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D R Critchley

Publications and source records attributed to D R Critchley.

At least 73 records · Page 4Linked to original sources
PubMed1989

Primary sequence and domain structure of chicken vinculin.

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.

Amino Acid Sequence↗
PubMed1989

An immunohistochemical and in situ hybridization study of c-myc and c-erbB-2 expression in primary human breast carcinomas.

In previous studies of the expression and organization of proto-oncogenes in human breast a significant correlation has been found between amplification of c-myc and c-erbB-2 genes in carcinomas and poor short-term prognosis. Gene expression was estimated by analysis of total RNA from tissues, and similarly assessment of gene organization relied upon extraction of DNA from tissues. The present study has compared the expression of c-myc and c-erbB-2 mRNA as determined by in situ hybridization, and c-myc and c-erbB-2 protein expression detected by immunohistochemistry in a group of carcinomas for which there was knowledge of genomic organization and/or expression. Formalin-fixed, paraffin-embedded tissues of 38 carcinomas were assessed for the presence of c-myc protein, and 13 of these were examined for c-myc mRNA by in situ hybridization. Similarly processed tissue from 14 tumours was tested for c-erbB-2 protein using the antiserum 21N and ten of these carcinomas studied for c-erbB-2 mRNA localization. There was a good correlation between gene amplification, the presence of c-erbB-2 protein and mRNA: both the latter were detected in six of the seven carcinomas with an amplification but in none without. For some carcinomas there was a good correlation between c-myc protein and mRNA levels. Three carcinomas with gene amplification had a lower percentage of cells with detectable protein than showed hybridization for mRNA. Other carcinomas had a lower level of mRNA expression than protein. Neither approach could predict which carcinomas had amplification of the c-myc gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1989

Comparison of the binding characteristics of two different preparations of tetanus toxin to rat brain membranes.

Two different preparations of tetanus toxin (HTT and WTT) were iodinated, and their binding to rat brain membranes characterized. Under optimal binding conditions (25 mM Tris-acetate, pH 6.0), both preparations bound to a large number of high affinity sites, thought to be gangliosides. Binding constants were identical. However, in a physiological buffer (Krebs-Ringer, pH 7.4) binding of the two toxin preparations showed a number of differences. Under these conditions we have previously shown that HTT binding is markedly reduced, and that there are two classes of sites, a small number of heat-, sialidase- and protease-sensitive high affinity sites, and a larger number of sialidase-sensitive, heat- and protease-resistant lower affinity sites, probably gangliosides (PIERCE et al. (1986) Biochem. J. 236, 845-852). Although WTT bound to these same two sites, it displayed a higher affinity for the protease-resistant site than did HTT. WTT also bound to free or immobilized trisialoganglioside with higher affinity than HTT, consistent with the view that the protease-resistant site represents binding to ganglioside. In contrast, both toxin preparations bound to the protease-sensitive site with similar affinities. These observations may explain the four to five-fold higher levels of WTT binding to brain membranes, and the fact that a smaller percentage of total WTT binding is protease sensitive. Despite their different ganglioside-binding properties, both toxin preparations showed comparable neurotoxic activities, and appeared identical on SDS gels.

Animals↗
PubMed1989

Structural analysis of homologous repeated domains in alpha-actinin and spectrin.

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.

Actinin↗
PubMed1989

Expression of the caeruloplasmin gene in the adult and neonatal rat liver.

1. It has been suggested that low levels of serum caeruloplasmin in Wilson's disease result from the failure to switch from a fetal to an adult mode of caeruloplasmin gene expression. To investigate postnatal expression of the caeruloplasmin gene, steady-state levels of caeruloplasmin messenger RNA in adult and neonatal rat liver were measured. 2. Copper parameters observed in neonatal rats were similar to those seen in Wilson's disease: hepatic copper concentration was significantly elevated (neonatal 164 +/- 35 micrograms/g, adults 50 +/- 8 micrograms/g, P less than .001) and serum copper and caeruloplasmin levels were low (neonatal 0.5 +/- 0.1 microgram/ml, adults 1.3 +/- 0.2 microgram/ml, P less than .001; neonatal 0.20 +/- 0.04 arbitrary units, adults 0.69 +/- 0.16 arbitrary units, P less than .001), respectively. 3. Caeruloplasmin messenger RNA levels were analysed by Northern and dot blotting using a 32P-labelled caeruloplasmin complementary DNA probe. A caeruloplasmin messenger RNA of approximately 4.4 kilobases was detected in both adult and neonatal rat liver, with no significant difference observed in steady-state levels. 4. A step subsequent to caeruloplasmin gene transcription must therefore be impaired in neonatal rats.

Animals↗
PubMed1989

Identification of a talin binding site in the cytoskeletal protein vinculin.

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

Amino Acid Sequence↗
PubMed1989

Expression of a muscle-type alpha-actinin cDNA clone in non-muscle cells.

We have previously isolated a chick smooth muscle-type alpha-actinin cDNA clone (C17) from a chick embryo fibroblast cDNA library. As part of an investigation into a possible role for a muscle isoform of alpha-actinin in non-muscle cells, we have cloned C17 into a eucaryotic expression vector, pKCR3, and examined the distribution of the expressed protein in non-muscle, monkey COS cells. We report here that the muscle isoform of chick alpha-actinin encoded by C17, was found in focal contacts and periodically distributed along actin filaments.

Actinin↗
PubMed1988

Tetanus toxin binding to mouse spinal cord cells: an evaluation of the role of gangliosides in toxin internalization.

