Biomedical subjects

D E Lawson

Publications and source records attributed to D E Lawson.

At least 37 records · Page 2Linked to original sources

PubMed1989

The human calbindin 27-kDa gene: structural organization of the 5' and 3' regions, chromosomal assignment, and restriction fragment length polymorphism.

The 5' and 3' regions of the human gene coding for calbindin 27 kDa were cloned and sequenced. Structural features of the 5' region included the presence of an Alu repeat and two elements regularly associated with eukaryotic promoters: an alternating purine-pyrimidine element and a homopurine-homopyrimidine box. The 3' region contained a second Alu family member and a degenerate 1.4-kb L1 repeat. A comparison with the chicken promoter was made in order to define regions conserved in evolution and potentially important in gene expression regulation. The greater similarity is located around the TATA box, but strongly conserved elements were not found. The gene was assigned to chromosome 8 by using human-rodent hybrid cell lines. Two restriction fragment length polymorphisms (HindIII and SacI) were detected with a cDNA probe recognizing the 3' end of the gene.

Amino Acid Sequence↗

PubMed1988

Loss of calbindin-28K immunoreactive neurones from the cortex in Alzheimer-type dementia.

An antibody raised against chick intestinal calbindin D28K was used to study the number and size of calbindin immunoreactive neurones in postmortem human brains from neurologically normal controls and from patients with neuropathologically diagnosed Alzheimer-type dementia (ATD). In the controls, calbindin immunoreactive neurones were observed in all cerebral cortex areas examined including the frontal, temporal and parietal cortices. When compared with the controls, the number and size of calbindin immunoreactive neurones were significantly reduced in the cortices of patients with ATD. These findings suggest that calbindin containing neurones are affected in ATD.

Aged↗

PubMed1988

Chick 28,000 Mr vitamin D-dependent calcium-binding protein in intestine, kidney and cerebellum. Purification using chromatofocusing.

A purification method has been developed for chick 28,000 Mr vitamin D-dependent calcium-binding protein, involving Blue Sepharose CL-6B column chromatography, heat treatment and chromatofocusing with a microparticulate anion exchanger (Mono P). It allowed the rapid and reproducible purification of milligram amounts of homogeneous calcium-binding protein with good yields from chick intestine, kidney and cerebellum. The calcium-binding proteins thus obtained have the same molecular weight of 28,000, heat stability, calcium binding capability and apparent isoelectric point of 4.0. These physico-chemical properties are in good agreement with those of proteins isolated by a previous procedure, which gave a low and variable yield of calcium-binding protein.

Animals↗

PubMed1988

Structure of chick chromosomal genes for calbindin and calretinin.

The chick chromosomal gene for calbindin (the 28,000 Mr intestinal calcium-binding protein) was cloned, and all the exons and flanking regions were sequenced. The promoter region contains typical ATAAA and GGGCGG boxes, the latter being unusual in "non-housekeeping" genes. Three polyadenylation signals are found in the calbindin gene that correspond to the three known mRNAs. Transcription termination is not efficient because homology with consensus sequences found downstream from the polyadenylation signal is weak. There are ten introns, most of which do not fall at homologous positions, neither with respect to the sixfold repeating structure of the calbindin protein, nor with respect to previously sequenced genes for calmodulin and other calcium-binding proteins. The gene for the related protein calretinin was cloned and partially sequenced. The introns are in the same positions in the calretinin and calbindin genes. The introns have apparently been inserted during the divergence of the calcium-binding protein superfamily.

Animals↗

PubMed1988

Relationship between the levels of calbindin synthesis and calbindin mRNA in chick intestine. Quantitation of calbindin mRNA.

An RNA-excess filter hybridization assay was established to measure the absolute amount of calbindin mRNA in chick tissues. The tissue with the highest level of mRNA is intestine, followed by kidney and cerebellum; the mRNA was not detected in liver and skin. Calbindin mRNA in intestine and kidney is vitamin D-dependent. The maximum concentration of calbindin and its mRNA found after dosing vitamin D-deficient chicks with dihydroxyvitamin D3 (1,25-(OH)2D3) is less than 5% of that found with vitamin D dosing. Secondary 1,25-(OH)2D3 stimulation produced greatly increased amounts of both calbindin mRNA and the protein, at least reaching levels similar to those found after vitamin D dosing. In this last case, each mucosal cell contains about 2000 calbindin mRNA molecules which are translated at a rate sufficient to account for the levels of calbindin found. Calbindin mRNA is translated most rapidly in the very short time periods after its release into the cytoplasm. 1,25-(OH)2D3 has two effects on calbindin mRNA formation: first, to permit the expression of the calbindin gene and a second effect, of slower onset but more persistent, which increases either the rate of calbindin gene transcription or the stability of calbindin mRNA.

