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D R Garrod

Publications and source records attributed to D R Garrod.

At least 55 records · Page 3Linked to original sources
PubMed1995

Induction of early stages of kidney tubule differentiation by lithium ions.

Kidney tubules develop by a mesenchyme-epithelium transition, normally induced by ureteric bud through a mechanism that remains obscure. Murine nephrogenesis in vitro has always required heterologous inducing cells. We have discovered that Li+ can elicit the early stages of epithelial differentiation in isolated nephrogenic mesenchyme. We have made detailed comparisons of the timing of morphoregulatory molecule expression between Li(+)-mediated induction and the traditional in vitro method using induction by spinal cord. Both followed the same program of early morphoregulatory molecule expression, though Li(+)-induced samples failed to progress into the later parts of the nephrogenic process. Mesenchymes induced by Li+ showed more DNA synthesis than controls, though less than those induced by spinal cord. Discovery of a chemical means to activate differentiation in the absence of heterologous tissue offers a new basis for studying molecular mechanisms regulating the early events of nephrogenesis, as well as for investigating transduction of inductive signals that initiate the process.

Animals↗
PubMed1995

A case of pemphigus vulgaris showing reactivity with pemphigus antigens (Dsg1 and Dsg3) and desmocollins.

Both pemphigus vulgaris antigen (PVA; Dsg3) and pemphigus foliaceus antigen (PFA; Dsg1) are members of the desmoglein subfamily of the cadherin supergene family. Another desmosomal cadherin, desmocollin, is occasionally recognized by certain pemphigus sera. We present a 38-year-old Japanese male who showed clinically and histopathologically typical features of pemphigus vulgaris, whose sera reacted with all PVA, PFA, and desmocollins using immunoblotting of both human epidermis and bovine snout epidermis. Studies using domain-specific fusion proteins of PFA and PVA suggested that this patient's serum reacted with the intracellular domain of PFA and the extracellular domain of PVA, the latter of which seems to be responsible for initiating the skin lesion. The patient's serum showed reactivity with human desmocollin and was shown to react with bovine Dsc2 fusion protein, further suggesting the significance of anti-desmocollin autoantibodies in pemphigus. These results indicate that certain pemphigus cases may produce antibodies against multiple antigen molecules, although the complex mechanism of the production of autoantibodies remains to be elucidated.

Adult↗
PubMed1995

Demonstration of antibodies to bovine desmocollin isoforms in certain pemphigus sera.

We have shown previously that IgG antibodies in certain pemphigus sera, particularly endemic Brazilian pemphigus foliaceus (BPF) sera, react with bovine desmocollins (Dsc), which are transmembranous glycoproteins of desmosome junctions. Desmocollins occur as three different isoforms (Dsc 1, 2 and 3), all of which are represented in the epidermis. In this study, we examined sera of various pemphigus types by immunoblotting purified bovine desmosomes and bovine Dsc 1, 2 and 3 fusion proteins, expressed in pGEX expression vectors. Six of 15 (40.0%) BPF sera, two of 18 (11.1%) non-endemic pemphigus foliaceus sera, eight of 39 (20.5%) pemphigus vulgaris (PV) sera, and two of 11 (18.2%) normal sera, showed reactivity with Dsc from desmosomes. Experiments with fusion proteins showed that no Dsc isoform was specifically recognized by sera of any individual pemphigus type. Our results indicate that the pathogenesis of pemphigus might be more complex than previously believed.

Animals↗
PubMed1995

Regulation of desmocollin transcription in mouse preimplantation embryos.

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527-539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

Animals↗
PubMed1995

Characterisation of a desmocollin isoform (bovine DSC3) exclusively expressed in lower layers of stratified epithelia.

Desmocollins are cadherin-like glycoproteins involved in cell adhesion and plaque formation in desmosome junctions. Three distinct isoforms, the products of different genes, have been found in bovine tissues. We have reported previously that one of these, DSC3, is expressed only in basal and lower suprabasal layers of stratified epithelia. Using RT-PCR we have now obtained the complete cDNA coding sequence of mature bovine DSC3. It has alternatively spliced 'a' and 'b' forms found in other desmocollins but is unique in having a 43 instead of a 46 base pair exon. We have characterised a monoclonal antibody, 07-4G, which is specific for the Dsc3 protein, recognising an epitope in the extracellular domain. Immunofluorescent staining with 07-4G confirms that this isoform is found only in stratified epithelia, being strongly expressed in the basal cell layers of these tissues. The intensity of expression fades gradually in the suprabasal layers and disappears completely below the upper limit of desmosome expression. These results suggest that Dsc3 plays an important role in cell epithelial differentiation.

