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D R Garrod

Publications and source records attributed to D R Garrod.

At least 19 recordsLinked to original sources
PubMed1992

The molecular biology of desmosomes and hemidesmosomes: "what's in a name"?

Desmosomes are junctions involved in intercellular adhesion of epithelial cells and hemidesmosomes are junctions involved in adhesion of epithelia to basement membranes. Both are characterised at the ultrastructural level by dense cytoplasmic plaques which are linked to the intermediate filament cytoskeleton of the cells. The plaques strongly resemble each other suggesting a relationship between the two kinds of junctions, as implied by their names. Recent characterisation of the molecular components of the junctions shows they are, in fact, quite unrelated implying that structural similarity is fortuitous. The molecular biology raises many fascinating problems relating to their structure and function.

Animals↗
PubMed1991

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains.

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.

Amino Acid Sequence↗
PubMed1991

An immunohistological study of desmosomes in Darier's disease and Hailey-Hailey disease.

The immunocytochemical distribution of desmosomal components was determined in involved skin from eight patients with Darier's disease, five patients with Hailey-Hailey disease and two patients with transient acantholytic dermatosis as well as skin from four normal controls. Sections were stained using monoclonal antibodies to the desmosomal proteins dp1 and dp2 (desmoplakins) and the desmosomal glycoproteins dg1 (desmoglein), and dg2 and dg3 (desmocollins). There was normal expression of desmosomal proteins and glycoproteins at the periphery of the keratinocytes in the perilesional skin in Darier's disease, in Hailey-Hailey disease and in transient acantholytic dermatosis. In the lesional skin there was reduced expression of desmosomal proteins and glycoproteins in the basaloid 'buds' at the base of the lesions, but there was bright diffuse staining of the acantholytic cells. Focal intracellular staining was detected within many of the acantholytic keratinocytes in Hailey-Hailey disease and within some of these cells in Darier's disease. Suction blisters were used to induce fresh acantholysis in lesional skin in Darier's disease and clinically uninvolved skin in Hailey-Hailey disease. The results indicated that acantholysis precedes the development of intracellular staining. Although there are immunopathological abnormalities in the distribution of desmosomal proteins and glycoproteins in both Darier's disease and Hailey-Hailey disease, the changes are probably secondary to internalization of desmosomal components with breakdown and redistribution of antigens rather than a primary deficiency in the synthesis of these proteins. Focal internalization was more widespread in Hailey-Hailey disease than in Darier's disease and the differences in the distribution of desmosomal components in these diseases confirm that they are distinct entities.

Blister↗
PubMed1991

Cell adhesion in Hailey-Hailey disease and Darier's disease: immunocytological and explant-tissue-culture studies.

The pathogenesis of Hailey-Hailey disease and Darier's disease was investigated using immunocytological and explant-tissue-culture techniques. There was breakdown of the intercellular adhesions between keratinocytes in explants from clinically uninvolved skin of patients with Hailey-Hailey disease or Darier's disease. The major desmosomal components were present in the cultures and were expressed in a punctate peripheral pattern at cell-cell contact sites, but there was diffuse staining of acantholytic cells. Plasminogen, which is expressed by basal keratinocytes in normal skin, was detected in association with suprabasal acantholytic cells in skin biopsies from these diseases. Plasminogen was reversibly displaced from the cells by 6-aminohexanoic acid, suggesting that binding is mediated by a reaction with the lysine receptor on the plasminogen molecule. Plasminogen was also detected in separating cells in explant cultures and there was cytoplasmic expression of the plasminogen activator urokinase by these cells. These abnormalities are not unique to either disease and do not account for the phenotypic differences between Darier's disease and Hailey-Hailey disease, but plasmin generation may have a role in perpetuating cell separation.

Adult↗
PubMed1991

Desmosome biogenesis in the mouse preimplantation embryo.