Animals↗

PubMed1988

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons.

Immunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.

Animals↗

PubMed1988

Localization of 28 kDa calbindin in human odontoblasts.

The presence of 28 kDa calbindin in human odontoblasts was studied by use of specific antibodies raised against chick duodenal 28 kDa calbindin, in immunofluorescence, immuno-peroxidase, and electron-microscopic labelling experiments. The calbindin-like protein was detected mainly in the cytoplasm of odontoblast cell bodies, in their processes and occasionally in their nuclei. Correspondingly, at the ultrastructural level, immunoreactive material was associated with the cytosol, microfilaments and cilia. These findings suggest that human odontoblasts express a 28 kDa vitamin D-dependent calcium-binding protein, unlike those of rats and mice in which ameloblasts are the only cells immunoreactive for the protein.

Animals↗

PubMed1987

Human 27-kDa calbindin complementary DNA sequence. Evolutionary and functional implications.

Human 27-kDa calbindin cDNA clones were selected by antibody screening from lambda gt11 brain libraries. The sequence revealed an open reading frame coding for a protein of 261 amino acids, containing four active calcium-binding domains, and two modified domains that had presumably lost their calcium-binding capability. Comparison with chick and bovine calbindins showed that the protein was highly conserved in evolution (evolutionary rate: 0.3 x 10(-9) amino acid-1 year-1) and that active and inactive domains were equally conserved. From the data we postulate that calbindin has an important physiological function involving protein--protein interactions. Comparison of calcium-binding domains from various proteins suggested that all members of the troponin C superfamily derive from a common two-domained ancestor, but that duplications leading to calbindin and to the four-domained calcium-binding proteins took place independently on different branches of the evolutionary tree. Preliminary data showed that another calcium-binding protein, homologous to calbindin, is present in the brain and encoded by a different gene.

Amino Acid Sequence↗

PubMed1987

Calbindin in vertebrate classes: immunohistochemical localization and Western blot analysis.

Calbindin immunoreactivity was investigated in various vertebrates. Positive labeling was observed in the absorptive cells of the duodenum of all birds and reptiles but not in mammals, amphibia, or fish. Staining was present in the kidney distal convoluted tubule from amphibia and higher vertebrates. Fish kidney was negative. In the central nervous system of all species investigated, cellular bodies and fibers were Calbindin positive. Their distribution was quite broad and correlates well with the previously reported mapping for chick and rat. Western blot analysis revealed two Calbindins in brain from mammals, birds, reptiles, and amphibia (27,000 and 29,000 Da). Only one band was detected in fish. We conclude that Calbindin from the evolutionary point of view is primarily a neuronal protein, with a highly conservative character.

Amphibians↗

PubMed1987

Etiology of rickets in Egyptian children.

Factors associated with the rickets found in children attending the Outpatients Department of Cairo Children's Hospital have been studied. All the children studied were small for their age with the rachitic children being significantly smaller than the controls. The clinical diagnosis of the disease was confirmed in 54 children by X-ray and plasma biochemistry. The best indicator of the disease was the enlarged epiphysis of the wrist and to a lesser extent that of the ankles. Some of the factors predisposing to rickets were assessed in the rachitic children and in age-matched controls. There was no difference in the nutrient intake of these two groups of children although intakes of energy, calcium and vitamin D, but not of protein, were lower than recommended dietary allowances. Although more of the rachitic children came from urban areas than the controls there were no significant differences in the environments to account for the occurrence of the disease. Factors contributing to the development of rickets are discussed with particular regard to aspects such as low body weight, low maternal vitamin D status and low calcium intakes which cause a high rate of utilization and metabolism of vitamin D.

Body Weight↗

PubMed1986

Ultrastructural localization of brain 'vitamin D-dependent' calcium binding proteins.

