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Biomedical subjects

B A Dale

Publications and source records attributed to B A Dale.

At least 109 records · Page 6Linked to original sources
PubMed1985

Expression of epidermal keratins and filaggrin during human fetal skin development.

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules.

Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized histologically by absent or reduced keratohyaline granules in the epidermis and mild hyperkeratosis. The basic defect in ichthyosis vulgaris is unknown. We have tested for the presence of filaggrin and its precursor, profilaggrin, in the epidermis of affected and unaffected individuals from 2 families with ichthyosis vulgaris and correlated its presence and relative quantity with ultrastructure findings in the same individuals. Filaggrin was present on stained sodium dodecyl sulfate gels and immunoblots of epidermal proteins from controls and unaffected family members. It was absent from the more severely affected individuals in each family and reduced in intensity in the less severely affected family members. Immunohistology in controls showed localization of filaggrin-related protein in the stratum corneum and within the granular layer. In contrast, tissue from affected individuals showed little or no reaction. Electron microscopic studies showed that keratohyaline granules were absent in 3 severely affected individuals, and reduced in number in the others. The relative amount of keratohyalin by electron microscopy correlated with the amount of filaggrin detectable on immunoblots. The stratum corneum was thicker than in normals but showed the typical "keratin pattern" staining suggesting that filaggrin is not essential for keratin filament aggregation and may have another function in vivo. We have demonstrated that the structural proteins, profilaggrin and filaggrin, are reduced or absent in 5 patients from 2 pedigrees with ichthyosis vulgaris. This biochemical abnormality correlates with the morphologic reduction in the amount of keratohyalin, and with the clinical severity of the disorder.

Adult↗
PubMed1985

Profilaggrin, a high-molecular-weight precursor of filaggrin in human epidermis and cultured keratinocytes.

Filaggrin is a histidine-rich matrix protein of keratinized epidermis. Filaggrin is synthesized as a high-Mr, phosphorylated precursor, profilaggrin, that is processed to form the lower-Mr product present in cornified cells. This study reports the identification of profilaggrin in human epidermis and in unusually well-differentiated cultured human keratinocytes with the use of a polyclonal antihuman filaggrin antiserum. Polyclonal antiserum raised against human filaggrin stained keratohyaline granules and stratum corneum in tissue sections of human skin. Analysis of epidermal extracts showed an immunoreactive low-Mr band (37,000), previously identified as filaggrin, and an immunoreactive high-Mr band (greater than 220,000). Both [32P] phosphate and [3H]histidine were incorporated into the high-Mr band after pulse labeling for 3 h. After a 15-h chase, [3H]histidine, but not [32P]phosphate appeared in filaggrin. Human foreskin keratinocytes cultured on a 3T3 feeder layer were unusually well differentiated. Numerous well-formed keratohyaline granules, complete desmosomes, lamellar granules, and cornified cell envelopes were observed. Immunofluorescence with antihuman filaggrin antiserum showed a granular staining pattern in the more stratified cells. Extracts of cultured cells contained a diffuse high-Mr immunoreactive band but no immunoreactive equivalent of filaggrin. These studies suggest that human skin filaggrin, like rodent filaggrin, is synthesized as a high-Mr, phosphorylated, histidine-rich precursor (profilaggrin) that is processed via posttranslational modification to filaggrin. In human keratinocyte cultures a similar high-Mr precursor is present, but evidence of processing to the lower-Mr product, filaggrin, is lacking.

Animals↗
PubMed1984

High-molecular-weight precursor of epidermal filaggrin and hypothesis for its tandem repeating structure.

Filaggrin is a histidine-rich protein that is intimately involved in mammalian epidermal keratinization. Using a combination of immunologic and in vivo pulse-chase studies with radiolabeled histidine and phosphate, we show that the phosphorylated precursor of both rat and mouse filaggrin has an apparent molecular weight much higher than previously realized (6 X 10(5) and 3.9 X 10(5), respectively). These high-molecular-weight filaggrin precursors can be rapidly labeled with histidine and extracted from the epidermis under denaturing conditions. More than half of the label incorporated in the precursor at 2 h is found in filaggrin at 24 h after injection, even though filaggrin is less than 10% of the size of the precursor. Limited proteolytic digestion of the precursor in vitro results in the formation of an oligomeric series of peptides based on a phosphorylated fragment slightly larger than filaggrin itself. More extensive digestion of this fragment shows that it is composed of filaggrin with few or no additional unrelated peptides, suggesting that the major part of the high-molecular-weight filaggrin precursor must be composed of repeated domains of filaggrin. Because the primary translation product of filaggrin mRNA is large, we propose that these domains are repeated in tandem. In addition, from molecular weight computations and peptide map analyses, we suggest that the filaggrins are themselves composed of multiple repeating units of an unidentified peptide of approximately Mr 8600. This value is derived from the molecular weights of filaggrin from several mammalian species that differ by integral multiples of 8600. A model for the structure of the high-molecular-weight precursor of filaggrin is presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1984

Abnormal keratinization in the pupoid fetus (pf/pf) mutant mouse epidermis.

