Biomedical subjects

R A Quinlan

Publications and source records attributed to R A Quinlan.

At least 19 recordsLinked to original sources

PubMed2004

Human keratin 8 mutations that disturb filament assembly observed in inflammatory bowel disease patients.

We have identified miss-sense mutations in keratin 8 in a subset of patients with inflammatory bowel disease (Crohn disease and ulcerative colitis). Inflammatory bowel diseases are a group of disorders that are polygenic in origin and involve intestinal epithelial breakdown. We investigated the possibility that these keratin mutations might contribute to the course of the disease by adversely affecting the keratin filament network that provides mechanical support to cells in epithelia. The mutations (Gly62 to Cys, Ile63 to Val and Lys464 to Asn) all lie outside the major mutation hotspots associated with severe disease in epidermal keratins, but using a combination of in vitro and cell culture assays we show that they all have detrimental effects on K8/K18 filament assembly in vitro and in cultured cells. The G62C mutation also gives rise to homodimer formation on oxidative stress to cultured intestinal epithelial cells, and homodimers are known to be polymerization incompetent. Impaired keratin assembly resulting from the K8 mutations found in some inflammatory bowel disease patients would be predicted to affect the maintenance and re-establishment of mechanical resilience in vivo, as required during keratin cytoskeleton remodeling in cell division and differentiation, which may lead to epithelial fragility in the gut. Simple epithelial keratins may thus be considered as candidates for genes contributing to a risk of inflammatory bowel disease.

Actin Cytoskeleton↗

PubMed2003

Tropomodulin binds to filensin intermediate filaments.

Tropomodulin (Tmod) is an actin filament pointed end capping protein found in the membrane skeleton of lens fiber cells. We demonstrate that Tmod4 is able to bind the lens-specific intermediate filament protein, filensin, in either co-sedimentation or solid phase binding assays in a saturable fashion, but with low affinity and stoichiometry. Furthermore, Tmod4 does not bind the 53 kDa rod domain of filensin, nor to CP49, the obligate assembly partner of filensin. Finally, the binding of filensin to Tmod4 does not inhibit the actin capping activity of Tmod4 in vitro, suggesting that the two functions are not mutually exclusive.

Actin Cytoskeleton↗

PubMed2001

Antiischemic effects of SB203580 are mediated through the inhibition of p38alpha mitogen-activated protein kinase: Evidence from ectopic expression of an inhibition-resistant kinase.

The aim of the present study was to determine whether the attenuation of myocardial ischemic injury by SB203580 is due to the inhibition of p38 mitogen-activated protein kinase (MAPK) or to other documented nonspecific effects of the drug. We made adenoviral vectors encoding the alpha isoform of p38 MAPK with or without site-directed mutations to prevent SB203580 binding and inhibition. In embryonal rat heart-derived cells and adult rat cardiocytes expressing wild-type p38alpha MAPK, injury was reduced significantly by SB203580 present during simulated ischemia. In contrast, SB203580 did not protect cells expressing the SB203580-resistant form of p38alpha MAPK. These observations suggest that SB203580-mediated protection depends on the inhibition of p38alpha MAPK.

Adenoviridae↗

PubMed2001

Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.

Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract.

Amino Acid Sequence↗

PubMed2001

Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6.

The transcription factor PAX6 plays a critical, evolutionarily conserved role in eye, brain and olfactory development. Homozygous loss of PAX6 function affects all expressing tissues and is neonatally lethal; heterozygous null mutations cause aniridia in humans and the Small eye (Sey) phenotype in mice. Several upstream and intragenic PAX6 control elements have been defined, generally through transgenesis. However, aniridia cases with chromosomal rearrangements far downstream of an intact PAX6 gene suggested a requirement for additional cis-acting control for correct gene expression. The likely location of such elements is pinpointed through YAC transgenic studies. A 420 kb yeast artificial chromosome (YAC) clone, extending well beyond the most distant patient breakpoint, was previously shown to rescue homozygous Small eye lethality and correct the heterozygous eye phenotype. We now show that a 310 kb YAC clone, terminating just 5' of the breakpoint, fails to influence the Sey phenotypes. Using evolutionary sequence comparison, DNaseI hypersensitivity analysis and transgenic reporter studies, we have identified a region, >150 kb distal to the major PAX6 promoter P1, containing regulatory elements. Components of this downstream regulatory region drive reporter expression in distinct partial PAX6 patterns, indicating that the functional PAX6 gene domain extends far beyond the transcription unit.

