The concept of scaffolds that can be equipped with artificial biochemically active sites has gained recent interest in the field of protein design. Members of the lipocalin protein family represent promising model systems in this respect. Especially prototypic lipocalins, such as the retinol-binding protein or the bilin-binding protein (BBP), exhibit a structurally simple one-domain fold with a conformationally well conserved beta-barrel as their central motif. This type of supersecondary structure is made of a cylindrically closed beta-sheet of eight antiparallel strands. At the open end of the barrel the beta-strands are connected by four loops in a pairwise manner so that a pocket for the ligand is formed. In a rational protein design study a metal-binding site was functionally grafted on the solvent-exposed surface of the beta-barrel, whereby the rigid backbone conformation permitted the spatially defined arrangement of three His side chains. In a combinatorial protein design approach, the natural ligand pocket of a lipocalin was reshaped. In this manner variants of the BBP were engineered which exhibit high affinity and remarkable specificity for haptens like fluorescein and digoxigenin. The so-called 'anticalins', i.e. artificial lipocalins recognizing prescribed ligands, could provide an interesting alternative to recombinant antibody fragments. Consequently, the use of lipocalins as a scaffold opens new applications for members of this functionally diverse protein family in biotechnology and medicine. (+info)
Fas and Fas ligand expressed on cells of the immune system, not on the target tissue, control induction of experimental autoimmune uveitis.
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The Fas-Fas ligand (FasL) interaction is important for maintaining lymphocyte homeostasis by signaling for activation-induced cell death. Mice homozygous for the lpr or gld mutations do not express functional Fas or FasL, respectively, and spontaneously develop progressive autoimmune symptoms. Recent studies implicated expression of FasL on immunologically privileged tissues in protection from immune-mediated damage. Conversely, tissue expression of Fas may facilitate damage. We evaluated the susceptibility of lpr and gld mice to induction of experimental autoimmune uveitis (EAU), a T cell-mediated autoimmune disease induced with retinal Ags, which targets the neural retina. gld as well as lpr mice immunized with a retinal Ag developed disease of lower incidence and severity than wild-type controls. Delayed hypersensitivity responses were not significantly different among immunized gld, lpr, or wild-type mice, although in vitro Ag-specific lymphocyte responses of the mutant mice were lower. To evaluate whether the diminished ability of gld and lpr mice to develop EAU was due to a defect at the level of the tissue or the immune system, radiation bone marrow chimeras constructed between wild-type and mutant mice were immunized to induce EAU. Mutant recipients of wild-type bone marrow, but not wild-type recipients of mutant bone marrow, developed normal disease scores. These results indicate that normal expression of Fas and of FasL on cells of the immune system is important for EAU expression. Unexpectedly, neither lack of Fas nor lack of FasL on the ocular tissues affected expression of EAU. (+info)
High dose of simvastatin normalizes postprandial remnant-like particle response in patients with heterozygous familial hypercholesterolemia.
