CRABP) to assess the presence and localization of this retinoid-binding protein in the lacrimal gland of the rat. Immunoblots of extracts of rat lacrimal gland showed specific immunostaining of lacrimal CRABP in the region 14-16 kDa. Sections of rat lacrimal glands that were stained with anti-CRABP antibodies showed reaction product in the cytoplasm of the acinar cells. Retinoic acid may play a role in maintaining the proper function of lacrimal gland cells.

Topological and epitope mapping of the cellular retinaldehyde-binding protein from retina.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinol or 11-cis-retinaldehyde as endogenous ligands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes. As a first approach to identifying functional domains and protein recognition sites in CRALBP, a low resolution topological and epitope map has been developed using monoclonal and polyclonal antibodies and limited proteolysis. Fifteen peptides of 8-31 residues spanning 99% of the 316-residue bovine CRALBP were synthesized and used to prepare 13 anti-peptide polyclonal antibodies. Using a competitive ELISA procedure, peptide epitopes were classified as either accessible or inaccessible in the native protein based on the extent of their recognition by these site-specific antibodies. Use of the synthetic peptides to map the epitopes of a polyclonal antibody to intact CRALBP confirmed that the amino terminus and carboxyl terminus are immunodominate regions and hence likely to be exposed, at least in part. Limited tryptic proteolysis of native CRALBP produced three major fragments which were shown by microsequence and Western analysis to be derived from sequential loss of short peptides from the amino terminus. None of these major fragments reacted with four monoclonal antibodies (mAbs) to intact CRALBP although each mAb immunoprecipitated native CRALBP. These results and the lack of mAb recognition of any of the synthetic peptides indicates that the amino terminus of the protein is exposed and contains part of an assembly epitope recognized by the mAbs. Overall this study indicates that residues 1-30, 100-124, and 257-285 contain highly exposed segments in the native protein and therefore constitute potential interaction domains for CRALBP and visual cycle enzymes. Residues 30-99 and 176-229 are inaccessible in the native structure and may be involved with retinoid binding. These results provide a basis for a systematic higher resolution mutagenesis study directed toward correlating CRALBP structural domains with function.

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas.

Stress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.

Somatostatin in rat retina: localization by in situ hybridization histochemistry and immunocytochemistry.

In order to analyse the mRNA-protein relationships of somatostatin containing neurons in the rat retina, we have used a combined method with in situ hybridization histochemistry using radio-labeled oligodeoxyribonucleotides complementary for rat somatostatin mRNA and immunocytochemistry using antiserum for somatostatin protein. We found double labeled amacrine cells in the innermost and outermost laminae of the inner nuclear layer as well as the displaced amacrine cells in the ganglion cell layer in the retina. Some cells in the inner nuclear layer had high levels of somatostatin mRNA. Radially immunostained interplexiform fibers were observed adjacent to the amacrine cells with high level of somatostatin mRNA. These data suggest the site of synthesis of somatostatin and post-translational migration of this neuropeptide within the rat retinal tissue.

Cellular localization of somatostatin mRNA in rat retina.

In an attempt to determine the localization of the messenger RNA (mRNA) encoding somatostatin in the rat retina, we studied Sprague-Dawley rats by in situ hybridization histochemistry using radiolabelled oligodeoxyribonucleotides complementary for rat somatostatin mRNA. Among the layers of retina, we found specific labelling in the soma of some cells in the innermost and outermost laminae of the inner nuclear layer and in the ganglion cell layer; no specific labelling was observed in the inner and outer plexiform layers or in the outer nuclear layer. These data indicate the major site of somatostain synthesis within the rat retina.

Immunolocalization of cellular retinoic acid binding protein to Müller cells and/or a subpopulation of GABA-positive amacrine cells in retinas of different species.