Rat brain vitamin D-dependent calcium-binding protein (D-CaBP) was assessed for vitamin D dependency, calcium binding and ultrastructural localization within neurons. No evidence of vitamin D dependency could be derived from the experiments on vitamin D-deficient rats. A 95% pure extract of the 27-kDa brain D-CaBP was shown to bind 45Ca on nitrocellulose membrane after sodium dodecyl sulphate-electrophoresis, specifically on the 27-kDa CaBP band. Immunogold staining with electron microscopy allowed detection of D-CaBP into Purkinje cells and climbing fibers of the cerebellum. The immunoreactivity was found to be hyaloplasmic and never membrane-bound. It was present in neuronal soma, neurites and postsynaptic as well as presynaptic terminals. These findings rule out D-CaBP as a possible neurotransmitter and bring further support to the hypothesis that the protein functions as a cytosolic calcium buffer. Immunohistochemical detection of D-CaBP is proposed as a means for morphologic detection of neurons with high calcium metabolism.

Animals↗

PubMed1986

Estimation of vitamin D3 and 25-hydroxyvitamin D3 in muscle and adipose tissue of rats and man.

An assay is described based on a high pressure liquid chromatography system for measurement of vitamin D3 and 25-hydroxyvitamin D3 concentration in adipose tissue and muscle. The sensitivity of the assay is less than 1 ng/g of tissue. Neither vitamin D3 nor 25-hydroxyvitamin D3 were found in tissues of vitamin D-deficient rats using this method. 25-Hydroxyvitamin D3 could not be detected in adipose tissue and very little was found in muscle of normal rats. The mean vitamin D3 concentrations in 15 samples of human perirenal adipose tissue was 45.3 ng/g +/- 22.2 (SD) and in 6 samples of axillary tissue 115.6 ng/g +/- 52.4 (SD). Human adipose tissue contains a substantial amount of vitamin D3 and its contribution to the maintenance of vitamin D status is discussed.

Adipose Tissue↗

PubMed1986

Interrelationships in rats of tissue pools of cholecalciferol and 25-hydroxycholecalciferol formed in u.v. light.

Vitamin D-deficient rats were irradiated with u.v. light three times weekly for 30 min for several weeks. D3 (cholecalciferol) and 25(OH)D3 (25-hydroxycholecalciferol) concentrations in skin, plasma, muscle and adipose tissue were measured. In other experiments, isolated skin or the whole animal was irradiated once and the cholecalciferol response monitored. Only a small fraction of the 7-dehydrocholesterol in skin is converted into D3 (less than 2%), and the presence of fur decreases the proportion converted into 20% of that occurring in shaved rat skin. D3 formed in the skin disappears relatively slowly, so that about 90% has gone after 7 days. In normal rats 10 micrograms of D3 formed over 2 h irradiation only caused a small rise in plasma D3 concentration over the following week, indicative of a high rate of clearance from this tissue. Irradiation of vitamin D-deficient rats for a prolonged period raised plasma D3 and 25(OH)D3 concentrations to a constant value. D3, but not 25(OH)D3, could be found in adipose tissue and muscle. Prolonged irradiation of normal rats showed these tissues and plasma could hold very large amounts of D3. Pharmacokinetic analysis of the changes in D3 concentration in rats showed that the disposition kinetics of D3 was explained by a two-compartment model with half-lives of 13.8 and 7.7 days. The volume of distribution of the more-slowly-turning-over compartment was 500 ml, which presumably reflects the large amounts of D3 that can accumulate in adipose tissue. Rat skin can synthesize about 0.85 ng of D3/mJ of u.v. light energy, but it seems that not all this is available to the rat. Adipose-tissue D3 is available for use by the rat, the t1/2 being 12.0 days.

Adipose Tissue↗

PubMed1986

Immunocytochemical localization of vitamin D-dependent calcium-binding protein in the growing chick lymphoid organs.

