Biomedical subjects

M A Chidgey

Publications and source records attributed to M A Chidgey.

17 recordsLinked to original sources

PubMed1997

Changing pattern of desmocollin 3 expression accompanies epidermal organisation during skin development.

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of 2 families, desmocollins and desmogleins. The desmosomal cadherins show distinct patterns of expression in adult epidermis, and we have suggested that the desmocollins have a functional role in regulating the differentiation and/or morphogenesis of that epithelium (North et al. [1996] Proc. Natl. Acad. Sci. USA 93:7701-7705.). To examine this hypothesis, we cloned murine desmocollins and examined the induction patterns of desmocollins 1 and 3 during skin and skin appendage development. Desmocollins 3 and 1 were first expressed in epidermis in highly regional patterns at embryonic days 13.0 and 13.5, respectively, and both were up-regulated in general body epidermis at day 14.5. At this stage, epidermis is undifferentiated and the desmocollins showed an unexpected expression pattern. However, by day 18.5 when skin had undergone terminal differentiation, desmocollin 1 and 3 expression resembled that found in the adult. Thus, the establishment of the adult pattern of desmocollin expression corresponds to the adult pattern of epidermal stratification. We suggest that it is the ratio of desmocollin 1 to desmocollin 3 expression at different levels in the epidermis that is fundamental in establishing this pattern of differentiation.

Amino Acid Sequence↗

PubMed1997

Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis type of IgA pemphigus.

IgA pemphigus showing IgA anti-keratinocyte cell surface autoantibodies is divided into subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic IgA dermatosis (IEN) types. We previously showed by immunoblotting that IgA from some IgA pemphigus patients reacted with bovine desmocollins (Dsc), but not human Dsc. To determine the antigen for IgA pemphigus, we focused on conformation-dependent epitopes of Dsc, because sera of patients with classical pemphigus recognize conformation-sensitive epitopes of desmogleins. We constructed mammalian expression vectors containing the entire coding sequences of human Dsc1, Dsc2, and Dsc3 and transiently transfected them into COS7 cells by lipofection. Immunofluorescence of COS7 cells transfected with single human Dscs showed that IgA antibodies of all six SPD-type IgA pemphigus cases reacted with the surface of cells expressing Dsc1, but not with cells expressing Dsc2 or Dsc3. In contrast, none of seven IEN-type IgA pemphigus cases reacted with cells transfected with any Dscs. These results convincingly indicate that human Dsc1 is an autoantigen for SPD-type IgA pemphigus, suggesting the possibility of an important role for Dsc1 in the pathogenesis of this disease. This study shows that a Dsc can be an autoimmune target in human skin disease.

Animals↗

PubMed1997

Desmosomes and disease.

Considerable progress has been made in our knowledge of desmosomes and their components. Molecular cloning of the desmosomal glycoproteins has established that desmoglein 1 and desmoglein 3 are targets for autoantibodies in the blistering diseases pemphigus foliaceus and pemphigus vulgaris respectively. New evidence suggests that another desmosomal glycoprotein, desmocollin 1, is the major target antigen in the upper epidermal form of intercellular IgA dermatosis (IgA pemphigus). In human cancer there is accumulating evidence which suggests a role for desmosomes in the prevention of invasion and metastasis. The possibility exists that a mutation in a desmosomal glycoprotein gene is responsible for an inheritable human disease, the striated form of palmoplantar keratoderma.

Animals↗

PubMed1996

Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of two families, desmocollins and desmogleins. Three isoforms of each are expressed in a tissue-specific and developmentally regulated pattern. In bovine nasal epidermis, the three desmocollin (Dsc) isoforms are expressed in overlapping domains; Dsc3 expression is strongest in the basal layer, while Dsc2 and Dsc1 are strongly expressed in the suprabasal layers. Herein we have investigated whether different isoforms are assembled into the same or distinct desmosomes by performing double immunogold labeling using isoform-specific antibodies directed against Dsc1 and Dsc3. The results show that individual desmosomes harbor both isoforms in regions where their expression territories overlap. Quantification showed that the ratio of the proteins in each desmosome altered gradually from basal to immediately suprabasal and upper suprabasal layers, labeling for Dsc1 increasing and Dsc3 decreasing. Thus desmosomes are constantly modified as cells move up the epidermis, with continuing turnover of the desmosomal glycoproteins. Statistical analysis of the quantitative data showed a possible relationship between the distributions of the two isoforms. This gradual change in desmosomal composition may constitute a vertical adhesive gradient within the epidermis, having important consequences for cell positioning and differentiation.

