Evidence against a Finno-Ugrian suicide gene.
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Europe↗
Biomedical subjects
H Melhus
Publications and source records attributed to H Melhus.
Growth hormone and insulin-like growth factor-I do not activate identical genes in normal human osteoblasts.
In this study, we have examined the expression of mRNAs induced by growth hormone (GH) and insulin-like growth factor-I (IGF-I) in normal human osteoblasts using the differential display PCR method. Cells were incubated in the presence of 10(-7)M GH or IGF-I, RNA isolated and reverse-transcribed using an oligonucleotide primer, T11AC, anchored to the polyadenylate tail of a subset of mRNAs. The cDNA was then amplified using T11AC and an arbitrary 10-mer in the presence of [alpha-35S] dCTP. The PCR products were subsequently separated on a 6% DNA sequencing gel. The obtained patterns demonstrate that most, but not all, mRNAs induced by GH or IGF-I are identical. We conclude that GH and IGF-I do not induce expression of identical mRNAs in human osteoblasts. Our results support the dual effector theory of growth hormone action.
Cells, Cultured↗
Epitope mapping of a monoclonal antibody that blocks the binding of retinol-binding protein to its receptor.
To define the receptor binding site of retinol-binding protein (RBP) we have generated monoclonal antibodies (mAbs) to human RBP and examined their ability to interfere with the receptor binding. MAbs to two conserved regions efficiently blocked the binding. No major conformational changes in the protein occurred upon mAb binding, since the mAbs could co-immunoprecipitate the RBP-transthyretin (TTR) complex. One blocking mAb showed reactivity to a synthetic peptide corresponding to one entrance loop of the retinol-binding pocket (amino acid residues 60-70). Thus, our results show that at least one of the entrance loops of the barrel of RBP is located in or close to the receptor binding site. It can also be concluded that the receptor and TTR binding sites involve different regions of RBP.
Antibodies, Monoclonal↗
Inhibitory effects of 9-cis and all-trans retinoic acid on 1,25(OH)2 vitamin D3-induced bone resorption.
The effects of retinoic acid (RA), and calcitriol are mediated by specific nuclear receptors (RARs and VDR, respectively). Induction of RAR and VDR responsive elements in target genes requires a cofactor, the retinoid-X-receptor (RXR), with its ligand 9-cis RA. We have previously demonstrated the expression of RARs and RXRs in osteoblasts, and herein investigated the effects of the retinoids all-trans RA and 9-cis RA alone and combined with calcitriol on bone resorption in vitro, measured by 45Ca-release from prelabeled neonatal mouse calvarial bones. All-trans RA and 9-cis RA were powerful stimulators of bone resorption and essentially equipotent. At threshold concentrations (1 nM) both 9-cis RA and at-RA markedly inhibited the resorption induced by calcitriol (1 pM). The findings are compatible with a physiological role for retinoids in bone metabolism.
Animals↗
Vitamin D receptor genotypes in primary hyperparathyroidism.
Vitamin D and parathyroid hormone (PTH) constitute the main regulators of systemic calcium homeostasis. As well as its calcaemic effects, active vitamin D3(1,25(OH)2D3) has a direct regulatory role on parathyroid cells. Active vitamin D3 acts via its receptor (VDR), and binding of the ligand-receptor complex to specific promoter regions of the PTH gene inhibits transcription. Active vitamin D3 constitutes a principal regulator of parathyroid cell growth, and polymorphism in the VDR gene has recently been related to bone mineral density and suggested as predisposing to osteoporosis. Impaired effects of active vitamin D3 may contribute to the relatively enhanced secretion and cell proliferation seen in hyperparathyroidism (HPT). Indeed, VDR dysfunction, of essentially unknown character, has been demonstrated in the pathological parathyroid tissue of primary HPT as well as HPT secondary to uraemia. Consistent with the essential role of active vitamin D3 in parathyroid regulation, the VDR gene polymorphism was studied in 90 postmenopausal women with primary hyperparathyroidism. The VDR genotype bb was found in 60.0% of HPT patients and in 33.3% of the postmenopausal female controls (P < 0.001). As the b allele has been linked to decreased transcriptional activity or messenger RNA stability, reduced VDR expression may impede regulatory actions of vitamin D and may contribute to parathyroid tumorigenesis in these patients.
Aged↗
[Vitamin D and osteoporosis].
