Search PubMed⌕ Search

Biomedical subjects

B A Dale

Publications and source records attributed to B A Dale.

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

Familial pityriasis rubra pilaris.

BACKGROUND: Familial pityriasis rubra pilaris is a rare autosomal dominant skin disorder. Four individuals from one family are described who demonstrate clinical features compatible with a diagnosis of familial pityriasis rubra pilaris. Results of light and electron microscopic, immunocytochemical, and biochemical analysis of skin biopsy specimens from three of these four individuals are presented. OBSERVATIONS: All affected individuals demonstrated erythematous scaly skin with follicular prominence and islands of sparing. Inheritance was consistent with an autosomal dominant trait. Light and electron microscopic findings were compatible with those reported in sporadic cases of pityriasis rubra pilaris. Immunocytochemistry showed suprabasal staining with monoclonal antibody AE1. Immunoblot analysis revealed abnormal keratins with K6/16 expression, the possibility of an abnormal K14 or K16, and a 45-kd acidic keratin not normally expressed in epidermis. Because similar biochemical analyses have not been reported previously in other cases of pityriasis rubra pilaris (familial or sporadic), comparisons cannot be made. CONCLUSIONS: The observations suggest that the cutaneous abnormality in this family extends beyond clinical and morphological alterations to abnormalities in biochemical markers of epidermal differentiation.

Adult↗
PubMed1994

Filaggrin expression in epidermolytic ichthyosis (epidermolytic hyperkeratosis).

To evaluate the role of filaggrin in keratin filament aggregation in epidermolytic ichthyosis (epidermolytic hyperkeratosis, EH), we studied EH skin by light and electron microscopic immunohistochemistry, and biochemical analysis using sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting. Immunohistochemical staining showed an increased number of filaggrin-immunoreactive cell layers, but the reaction was still confined to the mid- and upper epidermal layers, whereas an abnormal granular pattern of staining for K10 began in the lower suprabasal cell layers. This suggests that the aggregation of keratin filaments precedes, and occurs independently of, profilaggrin synthesis during epidermal differentiation. Although keratohyalin granules were frequently associated with clumped filaments, immunoelectron microscopy showed that K10 labelling was confined to keratin filaments (including clumped filaments), and that antifilaggrin antibodies stained only keratohyalin granules, at least in the living cells. Certain keratin aggregates in the cornified cells were still devoid of filaggrin staining. However, in some cells which appeared partially cornified, filaggrin immunoreactivity occurred over the aggregated keratin filaments. Immunoblotting showed a clear increase of filaggrin/profilaggrin expression, without evidence for a qualitative abnormality. It seems unlikely, therefore, that filaggrin is primarily involved in the keratin filament clumping in EH, but that in some EH cases it interacts with keratins in a defective manner, possibly due to premature cell death and profilaggrin processing and/or altered keratin filament structure involving the interaction points of keratin with filaggrin.

Adolescent↗
PubMed1993

Independent regulation of two cytoplasmic processing stages of the intermediate filament-associated protein filaggrin and role of Ca2+ in the second stage.

One of the final events in cornification of epidermal cells is processing of profilaggrin to the keratin-associated protein filaggrin. Processing involves several proteolytic events and occurs in two discrete proteolytic stages (Resing, K. A., Walsh, K. A., and Dale, B. A. (1984) J. Cell Biol. 99, 1372-1378; Resing, K. A., Walsh, K. A., Haugen-Scofield, J., and Dale, B. A. (1989) J. Biol. Chem. 264, 1837-1846). In a keratinocyte cell line derived from newborn rat epidermis, these two stages are independently regulated. Profilaggrin was expressed when the cells reached confluence; processing to intermediates began 24-36 h later (stage one), with filaggrin appearing at 48 h (stage two). Stage two processing required calcium in the medium with maximum processing occurring at 5-10 mM. Furthermore, stage two processing was inhibited by nifedipine, a calcium channel blocker, suggesting that calcium influx activates this event. Second-stage processing was also inhibited by the protease inhibitor leupeptin, implicating calpain. Confluent cells had higher levels of calpain I than subconfluent cells; in confluent cells, two immunoreactive bands were detected, comigrating with inactive (80 kDa) and activated (78 kDa) calpain I. In cells processing profilaggrin, most of the calpain I was in the 78-kDa form, implying extensive activation, supporting a role for calpain in processing.