Monospecific antiserum against chick duodenal vitamin D-dependent calcium-binding protein (D-CaBP) was used to localize this protein by the peroxidase-antiperoxidase method (PAP) in the thymus, spleen and bursa of Fabricius of normal growing chicks in 20 day old embryos; in normal growing chicks at 1, 2, 3, 4 and 6 weeks of age in chicks fed a rachitogenic diet for 4 weeks. In the normal chick thymus, D-CaBP was localized throughout the cytoplasm and in the nucleus of cortical epithelial reticular cells (ERC) and in Hassal's corpuscles of the medulla. In the normal spleen reticular cells of the marginal zones showed dense deposition of reaction product in the nucleus and throughout the cytoplasm. In the bursa of Fabricius, only a few scattered cells in the medulla showed some staining. Wide variation was encountered in D-CaBP staining in the thymus and spleen of 4 week old chicks from different broods. In 4 week old rachitic chicks, staining in the thymus and in the spleen was generally reduced in intensity and occasionally was entirely absent. The presence of D-CaBP in some reticular cells of the thymus, spleen and bursa of Fabricius, identifies these lymphoid organs as vitamin D3 targets. Thus, 1,25(OH)2D3 may have an important role in some aspects of the immune defence mechanism.

Animals↗

PubMed1986

Vitamin D-dependent calcium binding protein immunoreactivity in human retina.

Using immunohistochemistry, vitamin D-dependent calcium binding protein (D-CaBP) has been detected in human retina. In the photoreceptor layer the cones are positive but the rods are negative. In the inner nuclear layer, horizontal cells and some bipolar cells are D-CaBP. In the ganglion cell layer both small and large somata are immunoreactive for D-CaBP. Beaded fibres from the outer plexiform, inner plexiform and fibre layers are also positive.

Cells↗

PubMed1986

Response of rachitic rat bones to 1,25-dihydroxyvitamin D3: biphasic effects on mineralization and lack of effect on bone resorption.

Rachitic rats, maintained on a diet adequate in Ca and P, were treated daily with varying amounts of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. At low levels (1 ng/day) 1,25-(OH)2D3 sustained a healing response equivalent to that of 25-hydroxyvitamin D3 (100 ng/day) or the parent vitamin. Above 5 ng/day administration of 1,25-(OH)2D3 resulted in an accumulation of osteoid, giving a histological appearance similar to vitamin D deficiency. The effects of 1,25-(OH)2D3 on bone did not correlate with changes in plasma Ca or inorganic phosphorus; doses that were effective in raising bone ash and reducing the amount of osteoid failed to normalize plasma Ca, whilst the amount of sterol required to normalize plasma Ca was excessive in terms of the effect on bone. 1,25-(OH)2D3 did not stimulate any of the histological parameters of bone resorption. We conclude that 1,25-(OH)2D3 can effectively heal the bone lesions of vitamin D deficiency, but that, at high concentrations, the sterol can inhibit mineralization. Furthermore, these results question the accepted role of 1,25-(OH)2D3 as a regulator of bone resorption in vivo.

Animals↗

PubMed1985

Putative amino acid sequence of chick calcium-binding protein deduced from a complementary DNA sequence.

Two DNA fragments coding for chick CaBP have been isolated and sequenced. cDNA was prepared from enriched intestinal mRNA and cloned in pUC12. The recombinant clones were screened by differential hybridisation with 32P-cDNA probes synthesized from vitamin D replete and deficient chick intestinal mRNA. Two clones had outstanding affinity with the +D probe. Hybrid-arrested and hybrid-selected translation systems showed that both clones hybridised to mRNA coding for immunoprecipitable CaBP. The mRNA for CaBP has a 100 bp G,C rich sequence before a 786 bp coding region followed by 1250 nucleotides 3' untranslated region. Nucleotides coding for the Ca-binding sites show a high degree of homology for Ca-binding sites in chick calmodulin and rat intestinal CaBP. The amino acid sequence specified by the longest open reading frame contains five Ca-binding sites but is too large for the native CaBP; post-translational modification must therefore occur.

Amino Acid Sequence↗

PubMed1985

Rat brain synthesizes two 'vitamin D-dependent' calcium-binding proteins.

Two proteins from rat brain reacting against anti-chick intestinal vitamin D-dependent calcium-binding protein were characterized in terms of their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their molecular size. The proteins were present in the isolated cytoplasm and were produced following translation of brain mRNA in the rabbit reticulocyte lysate system. Their apparent molecular weight was 29,000 and 27,000 daltons whereas rat kidney contained only one protein cross-reacting with this antiserum and with a molecular weight of 27,000 daltons.

Animals↗