Animals↗

PubMed2001

Antagonistic action of Six3 and Prox1 at the gamma-crystallin promoter.

Gamma-crystallin genes are specifically expressed in the eye lens. Their promoters constitute excellent models to analyse tissue-specific gene expression. We investigated murine CRYGE/f promoters of different length in lens epithelial cell lines. The most active fragment extends from position -219 to +37. Computer analysis predicts homeodomain and paired-domain binding sites for all rodent CRYGD/e/f core promoters. As examples, we analysed the effects of Prox1 and Six3, which are considered important transcription factors involved in lens development. Because of endogenous Prox1 expression in N/N1003A cells, a weak stimulation of CRYGE/f promoter activity was found for PROX1. In contrast, PROX1 stimulated the CRYGF promoter 10-fold in CD5A cells without endogenous PROX1. In both cell lines Six3 repressed the CRYGF promoter to 10% of its basal activity. Our cell transfection experiments indicated that CRYG expression increases as Six3 expression decreases. Prox1 and Six3 act antagonistically on regulation of the CRYGD/e/f promoters. Functional assays using randomly mutated gammaF-crystallin promoter fragments define a Six3-responsive element between -101 and -123 and a Prox1-responsive element between -151 and -174. Since Prox1 and Six3 are present at the beginning of lens development, expression of CRYGD/e/f is predicted to remain low at this time. It increases as Six3 expression decreases during ongoing lens development.

3T3 Cells↗

PubMed2001

Expression of individual lamins in basal cell carcinomas of the skin.

In this study we used a unique collection of type specific anti-lamin antibodies to study lamin expression patterns in normal human skin and in skin derived from patients with basal cell carcinomas (BCCs). Lamin expression in serial sections from frozen tissue samples was investigated by single and double indirect immunofluorescence. In normal skin, lamin A was expressed in dermal fibroblasts and in suprabasal epithelial cells but was absent from all basal epithelial cells. Lamin C was expressed in dermal fibroblasts, suprabasal epithelial cells and a majority of basal epithelial cells. However, lamin C was not expressed in quiescent basal epithelial cells. Lamin B1 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all epithelial cells but was not expressed in dermal fibroblasts. Finally, lamin B2 was expressed in all cell types in normal skin. Lamin expression was also investigated in a collection of 16 BCCs taken from a variety of body sites. Based upon patterns of lamin expression the BCCs were classified into four groups: A-negative (10/16 tumours), C-negative (5/16 tumours), A/C-negative (1/16 tumours) and A/B2-negative (1/16 tumours). Lamin expression was also compared to cell proliferation index by staining serial sections with the proliferation marker Ki67. 9/10 of the lamin A negative tumours were highly proliferative, whereas 4/5 of the lamin C negative tumours were slow growing. Thus as a general rule absence of lamin A was correlated with rapid growth within the tumour, while absence of lamin C was correlated with slow growth within the tumour. Our data supports the hypothesis that lamin A has a negative influence on cell proliferation and its down regulation may be a requisite of tumour progression.

Antibodies↗

PubMed2000

Mapping of the human CP49 gene and identification of an intragenic polymorphic marker to allow genetic linkage analysis in autosomal dominant congenital cataract.

The CP49 protein is an intermediate filament protein expressed specifically in the lens fibre cells of the lens, where it is an important cytoplasmic structural component. Dominant-negative mutations in other intermediate filament proteins, such as keratins, cause disorders characterised by dense cytoplasmic aggregates in specific cell types. The CP49 gene is therefore a good candidate for dominantly inherited forms of cataract. To allow genetic linkage analysis of families with autosomal dominant cataract with respect to CP49, a highly polymorphic intragenic microsatellite marker for this gene has been developed. In addition, both low and high resolution radiation hybrid mapping of the CP49 gene has been completed, placing it very close to microsatellite marker D3S1290 on human chromosome 3q. Furthermore, using the intragenic CP49 microsatellite, linkage was excluded in four families with genetically uncharacterized forms of autosomal dominant congenital cataract.

Base Sequence↗

PubMed2000

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Animals↗

PubMed1999

The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Cardiomyopathies↗

PubMed1999

Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580.

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from 'non-specific' effects. Four events thought to occur upon activation of stress-activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen- and stress-activated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.