The molecular processes underlying the formation of the first desmosomes in the mouse early embryo have been examined by immunocytochemical and biochemical techniques using antibody probes recognising desmosomal proteins 1 and 2 (dp1 + 2, desmoplakins), dp3 (plakoglobin), desmosomal glycoprotein 1 (dg1, desmoglein) and dg2 + 3 (desmocollins). Immunofluorescence labelling of staged intact embryos and synchronised cell clusters indicates that dp1 + 2, dg1 and dg2 + 3 are first detectable on the lateral membrane contact sites between trophectoderm cells in early cavitating blastocysts, coincident with the onset of desmosome formation as seen in ultrastructural preparations. Membrane localisation of these antigens is predominantly punctate in appearance, occurs after division to the 32-cell stage and appears to be coincident with blastocoele formation since non-cavitated embryos/cell clusters of equivalent age/cell cycle are usually unlabelled. In contrast, dp3 is first detectable at the 32-cell stage at all internal membrane contact sites (including those with inner cell mass cells) in a continuous linear pattern, and appears in both cavitated and non-cavitated specimens. Subsequently during blastocyst expansion, dp3 localisation becomes punctate and restricted to trophectodermal membranes. Immunoprecipitation of desmosomal antigens following metabolic labelling indicates that synthesis of dp3 is underway from at least compaction in the 8-cell embryo, while dp1 + 2 synthesis is first evident in 16-cell morulae. Synthesis of dg1 and dg2 + 3 is not detectable until the early blastocyst stage. These results suggest that desmosome biogenesis in the preimplantation embryo might be regulated by transcription or translation of desmosomal glycoproteins and by maturational changes in the trophectoderm layer associated with blastocoele formation. The earlier expression and wider distribution of dp3 at cell contact areas may reflect non-desmosomal sites (eg, adherens junctions) for this protein and a possible role for dp3 in the development of intercellular junctions.

Animals↗
PubMed1990

A study of desmosomes in colorectal carcinoma.

Desmosomes are adhesive junctions of epithelial cells. Their expression may be altered or lost in carcinomas resulting in reduced cellular adhesiveness. The desmosomes of colorectal carcinomas have been studied by fluorescent antibody staining, immunoblotting and electromicroscopy. A series of 58 malignant specimens, comprised of primary tumours and metastases, were desmosome positive. There was no indication of a comparative reduction in desmosome expression that might give rise to reduced adhesiveness of tumour cells, although loss of polarised junctional distribution in poorly differentiated tumours might have such a consequence. Western blotting analysis of colorectal cancers and cultured carcinoma cells identified desmosomal polypeptides dp1 + 2, dg1 and dg2 + 3 with similar relative molecular weights to normal homologues. In addition, a polypeptide of 140,000 was recognised only in malignant epithelium by anti-dg2 + 3 antiserum. The significance of this polypeptide is not understood. Tumours and uninvolved epithelium were exposed to low extracellular [Ca2+] to test whether tumour desmosomes were of reduced stability. This caused much cellular degradation in tumours but some viable cell clumps possessed desmosomes resistant to disruption by low [Ca2+]. Desmosomes may thus have a positive role in metastasis by maintaining intercellular adhesion between metastasising cells.

Antibodies, Monoclonal↗
PubMed1990

Immunohistochemical staining with monoclonal antibody 32-2B to desmosomal glycoprotein 1. Its role in the histological assessment of urothelial carcinomas.

A series of transitional cell carcinomas of bladder were stained immunohistochemically with the monoclonal antibody, 32-2B, to desmosomal glycoprotein 1. All of the sections showed positive staining with the antibody. Assessment of staining intensity, by 3 independent examiners, revealed a strong negative correlation between density of desmosomal staining and degree of invasion (P = 0.012). Nests of strongly staining cells were identified in several invasive tumours, possibly indicating early squamous differentiation. Invasive tumour cells in the subepithelial stroma also stained strongly with the antibody. Correlation with clinical course, however, revealed no significant association between desmosomal staining and the incidence of recurrence or progression. It is suggested that staining with this antibody may be of value in detecting both stromal invasion and early squamous differentiation of transitional cell carcinomas. Both this and previous studies emphasise the value of this antibody as an epithelial marker in neoplasia.