Amino Acid Sequence↗
PubMed1995

Desmosomes and cancer.

Desmosome expression is downregulated in poorly differentiated transitional cell carcinomas and squamous cell carcinomas of the head and neck. In these tissues, desmosomes may have an invasion and metastasis suppressor function. In colorectal carcinoma, no downregulation is found. This raises the question of how tumours possessing intact desmosomes can invade and metastasize. Temporary modulation of adhesive affinity may be a possible mechanism. Desmosomes are widely distributed in epithelia and other tissues and desmoplakins are expressed in all. Thus, antibodies to desmoplakins are very reliable markers for immunohistochemical diagnosis of carcinomas. Intracranial tumours can also be distinguished because meningiomas possess desmosomes and vimentin intermediate filaments, but metastatic carcinomas possess desmosomes and keratin.

Animals↗
PubMed1995

Immunofluorescence and immunoblot studies on the reactivity of pemphigus vulgaris and pemphigus foliaceus sera with desmoglein 3 and desmoglein 1.

We have investigated expression of pemphigus vulgaris (PV) antigen, desmoglein 3 (Dsg3), and pemphigus foliaceus (PF) antigen, desmoglein 1 (Dsg1), in various tissues. Immunofluorescence studies suggested that Dsg1 and Dsg3 are preferentially expressed in the upper and lower epidermis, respectively. With immunoblotting of human epidermal extracts, all PV sera reacted with Dsg3 but not with Dsg1. By contrast, only half of PF sera reacted with Dsg1 but none reacted with Dsg3. These results confirm the distinct antibody specificity between PV and PF sera. They also suggest that PV sera contain antibodies against linear epitopes present even on the denatured antigen, while some PF sera contain antibodies only against conformational epitopes present on the native antigen. With immunoblotting of bovine desmosome preparations, certain PV sera and an anti-Dsg monoclonal antibody reacted with both Dsg1 and Dsg3. Affinity-purification of these PV antibodies suggested that the simultaneous reactivity with Dsg1 and Dsg3 was produced by two different subsets of antibodies and not by cross-reactivity of single antibodies. This study indicates that pemphigus serum is a useful probe with which we learn various aspects of keratinocyte biology.

Animals↗
PubMed1994

Detection of anti-desmocollins I and II autoantibodies in two cases of Hallopeau type pemphigus vegetans by immunoblot analysis.

Pemphigus foliaceus (PF) sera react with a 150 kDa antigen, desmoglein (DG), while pemphigus vulgaris (PV) sera react with a 130 kDa PV antigen. Recent molecular cloning studies have revealed that both DG and PV antigen are members of the cadherin family of cell adhesion molecules and that PV antigen shows very high homology to DG. We have recently noticed that other desmosoal cadherin molecules, desmocollins I and II (DC I/II), are also recognized by some pemphigus antibodies. In the present study we report two cases of Hallopeau type pemphigus vegetans. Immunoblot study revealed that the sera of these cases reacted not only with the PV antigen but also with both DG (PF antigen) and DC I/II. Although the significance of these findings is not known at present, further studies for the antigen profile for more Hallopeau type pemphigus vegetans patients may unravel the pathogenesis of this rare type of pemphigus.

Adult↗
PubMed1994

The bovine desmocollin family: a new gene and expression patterns reflecting epithelial cell proliferation and differentiation.