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Familial hypercholesterolemia (FH) and disturbances in postprandial lipoprotein metabolism are both associated with premature atherosclerosis. The effect of beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors on plasma cholesterol levels in patients with FH is well established; however, it is not known whether postprandial lipoproteins are also influenced. In this case-controlled intervention study, we investigated the effects of high-dose simvastatin on postprandial lipoproteins. We used a new method to analyze remnant lipoproteins based on the immunoseparation principle (remnant-like particle cholesterol [RLP-C] assay) and the well-established measurement of retinyl ester (RE) analysis in plasma and in the Svedberg flotation unit (Sf)<1000 fraction. Seven heterozygous FH patients and 7 control subjects matched for sex, age, body mass index, triglycerides, and apolipoprotein E genotype were enrolled in the study. An oral vitamin A (RE) fat-loading test was performed at baseline in both groups and after 3 months of high-dose simvastatin (80 mg/d) treatment in the FH patients. Before treatment, FH patients had significantly higher fasting and postprandial concentrations of lipoprotein remnants (plasma RLP-C 42+/-19 mg/dL and area under the RLP-C curve 415+/-82 mg. L(-1). h(-1), respectively) than did control subjects (7+/-3 mg/dL and 101+/-35 mg. L( -1). h(-1), respectively; P<0.05), suggesting a delayed clearance of chylomicron remnant particles in the FH patients. Treatment with simvastatin significantly reduced fasting and postprandial remnant lipoprotein cholesterol concentrations (13+/-3 mg/dL and 136+/-53 mg. L(-1). h(-1), respectively; P<0.05 for both). Postprandial RE in the Sf<1000 fraction, not total RE in plasma, was also significantly higher in FH patients than in control subjects (24+/-10 versus 6.3+/-5.9 mg. L( -1). h(-1), P<0.05), but treatment with simvastatin did not result in improvement of the postprandial RE response, either in the Sf<1000 fraction or in plasma. It is concluded that heterozygous FH patients have increased fasting and postprandial remnant lipoprotein concentrations. Treatment with simvastatin significantly reduced the fasting and postprandial RLP-C concentrations but did not result in improved postprandial RE response. (+info)
Characterization of an androgen-specific response region within the 5' flanking region of the murine epididymal retinoic acid binding protein gene.
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The epididymis provides the optimal milieu for sperm maturation and storage. Epididymal secretory proteins are believed to be involved in that process. Androgens are the major endocrine and paracrine regulatory signals that regulate gene expression in the epididymis. We have previously identified an androgen-dependent retinoic acid-binding protein (mE-RABP) that is secreted into the luminal fluid from the mouse mid/distal caput epididymidis. The mE-RABP protein belongs to the lipocalin superfamily and may be involved in the trafficking of retinoic acid within the epididymis. We have recently demonstrated that 5 kilobases of the 5' flanking region of the mE-RABP gene contained all the information for the hormonal regulation and the tissue-, region-, and cell-specific expression of the mE-RABP gene. In this study, we have identified a complex androgen-specific response region (ARR) within the first 600 base pairs of the mE-RABP gene promoter. Androgen (DHT) but not glucocorticoid (DEX) activates the ARR in HeLa and PC-3 cells. Two androgen receptor binding sites have been located at positions -445/-459 and -102/-88 and were named ARBS-1 and ARBS-0, respectively. Point mutations of ARBS-0 resulted in a slight decrease of the androgen response. However, mutations of ARBS-1 led to a total loss of the androgen responsiveness, suggesting that it was a major cis-acting element. When ARBS-1 is isolated from its promoter context, it serves as a weak androgen-responsive element that was activated by both androgens and glucocorticoids. Also, the -543/-88 DNA promoter fragment behaved as a poor androgen-responsive region, suggesting that regulatory elements located within the proximal mE-RABP promoter were required for a full androgen response. In conclusion, the mE-RABP ARR is a good model for the study of molecular mechanisms that lead to an androgen-specific responsiveness in vivo. (+info)
Mechanisms for vitamin A transfer from blood to milk in rhesus monkeys.
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In order to study vitamin transport during lactation tritium labeled vitamin A, attached to serum lipoproteins or retinol-binding protein (RBP), was injected intravenously into eight Rhesus monkeys. The turnover rate of vitamin A in plasma and its appearance in milk was quantitatively and qualitatively investigated. The turnover of plasma RBP-retinol, which was somewhat faster in lactating than in non-lactating animals, exceeded that of lipoprotein-vitamin A by a factor of three. Tritium--vitamin A appeared in milk mainly as retinylesters. The fractional rate of transfer was about 60% higher for vitamin bound to RBP than for other forms of plasma vitamin A. Consequently, about 90% of the vitamin A appearing in milk will normally be derived from the retinol-RBP complex. The importance of the lipoprotein-mediated transfer of vitamin A will probably increase during a high intake of the vitamin. (+info)
Tetracycline-inducible system for photoreceptor-specific gene expression.