Retinoic acid (RA) and its specific binding protein, cellular RA binding protein (CRABP), are found in relative abundance in bovine and rat retinas. Since RA does not participate in the visual cycle, the presence of RA and its binding protein in retina suggests that they may be involved in other aspects of retinoid action. As an initial step in identifying the role of RA and its binding protein in retina, monoclonal antibodies were prepared against CRABP purified from bovine retina and used to localize this antigen by immunocytochemistry in retinas of different species. Human and monkey retinas showed specific cytoplasmic labeling of Müller cells. Cat, bovine, rabbit, rat, turtle, and chick retinas showed specific cytoplasmic labeling of some somata in the inner nuclear and ganglion cell layers and characteristic strata in the inner plexiform layer. Cat and bovine retinas also showed cytoplasmic labeling of Müller cells. Immunoreactivity in these species was absent with nonimmune serum or abolished when the antibodies were preabsorbed with purified antigen. Chameleon, goldfish, and frog retinas were nonreactive. We used double-labeling immunofluorescence experiments to determine if the CRABP-positive cells were also positive for known neurotransmitters or associated enzymes. CRABP-positive amacrine cells of cat, cow, rabbit, rat, and chick represented a subset of the more numerous gamma-aminobutyric acid (GABA)-positive amacrine cells. However, turtle CRABP-positive amacrine cells were negative for GABA despite the fact that turtle retina contains many GABA positive cells. CRABP-positive amacrine cells in rat retinas were not immunoreactive for glycine, choline acetyltransferase, somatostatin, or tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of cellular retinoic acid-binding protein to amacrine cells of rat retina.

Monoclonal antibodies to performic acid-oxidized cellular retinoic acid-binding protein (CRABP) from bovine retina were prepared by fusion of spleen cells from immunized mice with mouse myeloma cells. Five antibodies were studied in detail. It was established by ELISA that the antibodies react with CRABP and oxidized CRABP, but not with other oxidized or unmodified retinoid-binding proteins. Competitive ELISA demonstrated that the antibodies react with heat-denatured antigen but not with native protein. Western blotting and immunostaining, following sodium dodecyl sulfate gel electrophoresis, provided evidence for recognition of a single component in retinal supernatants whose staining is prevented by preabsorption of the antibody with heat-denatured CRABP. The insoluble fraction from a retinal homogenate contains residual CRABP and two weakly-reacting components, whose staining is not affected by preabsorption of the antibody with antigen. Each antibody produces the same staining pattern on cryostat sections of rat retina by indirect immunofluorescence. Amacrine somata on both sides of the inner plexiform layer are labeled, as well as processes forming laminae within this layer. These results suggest that retinoic acid may play a functional role in the inner retina.

Immunolocalization of cellular retinol-, retinaldehyde- and retinoic acid-binding proteins in rat retina during pre- and postnatal development.

Cellular retinol-, retinaldehyde- and retinoic acid-binding proteins were localized in rat retina during pre- and postnatal development by indirect immunofluorescence. Cryostat tissue sections were prepared daily from embryonic day 11 until the day of birth (E11-22) and from postnatal days 1-32 (P1-32). Cellular retinaldehyde- and retinol-binding proteins were first detected in retinal pigment epithelium on E13 and E18, respectively, and in Müller cells at P1 and P15. Parallel studies showed that in adult retina cellular retinoic acid-binding protein is present in a subpopulation of GABAergic amacrine cells. During retinal differentiation, cellular retinoic acid-binding protein was first detected at E18 in cells sclerad to the developing inner plexiform layer, suggesting that this binding protein is expressed in amacrine cells very early during differentiation. During early ocular morphogenesis, cellular retinoic acid-binding protein was present in mesenchymal cells enveloping the eye (E12-15), in the neuroblastic layer of the retina (E13-15), in the nerve fibre layer (E14-15), and the developing optic nerve (E15). Our results suggest that retinoic acid, the natural ligand of cellular retinoic acid-binding protein, may be involved in neuronal differentiation in the inner retina. The studies further support a role for cellular retinoic acid-binding protein in mediating the effects of retinoic acid on developing neural crest cells and raise new questions about the role of cellular retinaldehyde-binding protein in the visual cycle and during development.