Animals↗
PubMed1993

Harlequin ichthyosis. Variability in expression and hypothesis for disease mechanism.

BACKGROUND: Harlequin ichthyosis is an inherited skin disorder that usually results in death shortly after birth. Although the clinical features of this disorder are well described, the underlying molecular basis is not understood. In this article, we discuss the results of the latest histologic, immunochemical, and Western immunoblotting studies done in our laboratory and propose a hypothesis for molecular basis of this disorder. OBSERVATIONS: Previous experiments done in our laboratory show suggestive evidence for defective lipid synthesis and protein dephosphorylation in harlequin ichthyosis. Our latest study shows that the catalytic subunit of one of the most prevalent protein phosphatase, type 2A protein phosphatase, appears to be altered in some cases of type 2 harlequin ichthyosis. CONCLUSIONS: Based on these observations and the known functions of protein phosphatase in keratinocytes, we hypothesize that the underlying molecular basis of harlequin ichthyosis may be related to mutations affecting protein dephosphorylation. We further describe approaches by which this hypothesis can be tested.

Cells, Cultured↗
PubMed1993

Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes.

Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.

Animals↗
PubMed1993

Identification of rat epidermal profilaggrin phosphatase as a member of the protein phosphatase 2A family.

The aggregation of cellular intermediate filaments is an important step in the terminal differentiation of keratinocytes. It has been shown that epidermal filaggrin can cause intermediate filaments to aggregate in vitro and may also have the same function in vivo. Filaggrin is derived via dephosphorylation and proteolysis from a highly phosphorylated precursor, profilaggrin, which is found in the granular layer of the epidermis. Using casein kinase II phosphorylated filaggrin as substrate, a profilaggrin phosphatase has been partially purified from rat epidermal homogenate by three chromatographic steps (DE52, hydroxylapatite and S200 gel filtration). Profilaggrin phosphatase activity eluted from the last column has a Km of 0.12 mM and a Vmax of 8 nmol/mg/min with respect to phosphofilaggrin. Results obtained by initial rate analysis showed that the enzymatic activity is not affected by phospho-tyrosyl phosphatase inhibitors and the active fractions preferentially dephosphorylate the alpha subunit of phosphorylase kinase which has been phosphorylated by cAMP-dependent kinase. These results suggest that epidermal profilaggrin phosphatase is not a phospho-tyrosyl phosphatase or a type 1 phospho-seryl/phospho-threonyl phosphatase. Dephosphorylation is not affected by EDTA, calcium or magnesium, but is very sensitive to okadaic acid inhibition (IC50 = 80 pM), suggesting that the enzymatic activity is related to that of the protein phosphatase 2A (PP2A).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.

Filaggrin is an intermediate filament-associated protein which functions to aggregate keratin intermediate filaments in the stratum corneum of mammalian epidermis. It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. In this report, we describe the characterization of cosmid genomic clones containing the human profilaggrin gene coding for 11 complete filaggrin repeats of 324 amino acids each. At the amino- and carboxyl-terminal ends of human profilaggrin are leader and tail peptide sequences of 293 and 157 amino acids, respectively, which differ from filaggrin. The leader peptide is composed of two distinct domains: an 81-residue segment which shows significant homology to the S-100 family of EF hand-containing calcium-binding proteins, and a hydrophilic second domain of 212 residues. The gene is divided into three exons, with one intron (approximately 9.6 kilobase pairs) in the 5' noncoding region and a second one of 570 base pairs between the EF hands. The position of intron 2 is identical to that of other members of the S-100-like family. The presence of an S-100-like domain suggests that profilaggrin binds calcium and that the calcium binding domain is functionally significant in the formation of keratohyalin and/or the subsequent processing of profilaggrin to filaggrin, both of which may be calcium-dependent events.