Animals↗

PubMed1996

Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells.

The rat pheochromocytoma cell line PC12 contains two distinct pathways of protein secretion. Proteins secreted via the regulated pathway are stored in secretory vesicles and exocytosed only in response to a specific signal, whereas proteins secreted via the constitutive pathway are exported continuously. Analysis of regulated secretion of a heterologous protein in this system often relies on comparison of secretion rates with those of endogenous proteins known to be secreted via the constitutive route. In order to improve these controls, we have evaluated a number of secreted enzymes, selected for the sensitivity and convenience of their assays, as transgenic markers for the constitutive pathway. We show that both human-secreted placental alkaline phosphatase (SEAP) and bacterial beta-lactamase operate in this way in transfected PC12 cells. In contrast, transfected human tissue plasminogen activator (tPA) is shown to be sorted to the regulated pathway.

Alkaline Phosphatase↗

PubMed1996

Expression of full-length desmosomol glycoproteins (desmocollins) is not sufficient to confer strong adhesion on transfected L929 cells.

Desmocollins are cadherin-like glycoproteins that are localized in desmosomes. They are thought to play a role in cell adhesion but direct evidence for this is currently unavailable. For this reason we have expressed cDNAs encoding full-length bovine desomocollin type 1a and type 1b in mouse fibroblast (L929) cells. This system has previously been used to demonstrate the adhesive properties of E-cadherin. E-cadherin-mediated cell-cell adhesion is thought to require interaction of the cytoplasmic domain with the catenins that are expressed in L-cells. Because L929 cells do not express cytoplasmic desmosomal components that may be required for desmocollin-mediated adhesion, we constructed a chimeric cDNA encoding the bovine type 1 extracellular domain linked to the mouse E-cadherin transmembrane and cytoplasmic domains. cDNAs were transfected into cells and clones that expressed heterologous protein at the cell surface were isolated. The full-length desmocollins apparently did not interact with any other molecules, but the chimeric protein did bind to endogenous mouse alpha- and beta-catenin. Surprisingly none of the desmocollin-transfected cell lines showed significant adhesive properties under conditions where cells transfected with E-cadherin exhibited strong adhesiveness. We conclude that desmcollin expression alone is not sufficient to confer adhesion on transfected cells and more than one desmosomal component may be required.

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

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↗

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↗

PubMed1993

Protein targeting to dense-core secretory granules.

Regulated secretory proteins are stored within specialized vesicles known as secretory granules. It is not known how proteins are sorted into these organelles. Regulated proteins may possess targeting signals which interact with specific sorting receptors in the lumen of the trans-Golgi network (TGN) prior to their aggregation to form the characteristic dense-core of the granule. Alternatively, sorting may occur as the result of specific aggregation of regulated proteins in the TGN. Aggregates may be directed to secretory granules by interaction of a targeting signal on the surface with a sorting receptor. Novel targeting signals which confer on regulated proteins a tendency to aggregate under certain conditions, and in so doing cause them to be incorporated into secretory granules, have been implicated. Specific targeting signals may also play a role in directing membrane proteins to secretory granules.

Biological Transport↗

PubMed1990

Renin is sorted to the regulated secretory pathway in transfected PC12 cells by a mechanism which does not require expression of the pro-peptide.