Anisomycin↗

PubMed1999

Molecular chaperones: small heat shock proteins in the limelight.

Small heat shock proteins have been the Cinderellas of the molecular chaperone world, but now the crystal structure of a small heat shock protein has been solved and mutation of two human homologues implicated in genetic disease. Intermediate filaments appear to be one of the key targets of their chaperone activity.

Animals↗

PubMed1999

Gap junctions containing alpha8-connexin (MP70) in the adult mammalian lens epithelium suggests a re-evaluation of its role in the lens.

A missense mutation in one of the three lens connexins, alpha8-connexin, has been recently shown to be the genetic basis of the zonular pulverant lens cataract. This connexin had been considered to be expressed only in lens fibre cells. The present studies show that alpha8-connexin is also expressed in the lens epithelial cell layer. For this study, the distribution of gap junctions in the adult bovine lens has been investigated by confocal immunofluorescence microscopy using antibodies against alpha8-connexin (MP70) and alpha1-connexin (Cx43). In addition to the anticipated localisation of alpha8-connexin to the broad faces of lens fibre cells as reported in other species, alpha8-connexin was also found colocalized with alpha1-connexin at plaques in the lateral epithelial-epithelial plasma membranes of the bovine lens. These data suggest that mixed alpha8-connexin/alpha1-connexin plaques are between epithelial cells at their apico-lateral plasma membranes, rather than between epithelial and fibre cells. Indeed, freeze fracture analyses of the epithelial-fibre cell interface failed to reveal gap junctions connecting the epithelium and the underlying fibre cells. Importantly, microdissection and subsequent immunoblotting of lens epithelium samples confirmed the immunolocalisation results. The data suggest mature mammalian lens epithelial cells could form either heteromeric, heterotypic and/or mixed homomeric-homotypic gap junctional complexes with unique physiological properties, an important point when considering the role of epithelial cell connexins in cataractogenesis.

Animals↗

PubMed1999

The eye lens cytoskeleton.

During lens cell differentiation there are a number of very characteristic morphological changes that occur. These include a 50- to 100-fold increase in cell length as the equatorial lens epithelial cells differentiate into fibre cells and the loss of the cellular organelles such as mitochondria, nuclei, Golgi apparatus and endoplasmic reticulum. Coincident with these changes are dramatic alterations in the organisation of the lens fibre cell cytoskeleton and in particular the lens-specific intermediate filament network comprising CP49 and filensin. Cell shape and cell polarisation as well as tissue integrity are all processes that depend upon the cytoskeleton and are therefore important to the lens. The unique aspects of the lenticular cytoskeleton are the subject of this review.

Animals↗

PubMed1999

Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin.

HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.

Cell Compartmentation↗

PubMed1998

cDNA cloning, expression, and assembly characteristics of mouse keratin 16.

There has been speculation as to the existence of the mouse equivalent of human type I keratin 16 (K16). The function of this keratin is particularly intriguing because, in normal epidermis, it is usually confined to hair follicles and only becomes expressed in the suprabasal intrafollicular regions when the epidermis is traumatized. Previous studies suggested that K16 is highly expressed in the skin of mice carrying a truncated K10 gene. We therefore used the skin of heterozygous and homozygous mice to create a cDNA library, and we report here the successful cloning and sequencing of mouse K16. Recent in vitro studies suggested that filaments formed by human K16 are shorter than those formed by other type I keratins. One hypothesis put forward was that a proline residue in the 1B subdomain of the helical domain was responsible. The data presented here demonstrate that this proline is not conserved between mouse and human, casting doubt on the proposed function of this proline residue in filament assembly. In vitro assembly studies showed that mouse K16 produced long filaments in vitro. Also, in contrast to previous observations, transfection studies of PtK2 cells showed that mouse K16 (without the proline) and also human K16 (with the proline) can incorporate into the endogenous K8/K18 network without detrimental effect. In addition, K16 from both species can form filaments de novo when transfected with human K5 into immortalized human lens epithelial cells, which do not express keratins. These results suggest that reduced assembly capabilities due to unusual sequence characteristics in helix 1B are not the key to the unique function of K16. Rather, these data implicate the tail domain of K16 as the more likely protein domain that determines the unique functions.

Amino Acid Sequence↗

PubMed1998

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.

Amino Acid Sequence↗

PubMed1998

Identification and functional analysis of the mouse lens filensin gene promoter.

Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.

Animals↗