Amino Acid Sequence↗
PubMed1992

CHILD syndrome: lack of expression of epidermal differentiation markers in lesional ichthyotic skin.

Congenital hemidysplasia with ichthyosiform erythroderma and limb defects (CHILD) syndrome is a rare genetic disorder. The epidermal abnormalities associated with the unilateral ichthyosis have previously been examined only by morphology. In order to describe these abnormalities more completely we analyzed the expression of markers of epidermal differentiation (keratins and filaggrin), grew keratinocytes in culture, and correlated the results with ultrastructural features. Expression of all differentiation markers was significantly reduced or absent, whereas keratins K5 and K14 and keratins K6 and K16 were strongly expressed in lesional epidermis, suggesting that basal cell keratin expression was not down-regulated as in normal epidermis and that lesional keratinocytes mature via an abnormal pathway. When removed from the tissue and grown in culture, keratinocytes from lesional and non-lesional biopsies had similar phase microscopic morphology as well as keratin and profilaggrin expression, in contrast to the extreme differences in vivo. Lesional keratinocytes also had similar contents of keratin filaments and keratohyalin, but showed abnormal accumulation of intercellular vesicles and debris and altered cell-cell and cell-substratum interaction. Comparison of the results in tissue and in culture suggests that systemic or dermal factors influence the abnormal structural protein expression and ichthyosiform epidermal differentiation seen in CHILD syndrome, but that lesional keratinocytes maintain abnormalities in the secretion and accumulation of extracellular material in vitro similar to the lesional tissue in vivo.

Abnormalities, Multiple↗
PubMed1992

Abnormal lamellar granules in harlequin ichthyosis.

Lamellar granules are specialized lipid-rich organelles present in epidermal granular cells. They fuse with the apical cell surface and discharge their contents into the intercellular space forming lamellar sheets. It was previously shown by electron microscopy that lamellar granules in biopsies of infants affected with harlequin ichthyosis are either absent or abnormal and no intercellular lamellae could be detected. A monoclonal antibody (AE17) directed against a protein component of lamellar granules was used for immunoblotting and immunohistochemical studies as an indication of both the presence and function of lamellar granules. Epidermal extracts from all harlequin and normal specimens tested showed an immunoreactive protein of 25-28 kD. Immunohistochemical staining of normal skin using AE17 showed apical cytoplasmic staining in the granular layer and intercellular staining between the granular and stratum corneum cells. Harlequin samples showed variable degrees of staining ranging from little to heavy apical cytoplasmic staining of granular cells. No intercellular staining was detected. The immunohistochemical staining pattern correlated with the electron microscopic localization of abnormal vesicles and the absence of intercellular lamellae in the affected samples. We conclude that the vesicles represent lamellar granules that contain the AE17 antigen but are structurally abnormal and defective in their ability to discharge both their lipid and protein contents into the intercellular space. We suggest that this defect in the lamellar granules represents the underlying basis for stratum corneum cell retention and subsequent accumulation of scale in harlequin ichthyosis.

Adult↗
PubMed1991

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in ichthyosis vulgaris.

Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo.

Animals↗
PubMed1990

Human oral epithelial cell culture. II. Keratin expression in fetal and adult gingival cells.

Epithelial cells from human fetal and adult gingiva were cultured in keratinocyte growth medium (KGM), a serum-free medium. The expression of keratin proteins in these cells was evaluated using immunohistochemistry and SDS-PAGE-immunoblot analysis and compared with expression in the tissue. Keratins 5, 6, 14, 16, and 19 were identified in cells cultured from both fetal and adult tissues. K19 was localized in basal cells of fetal oral tissue but was not seen in adult gingiva (except for scattered Merkel cells). K1 and K10 were expressed in tissue, but not in cultured cells. The keratin profiles of cultured epithelial cells from several adult donors were similar and were identical in cultures from primary through Passage 5. K13, a differentiation-specific keratin, was expressed in all suprabasal cells of fetal oral epithelium, but shows only spotty expression in adult gingival tissue. K13 was expressed in cultures of fetal cells, but very weakly or not at all in cultures of adult cells. K13 expression was greater in cultures grown with physiologic calcium concentrations (1.2 mM) than in those grown at 0.15 mM or less. Our findings are consistent with basal-like characters of these cells in 0.15 mM calcium growth conditions. Differentiation of fetal oral cells in culture to the suprabasal basal cell stage in 1.2 mM Ca2+ is shown by the expression of K13.