The rat pheochromocytoma cell line PC12 targets secretory proteins into two distinct pathways. When DNA encoding human prorenin was transfected into PC12 cells, the protein was sorted into the regulated secretory pathway and released with similar kinetics to noradrenaline upon carbachol stimulation. To determine whether information for targeting prorenin lies within the pro-peptide we have transfected PC12 cells with a construct lacking the pro-peptide coding sequence. The transformed line secretes an apparently fully active enzyme and responds to carbachol stimulation with a rapid release of renin activity. We conclude that the pro-peptide of renin is not essential for targeting the protein to the regulated pathway in PC12 cells.

Adrenal Gland Neoplasms↗

PubMed1990

Percutaneous absorption of benzyl acetate through rat skin in vitro. 1. Validation of an in vitro model against in vivo data.

The percutaneous absorption in vitro of the fragrance agent benzyl acetate has been evaluated in flow-through diffusion cells using shaved full-thickness skin from male Fischer 344 rats. After the application of neat [methylene-14C]benzyl acetate to the epidermal surface of the skin and occlusion with parafilm 1.3 cm above the skin surface, the absorption of the chemical across the skin and into the receptor fluid was rapid and extensive, commencing within 1 hr of application, and reaching 49.8 +/- 3.2% (mean +/- SD, n = 4) of the applied dose after 48 hr. The coefficient of variation for absorption at 48 hr between four identical experiments was 6.4%. The extent of absorption at 48 hr of benzyl acetate applied in 50% (v/v) ethanol was not significantly different from that after application neat, although absorption at earlier times was enhanced, with a maximum increase of 8.5% of the applied dose at 12 hr. Over the dose range studied there was a linear relationship (r = 0.996) between the amount of benzyl acetate applied to the skin (1.66-33.13 mg benzyl acetate/cm2) and the amount absorbed into the receptor fluid at 24 hr (0.66 +/- 0.04-10.27 +/- 0.51 mg/cm2). The extent of absorption of benzyl acetate through rat skin in vitro was compared with the extent of absorption in vivo at 24 hr and a correlation coefficient of 0.993 was obtained. These data support the use of this in vitro system as a model to predict in vivo absorption and indicate the suitability of the system to study factors influencing the disposition of topically applied benzyl acetate.

Animals↗

PubMed1989

The pro-peptide is not necessary for active renin secretion from transfected mammalian cells.

Cultured mouse myeloma cells were transfected with expression vectors encoding the aspartyl proteinase, human renin. The full construct, encoding the renin precursor prorenin, allows transfected cells to secrete the enzymically inactive pro-protein. Activity is detectable only following trypsin treatment which mimics the physiological activation step. Accordingly, it appears that myeloma cells do not contain detectable levels of an appropriate activating proteinase. However, when these cells are transfected with a construct from which the pro-peptide coding sequence has been deleted, they secrete an apparently fully active enzyme which is indistinguishable from mature renin. We conclude that expression of the pro-peptide is not necessary to allow correct folding of the molecule and its passage through the secretory pathway.

Amino Acid Sequence↗

PubMed1987

Studies on benzyl acetate. III. The percutaneous absorption and disposition of [methylene-14C]benzyl acetate in the rat.

[methylene-14C]Benzyl acetate was applied over an area of 6.25, 12 or 18 cm2 to the shaved backs of male Fischer 344 rats under an occlusive dressing at dose levels of 100, 250 and 500 mg/kg. The compound was administered either as the neat substance or as a 50% (v/v) solution in ethanol. After 6 hr the dressing was removed, the shaven area was washed with ethanol and the dressing and washings were counted for 14C. Urine and faeces were collected for 72 hr from the start of treatment and urinary metabolites were assayed by radio-TLC and HPLC. Following administration of the neat compound, a significant proportion of the dose was recovered from the application site (28-48%) and a similar proportion (28-46%) was absorbed and excreted in the 0-24-hr urine. Excretion of 14C in the urine over 0-24 hr accounted for c. 95% of absorbed 14C in all cases, and total recovery of radioactivity was 79-84% with less than 2% of the dose present in the carcass at the end of the experiments. The extent of absorption of benzyl acetate per unit area of skin, as assessed by the recovery of its metabolites in urine, rose with increasing concentration (mg/cm2) of the test compound on the skin. The absorption of topically applied benzyl acetate was essentially the same when the dose was administered in a 50% ethanolic solution. In all cases, the major urinary metabolite was hippuric acid (c. 95% of urinary 14C), together with much smaller amounts of benzoyl glucuronide, benzoic acid and benzylmercapturic acid. The distribution of 14C in the tissues was examined 6 and 24 hr after the topical application of 5 mg [methylene-14C]benzyl acetate/kg as a 1% (v/v) solution in ethanol to rats. Radioactivity in all carcasses was less than 4% of the administered dose and levels in all the organs examined were lower at 24 than at 6 hr.