We have discovered a third bovine desmocollin gene, DSC3, and studied expression of all three desmocollin genes, DSC1, 2, and 3, by Northern blotting, RT-PCR and in situ hybridization. DSC1 is strongly expressed in epidermis and tongue papillae, showing a "skin"-type pattern resembling that previously described for keratins 1 and 10. Expression is absent from the epidermal basal layer but appears in the immediate suprabasal layers and continues uniformly to the lower granular layer. In tongue epithelium, expression is suprabasal and strictly localized to papillae, being absent from interpapillary regions. In other epithelial low level DSC1 expression is detectable only by RT-PCR. The distribution of Dsc1 glycoproteins, detected by an isoform-specific monoclonal antibody, closely reflects mRNA distribution in epidermis and tongue. DSC2 is ubiquitously expressed in epithelia and cardiac muscle. In stratified epithelia, expression appears immediately suprabasal, continuing weakly to the lower granular layer in epidermis and to just above half epithelial thickness in interpapillary tongue, oesophageal, and rumenal epithelia. DSC3 expression is restricted to the basal and immediately suprabasal layers in stratified epithelia. In deep rete ridges DSC expression strikingly resembles the distribution of stem, transit-amplifying, and terminally differentiating cells described by others. DSC3 expression is strongly basal, DSC2 is strong in 5-10 suprabasal layers, and then weakens to be superseded by strong DSC1. These results suggest that desmocollin isoform expression has important functional consequences in epithelial proliferation, stratification, and differentiation. The data also provide a standard for nomenclature of the desmocollins.

Alternative Splicing↗
PubMed1994

Demonstration of desmosomal antigens by electron microscopy using cryofixed and cryosubstituted skin with silver-enhanced gold probe.

In a previous post-embedding immunogold electron microscopic (EM) studies, localization of various desmosomal antigens was possible at high but not at low magnification. We developed a method for simultaneous demonstration of epidermal desmosomal antigens at both low- and high-power EM magnifications by a method based on cryofixation and acetone cryosubstitution and the use of a 1-nm gold probe with silver enhancement. Ultra-thin sections of Lowicryl K11M were incubated with primary antibodies against desmoplakin, desmocollin, or desmoglein, followed by 1-nm gold-conjugated secondary antibody. Silver enhancement for 12 min provided the ideal labeling size for low-power visualization, whereas silver enhancement for 4-6 min was ideal for high-power EM observation. Each desmosome immunolabeled with the gold probe was clearly demonstrated, even at very low-power magnification. The level of background labeling could be determined easily and the area of interest for high-power observation selected accurately. The fine ultrastructural appearance of desmosomal molecules was precisely demonstrated on high-power observation. This system should be useful for the immunocytochemical study of a variety of desmosomal antigens as well as other molecules of interest.

Adult↗
PubMed1993

Desmosomes and hemidesmosomes.

Desmosomes and hemidesmosomes are extremely different in their molecular composition. Most of the protein and glycoprotein components are products of members of multigene families, but show specialization for plaque formation and intermediate filament attachment. Desmosomal glycoproteins are more heterogeneous than previously suspected, with different isoforms showing tissue-specific and differentiation-related expression. Both types of junctions can be modulated in response to extracellular signals and may turn out to be involved in signal transduction.

Amino Acid Sequence↗
PubMed1993

Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus.

Recently, it has been shown that desmoglein, pemphigus foliaceus target antigen, and a 130-kD pemphigus vulgaris antigen belong to the cadherin family of cell adhesion molecules. We tried to determine whether desmocollins I/II, other cadherin-like transmembranous glycoproteins present in desmosomes, are also recognized by pemphigus autoantibodies of the IgG class. We examined 16 pemphigus vulgaris sera, 15 pemphigus foliaceus sera, 15 Brazilian pemphigus foliaceus sera, five bullous pemphigoid sera, and 65 normal sera. Four (25%) pemphigus vulgaris sera, one (7%) pemphigus foliaceus serum, eight (53%) Brazilian pemphigus foliaceus sera, and three (5%) normal sera reacted with desmocollins I/II on immunoblots of bovine desmosome preparation. The affinity-purified desmocollins I/II pemphigus autoantibodies were shown to bind the epidermal cell surface by indirect immunofluorescence. Immunoblot analysis revealed one pemphigus vulgaris serum, one Brazilian pemphigus foliaceus serum, and one normal serum recognizing a recombinant protein produced by a desmocollin cDNA clone. Moreover, immunoblot analysis of reactivity of a Brazilian pemphigus foliaceus serum with recombinant proteins produced by deletion mutants of the desmocollin cDNA clone showed that the extracellular portion of desmocollin is immunogenic in this pemphigus patient. We conclude that desmocollins I/II are recognized by certain sera from patients with various types of pemphigus, particularly Brazilian pemphigus foliaceus. However, the significance of this reactivity remains to be defined.

Animals↗
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↗