Antibodies, Monoclonal↗
PubMed1990

Early expression of desmosomal components during kidney tubule morphogenesis in human and murine embryos.

Developing kidneys of human and murine fetuses have been stained with monoclonal antibodies to desmosomal proteins 1 and 2 (desmoplakins) (dp 1&2), desmosomal glycoprotein 1 (desmoglein) and a polyclonal antiserum to desmosomal glycoproteins 2 and 3 (desmocollins). All three antibodies stain the mesenchymal condensates that represent the first stage in kidney tubule development, indicating that desmosomal antigens are expressed very early in tubule morphogenesis. Desmosomal antigens are continuously expressed throughout the developing tubule being concentrated at the apical and basal regions of the lateral membranes of cells. Staining is also present in both visceral and parietal membranes of the developing Bowman's capsule. In the mature tubule, desmosomal staining becomes restricted to a discontinuous apico-lateral ring around the cells. Staining is completely lost from the visceral membrane of the mature Bowman's capsule (the podocytes) but persists in the parietal membrane. At the condensate stage, staining for dp1&2 is much more intense than staining for simple epithelial keratin. Electron microscopy showed the presence of small (ca 0.1 microns) punctate junctions in the developing tubule. These may be immature desmosomes. No fully mature desmosomes such as are present in mature kidney were found. The results suggest that desmosomal proteins and glycoproteins are involved in the early development of adhesive contacts between cells of the kidney tubule. The changing pattern of antigen expression, the loss of desmosomal staining from the podocytes and the immaturity of junctions suggest that desmosomal adhesion is labile during tubule morphogenesis, perhaps in order to facilitate changes of cell-cell contact.

Animals↗
PubMed1990

Size heterogeneity, phosphorylation and transmembrane organisation of desmosomal glycoproteins 2 and 3 (desmocollins) in MDCK cells.

Metabolic labelling with [35S]methionine and immunoprecipitation with specific antibodies to bovine desmosomal glycoproteins 2 and 3 (dg2 and dg3: desmocollins) reveals a triplet of polypeptides of Mr 115,000, 107,000 and 104,000 in MDCK cells. Tunicamycin treatment shows that this heterogeneity does not arise through differential N-linked glycosylation. Under conditions in which cells are actively forming desmosomes, the largest polypeptide, dg2, becomes phosphorylated on serine, but the two smaller polypeptides, dg3a and 3b, do not. Controlled trypsinisation of intact cells yields three membrane-protected fragments (Mr 28,000, 24,000 and 23,000) derived from these glycoproteins. The largest of these fragments is phosphorylated but the two smaller fragments are not. A monoclonal antibody to bovine dg2 and dg3 stains MDCK cells cytoplasmically. In immunoblotting of MDCK cells the monoclonal antibody recognises dg2 strongly and shows a weaker reaction with a band of lower Mr corresponding to dg3a. It also recognises the immunoprecipitated 28,000 Mr fragment from trypsinised cells and a smaller fragment of 24,000 Mr. The simplest interpretation of these data is that all three glycoproteins have a transmembrane configuration with a single membrane-spanning domain, and show heterogeneity of size and phosphorylation in their cytoplasmic domains. The data are discussed in relation to the known structures of some cell adhesion molecules. Questions about the relative roles and distributions of the different polypeptides in desmosomal organisation are raised.

Amino Acids↗
PubMed1990

Desmosomal glycoproteins 2 and 3 (desmocollins) show N-terminal similarity to calcium-dependent cell-cell adhesion molecules.