Vitamin D constitutes a complex endocrine-regulated system, and is both a prohormone for the endogenous synthesis of the active hormone, calcitriol, and a vitamin which may be administered to supply the organism's requirements. No single test or investigation is available for the demonstration of vitamin D deficiency. Both vitamin D intake and ability to synthesise vitamin D decrease with increasing age, and particularly the elderly in institutionalised care are at risk of developing vitamin D deficiency. Iceland excepted, mean daily vitamin D consumption in the Nordic countries is less then 5 micrograms; and in approximately 10-25 per cent of the population, daily intake is less than 2.5 micrograms which is insufficient to maintain an adequate serum calcidiol concentration in individuals unexposed to sunlight. The recommended daily intake of 5 micrograms, currently adopted in the Nordic countries, may be too low-an intake of 10 micrograms is probably necessary to satisfy requirements in the elderly.
Adrenal Cortex Hormones↗
The loop region around amino acid residue 50, the N-terminal part of the alpha-helix, and the C-terminus of human retinol-binding protein are not located in or close to the transthyretin binding site.
Although the three-dimensional structures of both human retinol-binding protein (RBP) and transthyretin (TTR) are known, the binding sites have not been defined. In this study we have epitope-mapped a rabbit antiserum against human RBP using synthetic peptides corresponding to all potentially antigenic sites. Immunoreactivity was seen with peptides corresponding to amino acid residues 46-54, 137-146, 143-153, and 172-182 of RBP. Since previous studies have demonstrated that these antibodies bind equally well to free RBP and to the RBP-TTR complex, we conclude that neither the loop region around amino acid residue 50, the N-terminal part of the alpha-helix, nor the C-terminus of RBP is located in or close to the TTR binding site. Our results support the hypothesis that one of the entrance loops is involved in the TTR binding.
Animals↗
Vitamin D receptor genotypes in osteoporosis.
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Aged↗
Competitive PCR demonstrates that 9-cis retinoic acid induces cellular retinoic acid-binding protein-II more efficiently than all-trans retinoic acid in human osteosarcoma cells.
Cellular retinoic acid-binding proteins (CRABPs) are thought to play a key role in the regulation of the levels of retinoic acid (RA) available to interact with the nuclear receptors. In this study we have used reverse transcription (RT) and competitive polymerase chain reaction (cPCR) to investigate the effects of RA on CRABP-II expression levels in the human osteosarcoma cell line MG-63. Two different isomers of RA, all-trans (at) and 9-cis RA, were chosen since at-RA but not 9-cis RA binds to CRABP-II. Cells were treated with 10(-6) M at-RA or 9-cis RA for 24 h. Following RNA preparation and RT, cPCR was performed using constructed internal standards for CRABP-II and beta-actin. While no change occurred in the beta-actin control, an approximate two-fold increase of CRABP-II mRNA levels was seen in cells treated with at-RA and at least a four-fold increase with 9-cis RA. Thus, although 9-cis RA does not bind to CRABP-II, it induces CRABP-II expression more efficiently than at-RA in human osteosarcoma cells.
Base Sequence↗
Adult human osteoblast-like cells do not express insulin-like growth factor-I.
Previous studies using cultures of fetal rodents calvaria have indicated an important role for insulin-like growth factor-I (IGF-I) in the local control of bone formation. In this study, we have examined the expression of IGF-I in adult human osteoblast-like (hOB) cells. To detect very low levels and to distinguish between the two IGF-I mRNA transcripts Ea and Eb, which are formed by alternate splicing, we have used the reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of both Ea and Eb IGF-I mRNA transcripts were found in human liver and in placenta. However, neither Ea nor Eb IGF-I mRNA could be detected under basal conditions or after stimulation with growth hormone in normal hOB cells and two human osteosarcoma cell lines with osteoblastic properties, SaOS-2 and MG-63. We conclude that adult hOB cells do not synthesize IGF-I. Thus, in contrast to its crucial role as a local regulator of skeletal remodeling in fetal rodent bone, IGF-I does not appear to have this autocrine function in adult human bone.
Base Sequence↗
Reverse transcription-polymerase chain reaction assay demonstrates that the 9-cis retinoic acid receptor alpha is expressed in human osteoblasts.
The 9-cis retinoic acid receptor (RXR) alpha, a co-regulator of the thyroid hormone and vitamin D receptors, has previously been shown to be expressed predominantly in metabolic organs such as the liver and the kidney. In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to examine the expression of retinoic acid (RA) nuclear receptors in primary human osteoblasts and in SaOS-2, a human osteosarcoma-derived cell line with osteoblastic characteristics. Our results demonstrate that human osteoblasts express RXR alpha, as well as the all-trans RA receptors (RAR) alpha, beta, and gamma. These data further establish bone as a major target for retinoids and suggest that RA can regulate vitamin D and thyroid hormone actions in osteoblasts.
Base Sequence↗
Cellular retinoic acid-binding protein type II is expressed in adult human osteoblasts and in adult liver.
