A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.
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Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism. (+info)
Stage-dependent responses of the developing lung to retinoic acid signaling.
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Morphological analysis of vitamin A-deficient rat fetuses and of retinoic acid receptor (RAR and RXR) mutant mice have demonstrated that retinoic acid (RA) is essential for lung development. To gainfurther insight into RA signaling pathways during primary lung budformation and lung branching, we have investigated the effects of RA and of a pan-RAR antagonist in cultures of whole embryos and lung explants. Treatment of E8.0 embryos with the pan-RAR antagonist inhibits the formation of the primitive respiratory system. On the other hand, treatment of E11.75 and E12.5 lung explants with RA inhibits branching morphogenesis, whereas treatment with the pan-RAR antagonist at the same developmental stages stimulates formation of distal buds. The inhibitory effect of RA on branching is strongly decreased in RARbeta null lungs, while enhancement of budding by the pan-RAR antagonist is not affected by an RARgamma null mutation. Additionally, cellular retinol binding protein one (CRBPI) null lungs are more sensitive than wild type lungs to the pan-RAR antagonist-induced stimulation of branching. These data indicate that retinoid signaling is indispensable for the formation of primary lung buds and the oesophagotracheal septum from the primitive foregut. They also suggest that at the pseudoglandular stage, RA signaling through RARbeta, but not RARgamma, inhibits distal bud formation thereby promoting the formation of conducting airways. Moreover, the level of CRBPI in the pseudoglandular lung appears to participate in the control of branching morphogenesis. (+info)
Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.
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Transthyretin (TTR) acts physiologically in the transport of retinol in the circulation. We previously reported the generation and partial characterization of TTR-deficient (TTR(-)) mice. TTR(-) mice have very low circulating levels of retinol and its specific transport protein, retinol-binding protein (RBP). We have examined the biochemical basis for the low plasma retinol-RBP levels. Cultured primary hepatocytes isolated from wild type (WT) and TTR(-) mice accumulated RBP in their media to an identical degree, suggesting that RBP was being secreted from the hepatocytes at the same rate. In vivo experiments support this conclusion. For the first 11 h after complete nephrectomy, the levels retinol and RBP rose in the circulations of WT and TTR(-) mice at nearly identical rates. However, human retinol-RBP injected intravenously was more rapidly cleared from the circulation (t(12) = 0.5 h for TTR(-) versus t(12) >6 h for WT) and accumulated faster in the kidneys of TTR(-) compared with WT mice. The rate of infiltration of the retinol-RBP complex from the circulation to tissue interstitial fluids was identical in both strains. Taken together, these data indicate that low circulating retinol-RBP levels in TTR(-) mice arise from increased renal filtration of the retinol-RBP complex. (+info)
Blockade of costimulation through B7/CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance.
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It has been reported that costimulation blockade can result in T cell anergy. We investigated the effects of blocking costimulatory molecules in vivo on the development of experimental autoimmune uveoretinitis (EAU), a model for autoimmune uveitis in humans that is induced in mice by immunization with the retinal Ag interphotoreceptor retinoid binding protein. B10.A mice immunized with a uveitogenic regimen of interphotoreceptor retinoid-binding protein were treated with Abs to B7.1 and B7.2 for 2 wk. Evaluation of EAU and immunological responses 1 wk later showed that disease had been abrogated, and cellular responses were suppressed. To determine whether the costimulation blockade resulted in tolerance, adult-thymectomized mice immunized for uveitis and treated with anti-B7 or anti-CD28 were rechallenged for uveitis induction 5 wk after the initial immunization. Although confirmed to be disease free after the initial immunization, both anti-B7- and anti-CD28-treated mice developed severe EAU and elevated cellular responses after the rechallenge, equivalent to those of control mice. We conclude that in this model costimulatory blockade in vivo prevents the development of autoimmune disease, but does not result in long-term tolerance. The data are compatible with the interpretation that B7/CD28 blockade prevents generation of effector, but not of memory, T cells. (+info)
Circadian-dependent retinal light damage in rats.
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PURPOSE: To determine the relative susceptibility of rats to retinal light damage at different times of the day or night. METHODS: Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure. RESULTS: When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light. CONCLUSIONS: These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility. (+info)
Evolution of the lipocalin family as inferred from a protein sequence phylogeny.
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The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency. (+info)
Plasma retinol binding protein: structure and function of the prototypic lipocalin.
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In terms of both structure and biological function, retinol binding protein (RBP) is one of the best characterized members of the lipocalin superfamily. The molecular interactions in which RBP participates are described herein. (+info)
The transthyretin-retinol-binding protein complex.
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Transthyretin (TTR, formerly called prealbumin), one of the transporters of the hormone thyroxine and the lipocalin retinol-binding protein (RBP), the specific carrier of the vitamin, are known to form, under physiological conditions, a macromolecular complex that is believed to play an important physiological role: prevention of glomerular filtration of the low molecular weight RBP in the kidneys. The physiological significance of complex formation is discussed first, followed by a brief description of the three-dimensional structure of the two participating proteins. The two X-ray models of the complex available are subsequently discussed and compared and finally the non-crystallographic evidence that supports these models is reviewed. (+info)