The N-terminal sequence of a mixture of desmosomal glycoproteins 2 and 3 (dg2/3, desmocollins) from bovine nasal epidermis, prepared by electro-elution from polyacrylamide gels, was determined by solid-phase Edman degradation. A sequence of 23 amino acids was obtained. This showed 43% identity with that of the N terminus of the calcium-dependent cell adhesion molecule, N-cadherin. A lesser degree of identity with other members of the cadherin-uvomorulin-L-CAM family was also found. In order to confirm that the sequence was derived from the dg2/3 molecules a rabbit antiserum was raised against a synthetic peptide corresponding to the sequence, conjugated to keyhole limpet haemocyanin (KLH). The antiserum obtained showed high (titre) activity against both the peptide and KLH in ELISA. Each activity could be specifically adsorbed with the appropriate ligand. The antiserum reacted specifically with both dg2 and dg3 of bovine nasal epidermis on immunoblots, this binding was blocked by the N-terminal peptide but was unaffected by KLH. The identity of dg2 and -3 in these preparations was confirmed by immunoblotting with two monoclonal antibodies and one polyclonal antiserum raised against the whole molecules. The N-terminal peptide antiserum was shown to bind to the intercellular space of desmosome profiles by immunoelectron microscopy on ultra-thin frozen sections. One of the two monoclonal antibodies (07-4D) also reacted with the desmosomal intercellular space. dg2 and -3 were shown by Staphylococcus aureus V8 protease digestion to have identical one-dimensional peptide maps. Both the N-terminal antiserum and 07-4D reacted with a V8 fragment of 19,000 Mr derived from dg2 and dg3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Development of desmosomal adhesion between MDCK cells following calcium switching.

The development or maturation of intercellular adhesions following their initiation has received very little attention even though this is an area of significance for a variety of in vivo processes. Using Ca2(+)-induced desmosome formation in MDCK cells as a study system it is shown that, following its initiation, desmosome formation continues for many hours. Following Ca2+ switching the major desmosomal glycoproteins, dg2/3a,b (desmocollins), accumulate progressively at the cell surface. Accumulation is first detectable within 45 min, but continues linearly for approximately 16 h, reaching a plateau at 24-32 h at 15 times the amount present in low-Ca2+ medium (LCM). Desmosomes do not increase in size during this time, but appear to become more numerous. These results suggest that cells progressively increase their desmosome-mediated adhesion over this period of time. Cycloheximide treatment shows that approximately 93% of the total dg2/3a,b accumulation is dependent upon protein synthesis after Ca2+ switching and only approximately 7% on assembly of pre-synthesised material. Thus, although triggering of desmosome formation is rapid, protein synthesis makes a major contribution to the gradual development of desmosomal adhesion in these cells. The initial assembly phase itself can be inhibited by treating cells in LCM with chloroquine, which reduces the cell surface concentration of dg2/3a,b by 40-50%. However, slow dg2/3a,b accumulation does take place in chloroquine and, if protein synthesis is permitted, desmosome formation occurs. It is suggested that when cell contacts are formed in vivo, maximisation of intercellular adhesiveness may take many hours and is dependent on the synthesis and accumulation of adhesive components.

Animals↗
PubMed1987

Recognition, calcium and the control of desmosome formation.

Since desmosome formation requires the participation of two adjacent cells, a crucial initiating event must be recognition between desmosomal adhesion molecules. Studies of mutual desmosome formation between different cell types suggest that the recognition mechanisms are highly conserved between different tissues and different species of animals. A further requirement for desmosome formation is an adequate extracellular concentration of Ca2+ (greater than 0.1 mM). Keratinocytes, MDCK cells and MDBK cells all show Ca2+-induced desmosome formation. The desmosomes of these cells also show variable stability to reduction in [Ca2+] and Ca2+ chelation. Desmosome formation at low [Ca2+] is triggered by tunicamycin in keratinocytes, suggesting that the carbohydrate moieties of desmosomal glycoproteins may be involved in the Ca2+ control mechanism. The desmosomal glycoproteins appear to bind Ca2+, while the desmosomal adhesion molecules known as desmocollins, like other Ca2+-dependent adhesion molecules, yield a soluble fragment on trypsinization in the presence of Ca2+. For desmocollins the soluble fragment has a relative molecular mass of 42,000.