Animals↗

PubMed1986

Studies on benzyl acetate. I. Effect of dose size and vehicle on the plasma pharmacokinetics and metabolism of [methylene-14C]benzyl acetate in the rat.

Male Fischer 344 rats received [methylene-14C]benzyl acetate by gavage in a dose of 5,250 or 500 mg/kg, as the neat substance, in corn oil or in propylene glycol. Urine and faeces were collected and urinary metabolites were assayed by radio-TLC and HPLC. Other animals were killed at various times and exsanguinated, and plasma levels of 14C in Plasma occurred earliest and were highest when benzyl acetate was given neat. Peak levels were lower and absorption was delayed with the propylene glycol vehicle. The use of corn oil as the dose vehicle at the higher doses (250 and 500 mg/kg) led to the maintenance of plateau plasma levels, at about one half of the peak levels seen with the neat compound, for up to 8 hr after administration. At the 5 mg/kg dose, the plasma levels of 14C were essentially the same whether the dose was given in corn oil or propylene glycol. At the 250- and 500-mg/kg doses, at all time points, the major metabolite in plasma was benzoic acid, accompanied by smaller amounts of hippuric acid. Benzyl alcohol was also detected in some plasma samples. At the 5-mg/kg dose, the major plasma metabolite was hippuric acid, together with a smaller amount of benzoic acid. When propylene glycol was used as the vehicle at this dose level, benzylmercapturic acid was also present in the plasma. The major urinary metabolite was hippuric acid (c. 66% of the dose), with benzoic acid (2%) and benzylmercapturic acid (1%) also present. The elimination of benzoyl glucuronide increased with increasing dose, from c. 3 to 11% of the dose.

Animals↗

PubMed1986

Studies on benzyl acetate. II. Use of specific metabolic inhibitors to define the pathway leading to the formation of benzylmercapturic acid in the rat.

Specific metabolic inhibitors were used to define the route of metabolism of benzyl acetate leading to the formation of benzylmercapturic acid. Male Fischer 344 rats were dosed by gavage with [methylene-14C]benzyl acetate (500 mg/kg) alone or together with pyrazole (200 mg/kg), pentachlorophenol (10 mg/kg) or both pentachlorophenol (10 mg/kg) and pyrazole (200 mg/kg), given in each case ip. Urine and faeces were collected and urinary metabolites were assayed by radio-TLC and HPLC. The excretion of 14C was rapid in all cases, with most of the dose excreted in the urine within 24 hr. Co-administration of pyrazole (an inhibitor of alcohol dehydrogenase) with benzyl acetate caused an 11-fold increase in the excretion of benzylmercapturic acid and halved the percentage of the dose excreted as benzoyl glucuronide. Pretreatment with pentachlorophenol, an inhibitor of sulphotransferase activity in vivo, abolished the excretion of benzylmercapturic acid, while excretion of the mercapturate following treatment with both pyrazole and pentachlorophenol was higher than in control or pentachlorophenol-treated rats, but much lower than in the animals given pyrazole alone. Taken together, these results suggest very strongly that the formation of benzylmercapturic acid involves the sulphate ester of benzyl alcohol as an obligatory intermediate and does not appear to involve a metabolic intermediate with sufficient reactivity to have toxicological relevance.

Acetylcysteine↗