Studies on the binding of 125I-labelled tetanus toxin to mouse spinal cord cell cultures revealed two classes of toxin acceptor, one a high-affinity protease-sensitive site, the other a lower affinity protease-resistant site. The latter was shown to be sialidase-sensitive and may represent binding to gangliosides. Tetanus toxin internalization by spinal cord cells is rapid and coated pits have been implicated in the process (Parton et al. J. Neurochem., 49 (1987) 1057-1068). To investigate the role of gangliosides in the internalization process, trisialoganglioside was incorporated into the plasma membrane of Balb/c 3T3 cells which lack endogenous toxin-binding activity. 125I-labelled tetanus toxin bound specifically to ganglioside treated cells at 4 degrees C. On warming cells to 37 degrees C, a proportion of bound toxin was degraded indicating that some internalization of toxin had occurred. The mechanism of internalization was studied using tetanus toxin adsorbed to colloidal gold. At 4 degrees C, toxin-gold was concentrated in non-coated cell surface membrane invaginations. On warming cells to 37 degrees C, toxin gold was internalized via non-coated vesicles and accumulated within multivesicular bodies and lysosomes. The relative roles of coated and non-coated vesicular uptake systems in the mechanism of action of tetanus toxin are discussed.

Animals↗
PubMed1988

The localization of laminin mRNA and protein in the postimplantation embryo and placenta of the mouse: an in situ hybridization and immunocytochemical study.

In situ hybridization (ISH) and immunocytochemistry were used to localize sites of synthesis and deposition of the basement membrane glycoprotein laminin during development in the postimplantation mouse embryo and extraembryonic membranes. In addition, similar studies were performed on postnatal viscera during the first 20 days after birth. Up to 10 days post coitum, embryonic laminin synthesis was confined to parietal endoderm. In maternal tissue, intense laminin mRNA expression was detected in decidual cells in the mesometrial and antimesometrial endometrium at 5-7 days. At 10 days, uniform expression was still seen within the mesometrial endometrium, with higher levels around migrating trophoblast, but in the antimesometrial aspect expression was restricted to the basal zone. High levels of mRNA expression persisted in parietal endoderm throughout gestation but much lower levels were detected in visceral yolk sac. In the mature placenta, laminin mRNA expression was also found associated with fetal vessels in the labyrinth and giant cells at the fetal/maternal boundary. In the embryo, the external limiting membrane of the cerebral vesicles and spinal cord stained for laminin protein and detectable mRNA was found in the pia mater. Growing peripheral nerves and dorsal and ventral root fibres expressed laminin mRNA and stained for laminin protein. Laminin mRNA expression was found in ureteric buds and nephrogenic vesicles (but not in metanephric blastema) during early prenatal kidney development, and in glomeruli, Bowman's capsule, loops of Henle and collecting duct cells at later stages of development, and after birth. All these structures possessed laminin-rich basement membrane (BM). Laminin mRNA expression fell to below detectable levels in the kidney around weaning. In the gut, laminin expression and protein staining was confined to the muscularis externa and the lamina propria during embryogenesis. After birth, the muscularis externa, muscularis mucosa and lamina propria cells corresponding to fibroblasts had detectable laminin mRNA, but in adult gut no laminin mRNA could be demonstrated in any cell type. In liver, low levels of laminin mRNA were seen in the capsule and in periportal connective tissue. After birth, laminin mRNA was associated with intrahepatic bile channels; no laminin mRNA was detected in the parenchyma and protein deposition was restricted to blood sinus BM. In the adult liver, no laminin mRNA was detected in any cell type. The developing heart showed uniform expression of laminin mRNA from 12 days to before birth. Postnatally, labelling was restricted to connective tissue cells.

Animals↗
PubMed1987

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin.

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.

Actinin↗
PubMed1987

Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts.

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of a cDNA encoding a chick alpha-actinin.

We have isolated and sequenced a 2.1-kilobase cDNA encoding 86% of the sequence of alpha-actinin. The cDNA clone was isolated from a chick embryo fibroblast cDNA library constructed in the expression vector lambda gt11. Identification of this sequence as alpha-actinin was confirmed by immunological methods and by comparing the deduced protein sequence with the sequence of several CNBr fragments obtained from adult chicken smooth muscle (gizzard) alpha-actinin. The deduced protein sequence shows two distinct domains, one of which consists of four repeats of approximately 120 amino acids. This region corresponds to a previously identified 50-kDa tryptic peptide involved in formation of the alpha-actinin dimer. The last 19 residues of C-terminal sequence display an homology with the so-called E-F hand of Ca2+-binding proteins. Hybridization analysis reveals only one size of mRNA (approximately 3.5 kilobases) in fibroblasts, but multiple bands in genomic cDNA.

Actinin↗
PubMed1987

A study of the mechanism of internalisation of tetanus toxin by primary mouse spinal cord cultures.

The fate of tetanus toxin bound to neuronal cells at 0 degree C was followed using an anti-toxin 125I-protein A assay. About 50% of surface-bound toxin disappeared within 5 min of warming cells to 37 degrees C. Experiments with 125I-toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilizing cells with Triton X-100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin-gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin-gold particles bound to the cell body at 4 degrees C showed that it was concentrated in coated pits. After 5 min at 37 degrees C, toxin-gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti-toxins, and protein A-gold. Toxin-gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and intraaxonal uncoated vesicles, respectively.

Adsorption↗
PubMed1986

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.

Animals↗