Animals↗
PubMed1987

Antidesmosomal monoclonal antibody in the diagnosis of intracranial tumours.

Immunocytochemistry has been applied extensively to the diagnosis of intracranial tumours, but meningiomas still present a diagnostic problem. However, desmosomes have been shown by electron microscopy to be present in meningiomas, and this distinguishes them from gliomas. This paper describes a new monoclonal antibody, 11-5F, against desmosomal proteins 1 and 2 (desmoplakins) and assesses its usefulness in the diagnosis of meningiomas and other intracranial tumours. A total of 74 surgically removed intracranial tumours were examined by fluorescent antibody staining with 11-5F on frozen sections. In addition, a panel of antibodies against cytokeratin, vimentin, glial fibrillary acidic protein, and S100 protein was used. 11-5F stained 30/30 meningiomas and 14/14 metastatic carcinomas but 0/30 gliomas, thus distinguishing meningiomas and metastatic carcinomas from gliomas. The distinction between meningiomas and metastatic carcinomas on the basis of intermediate filaments staining was more difficult because neither the anticytokeratin nor the antivimentin antibody was specific for either tumour type. This study emphasizes the value of antidesmosomal antibodies as an important adjunct to the diagnosis of intracranial tumours.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal antibody to desmosomal glycoprotein 1--a new epithelial marker for diagnostic pathology.

Desmosomes are intercellular adhesive junctions that occur in almost all epithelia and should therefore be useful as epithelial markers in tumour diagnosis. Here, we describe a monoclonal antibody, 32-2B, to a major desmosomal glycoprotein (dgl) which reacts with human tissues in paraffin sections. This antibody was tested for its ability to stain epithelia and tumours. It reacted with all epithelia tested and with every specimen of a wide range of carcinomas. It also stained meningiomas, another desmosome-containing tumour. It did not stain other types of tumours including lymphomas, melanomas, and various sarcomas, or normal tissues which lack desmosomes. These characteristics demonstrate that 32-2B is a reliable epithelial marker that may have a useful role in diagnostic histopathology.

Animals↗
PubMed1987

Transplantable colonic adenocarcinomas in rats.

The induction of colonic adenocarcinoma using two different regimens of dimethylhydrazine (DMH) in Fischer F344 rats is described. Rats receiving 20 mg/kg of DMH per week for 20 weeks developed primary tumors with metastases, whereas rats receiving the same weekly dose for 15 weeks developed primary tumors only. The most common route of metastases was transcelomic which often was associated with ascites. The epithelial origin of malignant ascites cells was confirmed by immunofluorescent staining with antidesmosomal antibodies and demonstration of desmosomes by electron microscopy. When transplanted into syngeneic rats, the cells of the malignant ascites resulted in the development of adenocarcinomatous metastases.

Adenocarcinoma↗
PubMed1986

Hemidesmosome formation by embryonic chick corneal epithelium in vitro.

This study was undertaken in order to determine whether 15-day embryonic chick corneal epithelial cells can form hemidesmosomes when cultured on a variety of substrata. It was found that hemidesmosomes were formed on gelatin films, hydrated collagen gels, lens capsule, scraped corneal stroma, matrix produced by corneal endothelial cells and untreated tissue culture plastic. Hemidesmosomes were found after 5 days in cultures produced from either dissociated epithelial cells or whole epithelial explants. Hemidesmosomes occurred both singly and in groups and their morphology varied between well-defined structures with attachment plaques, sub-basal dense plates and connections to intracellular filamentous networks, and more rudimentary forms. The presence of extracellular material was often associated with the hemidesmosomes, although it was also possible to find hemidesmosomes where this material was absent. This work suggests that, in the embryonic chick cornea, extracellular structures such as anchoring filaments and anchoring fibres often associated with mature hemidesmosomes are not essential for hemidesmosome formation.

Animals↗