(70/1000)
PURPOSE: To develop a system for inducible photoreceptor-specific gene expression in transgenic mice. The tetracycline regulatory system was chosen because it possesses the useful property of direct control of gene expression through use of an exogenous agent, doxycycline, a tetracycline derivative. METHODS: Transgenic mice were generated that carried the reverse tetracycline-controlled transactivator under the control of the photoreceptor-specific promoters for rhodopsin and interphotoreceptor retinoid-binding protein. These animals were crossed with transgenic mice carrying the lacZ reporter gene under control of the tetracycline operator cassette, creating doubly transgenic mice. Doxycycline was administered to induce expression of the reporter gene. Reporter assays were then performed to evaluate lacZ expression. RESULTS: Doxycycline administration led to photoreceptor-specific expression of the lacZ reporter gene in the doubly transgenic mice. X-gal staining was restricted to photoreceptor inner segments and synaptic termini. Induction could be achieved by addition of the drug to the animals' drinking water or by intravitreal injection. Induction was noted within 24 hours of doxcycline administration. Because of variability among animals, there was an approximate correlation, but not a clean dose-response curve relating drug dose to level of reporter expression. CONCLUSIONS: A transgenic system for inducible photoreceptor-specific gene expression has been developed. This system is currently being exploited to study the effects of regulated expression of genes of biological interest. (+info)
Association between increased levels of TNF-alpha, decreased levels of prealbumin and retinol-binding protein, and disease outcome.
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We determined whether there is an association between tumor necrosis factor-alpha (TNF-alpha), undernutrition [prealbumin (PA) <160 mg/L, retinol binding protein (RBP) <30 mg/L], disease stage, outcome (death or survival), and race in children with leukemia. TNF-alpha, PA, and RBP were measured in 52 patients (0.8 to 17 years old): 18 African Americans, 34 whites; 27 newly diagnosed (ND), and 25 in clinical remission (CR). Mean levels of TNF-alpha were higher in patients than in 46 healthy children (p < 0.05), but were not different between ND and CR groups. Mean acute phase proteins (APP) were different among groups: ND > CR > controls (p < 0.05). Mean levels of PA and RBP were lower in patients than in controls (p < 0.051, and tended to be higher in CR than in ND patients. African-American patients had lower concentrations of TNF-alpha, PA, and RBP but higher APP than white patients (p < 0.05). CR patients and African-American patients who died tended to have higher levels of TNF-alpha and APP, but lower PA and RBP than those who survived. A higher percentage of ND African Americans (45%) than of ND whites (13%) died. Results suggest that undernutrition and inflammation in CR patients and African Americans were associated with poor survival, and that ND African Americans have a poorer outcome than whites independently of TNF-alpha levels. (+info)
The distribution of vitamin A and retinol-binding protein in the blood plasma, urine, liver and kidneys of carnivores.
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The contents of retinol and retinyl esters as well as retinol-binding protein (RBP) in the plasma, urine, liver and kidneys of dogs, raccoon dogs and silver foxes were investigated. In the plasma and urine of all three species, vitamin A was present as retinol and retinyl esters. Vitamin A levels (1376+/-669 microg x g(-1)) were significantly higher in the livers of dogs than in the kidneys (200+/-217 microg x g(-1), P < 0.001 ). However, vitamin A levels in the kidneys of raccoon dogs (291+/-146 microg x g(-1)) and silver foxes (474+/-200 microg x g(-1)) were significantly higher than in the liver (67+/-58 microg x g(-1) and 4.3+/-2.4 microg x g(-1), respectively, both P < 0.001). RBP was immunologically detected in the blood plasma of all species, but never in the urine. In the liver, immunoreactive RBP was found in hepatocytes. In the kidneys of all species, RBP was observed in the cells of the proximal convoluted tubules. The levels of vitamin A in the livers of raccoon dogs and silver foxes were extremely low, which would be interpreted as a sign of great deficiency in humans. This observation might indicate that the liver status cannot be used as an indicator of vitamin A deficiency in canines. The high levels of vitamin A in the kidneys in all three species may indicate a specific function of the kidney in the vitamin A metabolism of canines. (+info)