Adult↗
PubMed1990

Filaggrin, an intermediate filament-associated protein: structural and functional implications from the sequence of a cDNA from rat.

Filaggrin is an intermediate filament-associated protein that is involved in aggregation of keratin filaments in fully cornified cells of the mammalian epidermis, and is an important marker for epidermal differentiation. In this report, the sequence of a rat cDNA clone coding for a portion of the polymeric precursor, profilaggrin, is presented. The cDNA is 2,314 bp long with 1,875 bp of coding region ending with an A-T-rich 3' noncoding region. Genomic analysis indicates that the profilaggrin gene consists of 20 +/- 2 repeats of 1,218 bp of sequence coding for 406 amino acids, making the mRNA at least 25-27 kb in length. Each repeat consists of a filaggrin domain and a linker sequence with an estimated size of 380 and 26 amino acids, respectively. High levels of profilaggrin mRNA are found only in keratinizing epithelia. Comparison of the rat filaggrin sequence with that of mouse and human filaggrin and with the sequence of phosphorylated peptides from mouse profilaggrin indicates that the proteins share extensive amino acid sequence similarities, especially in the two phosphorylated regions. Proteolytic processing sites are also quite similar in rat and mouse. The three species show blocks of sequence that are similar in length and composition which alternate with sequences that are variable in length. This analysis suggests that the evolution of the present-day filaggrins has been constrained by maintenance of phosphorylation sites and overall amino acid composition. The cDNAs for the profilaggrins are similar in structure, reflecting genes that have simple repeating structures and lack introns within their coding regions. Mouse and rat profilaggrin terminate with a nonpolar sequence atypical of the rest of the coding region, and have similar 3' noncoding regions. To explain these observations, a novel evolutionary model is proposed.

Amino Acid Sequence↗
PubMed1990

Heterogeneity in harlequin ichthyosis, an inborn error of epidermal keratinization: variable morphology and structural protein expression and a defect in lamellar granules.

Skin biopsies and scale samples from nine infants and one fetus affected with harlequin ichthyosis (HI) were obtained from eight families. Epidermal differentiation was examined by morphologic and biochemical criteria and cell culture studies. Two striking abnormalities were identified; first, keratin and filaggrin expression were abnormal and varied between cases, and, second, in all cases lamellar granules were absent or abnormal, and intercellular lamellae within the stratum corneum were absent. Three HI phenotypes were distinguished by variable expression of epidermal structural proteins. Cases were classified by the absence (type 1) or presence (types 2 and 3) of keratins K6 and K16 ("hyperproliferative" keratins) and by the presence of profilaggrin in the interfollicular epidermis (types 1 and 2 only). Profilaggrin is apparently not converted to filaggrin, but it is retained in the scale. The block in profilaggrin processing may be due to an inactive phosphatase. Siblings in two families (presenting with types 1 and 2) showed the same type classification suggesting that expression of the phenotype is consistent within families but differs between families. Cultured HI keratinocytes were normal by phase microscopy, but abnormal by electron microscopy with no lamellar granules and extensive stacking of the upper layers. We conclude that harlequin ichthyosis is a genetically heterogeneous group of disorders with altered lamellar granules, intercellular lipids, and variation in expression and/or processing of structural protein markers of normal epidermal keratinization. Furthermore, the lamellar granule and structural protein defects may be indirectly related via a mechanism involving phosphorylation/dephosphorylation.

Biopsy↗