In this study we have used a reverse transcription polymerase chain reaction (RT-PCR) to demonstrate that adult primary human osteoblasts and SaOS-2, a human osteosarcoma-derived cell line with osteoblastic properties, express cellular retinol-binding protein I (CRBP I), cellular retinoic acid-binding protein II (CRABP II), and very low levels of CRABP I. We also show that CRABP II is expressed in the adult liver, which does not express CRABP I. The results suggest that CRABP II is the important isoform in the adult bone as well as in the adult liver. Since the 9-cis retinoic acid receptor (RXR) alpha previously has been shown to be expressed predominantly in the liver, CRABP II might be involved in the transport of 9-cis retinoic acid to its nuclear receptor.
Adult↗
Ligand-dependent secretion of rat retinol-binding protein expressed in HeLa cells.
A minigene encoding rat retinol-binding protein (RBP) was transfected into HeLa cells, which do not express endogenous RBP, transthyretin, or cellular retinol-binding protein. The HeLa cells manufactured and secreted the transfected gene product, demonstrating that RBP-transthyretin assembly is not a requirement for the secretion of RBP. When HeLa cells were grown under vitamin A-deficient conditions, RBP accumulated in the endoplasmic reticulum. Both serum and retinol stimulated secretion of RBP in a concentration-dependent manner. The retinol-regulated secretion occurred also after protein synthesis had been blocked by cycloheximide. Addition of holo-RBP or retinal, but not retinoic acid, stimulated secretion of RBP. Thus, an in vitro model system that resembles the rat hepatocyte in vivo with regard to the known regulation of RBP secretion has been established in a human cell line of extrahepatic origin. It can be concluded that cellular retinol-binding protein is not required for the transfer of retinol to RBP and that the mechanism whereby retinol controls the intracellular transport of RBP is neither specific for tissues synthesizing RBP nor species-specific. To investigate the structural properties responsible for the endoplasmic reticulum retention of RBP in the absence of its ligand, a cDNA encoding chicken purpurin, a protein that is 50% identical to RBP and that binds retinol, was expressed in HeLa cells. In contrast to RBP, purpurin was not retained in vitamin A-deficient HeLa cells.
Animals↗
A genetically engineered purpurin/retinol-binding protein hybrid that binds to transthyretin.
A mini-gene encoding rat retinol-binding protein (RBP) and a cDNA encoding chicken purpurin were separately transfected into HeLa cells. In contrast to RBP, expressed purpurin did not bind to transthyretin (TTR). A purpurin/RBP hybrid protein was constructed by substituting the cDNA sequence encoding the N-terminal 29 amino acids of purpurin for the corresponding part of RBP. The expressed hybrid molecule bound to the TTR-Sepharose. These results demonstrate that purpurin does not bind to TTR, that a functional purpurin/RBP hybrid can be constructed, and that the N-terminal coil of RBP is not required for TTR binding.
Amino Acid Sequence↗
Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol.
To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.
Anthraquinones↗
Cellular retinoid binding proteins.
The cellular retinol-binding protein (CRBP) and the cellular retinoic acid binding protein (CRABP) have similar physicochemical characteristics. The amino acid sequences of rat CRBP and bovine CRABP have been elucidated and they display 40% sequence identity. Both protein sequences appear to be evolutionarily highly conserved. The amino acid sequence of human CRBP, deduced from a cDNA-clone, is 96% identical to the rat CRBP sequence. CRBP and CRABP are members of a protein family, all members of which may bind hydrophobic ligands and interact with membrane components. All members of the protein family are probably related in tertiary structure and might interact with membrane components through two regions with a high probability for alpha-helix. The tissue distribution of CRBP and CRABP, together with their relation to lipid transporting proteins suggests that CRBP and CRABP are cellular transporting proteins for retinol and retinoic acid, respectively.
Amino Acid Sequence↗
The primary structure of rabbit and rat prealbumin and a comparison with the tertiary structure of human prealbumin.
The primary structures of rabbit and rat prealbumin have been determined. The amino acid sequence of rabbit prealbumin was determined by analyses of peptides obtained by trypsin and Staphylococcus aureus protease digestions. The rat prealbumin sequence was deduced by analyses of tryptic peptides as well as by nucleotide sequencing of cDNA clones. Both amino acid sequences contain 127 amino acid residues, the same as human prealbumin. Pairwise comparisons show that the three sequences are more than 80% identical. All three prealbumins were found to display significant sequence homology with human thyroxine-binding globulin. A comparison of the primary structures of the prealbumins with the tertiary structure of human prealbumin shows that amino acid replacements are preferentially located at the surface of the molecule and in the loops connecting the beta-strands. The locations of the replacements are discussed as regards the different molecular interactions in which prealbumin is involved.
Amino Acid Sequence↗