During its development the epidermis of the pf/pf mutant mouse is invaded by cells from the underlying dermis. These invading cells establish a network of cells including fibroblasts, endothelial cells, and nerve fibers, throughout the epidermis. Subsequent to these events the keratohyalin protein, filaggrin, is drastically reduced and keratinization fails to occur. Heterotypic tissue recombinations indicate that the pf gene is not expressed in the skin. After simply grafting whole mutant dorsal skin, filaggrin synthesis is initiated and an orderly process of epidermal differentiation is achieved. These results suggest that the pf gene acts systemically and that the failure of epidermal differentiation in the mutant occurs secondary to abnormal epidermal organization.

Animals↗
PubMed1984

Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis.

A major event in the keratinization of epidermis is the production of the histidine-rich protein filaggrin (26,000 mol wt) from its high molecular weight (greater than 350,000) phosphorylated precursor (profilaggrin). We have identified two nonphosphorylated intermediates (60,000 and 90,000 mol wt) in NaSCN extracts of epidermis from C57/Bl6 mice by in vivo pulse-chase studies. Results of peptide mapping using a two-dimensional technique suggest that these intermediates consist of either two or three copies of filaggrin domains. Each of the intermediates has been purified. The ratios of amino acids in the purified components are unusual and essentially identical. The data are discussed in terms of a precursor containing tandem repeats of similar domains. In vivo pulse-chase experiments demonstrate that the processing of the high molecular weight phosphorylated precursor involves dephosphorylation and proteolytic steps through three-domain and two-domain intermediates to filaggrin. These processing steps appear to occur as the cell goes through the transition cell stage to form a cornified cell.

Animals↗
PubMed1984

Intracranial suppuration 1968-1982--a 15 year review.

The CT scanner and the recognition of anaerobic organisms has altered the investigation and management of intracranial suppuration. Improved treatment of acute and chronic middle ear infection has also occurred. A 15 year retrospective review was undertaken to assess the effects of these changes. The ears and sinuses were confirmed as the major source of intracranial suppuration (69%) although initial presentation to ENT surgeons was less common (14%). Ear complications occur in childhood and in the fifth and sixth decades; sinus complications predominantly in the second and third decades. The incidence of sinus infection appears to be rising. With the introduction on the CT scanner cerebral abscess mortality declined from 27.5% to 6.5% and subdural abscess mortality halved. The introduction of metronidazole contributed to a drop in mortality. The use of the CT scanner and more accurate antibacterial treatment, including metronidazole, gives hope for a more successful outcome than was previously possible.

Adolescent↗
PubMed1984

Abnormal corneal and eyelid development in the repeated epilation mouse.

Development of the eyelids and cornea in repeated epilation (Er/Er) mice is characterized by fusion of the tarsal conjunctiva to the epithelia of the bulbar conjunctiva and cornea. We investigated the ultrastructural features of this malformation and tested for abnormal expression of filaggrin by immunofluorescence. Heterozygous (Er/+) breeding stock were mated and 13,14,15, and 19-day-old embryos were studied by light and electron microscopy. Fusion occurred in all Er/Er specimens and was associated with abnormal migration of surface ectodermal cells onto the cornea. Immunofluorescence studies with antimouse filaggrin antibody on day 13 and day 15 revealed the presence of filaggrin precursors in the fused epithelia of mutants, but not in normal corneal or conjunctival epithelia. The results suggest defective regulation of the synthesis of cellular proteins and altered cell surface properties in the Er/Er ocular epithelia.

Abnormalities, Multiple↗
PubMed1983

Epidermal filaggrin is synthesized on a large messenger ribonucleic acid as a high-molecular-weight precursor.

Filaggrin is a keratin filament associated protein found in the differentiated cells of the epidermis. The mouse protein has a molecular weight of 26.5 X 10(3), while the molecular weight of rat filaggrin is 49 X 10(3), when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In order to clarify our understanding of the precursor form of filaggrin, RNA was isolated from mouse and rat epidermis and the poly(A+) fraction was translated in a cell-free protein synthesis system. Immunoprecipitation of translated proteins with specific antibody against rat filaggrin revealed a diffuse radiolabeled band with a molecular weight of approximately 250 X 10(3) on SDS-polyacrylamide gels. The size of filaggrin mRNA was estimated by sedimenting poly(A+) RNA through isokinetic sucrose gradients. Mouse filaggrin mRNA activity was located in the 30S region of the gradient, while rat filaggrin mRNA was located in the 34S region. The molecular weight of rat filaggrin mRNA was estimated to be 5 X 10(6) by electrophoresis in denaturing agarose gels containing methylmercury(II) hydroxide. A messenger RNA of this size could code for a polypeptide as large as approximately 600 kilodaltons. We conclude that filaggrin is synthesized as a large molecular weight precursor that must undergo substantial processing prior to formation of the mature form of the protein.

Animals↗
PubMed1983

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein.

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.

Amino Acids↗
PubMed1983

The complications of sphenoid sinusitis.

Five patients are described who presented with various complications of infective sphenoid sinusitis. Their clinical course and management is narrated and the importance of making this diagnosis and its simple effective treatment is stressed.

Adult↗
PubMed1983

Epidermolytic hyperkeratosis: ultrastructure and biochemistry of skin and amniotic fluid cells from two affected fetuses and a newborn infant.

Skin biopsy samples and amniotic fluid cells obtained in utero from two fetuses at risk for epidermolytic hyperkeratosis were examined by light and electron microscopy. Both fetuses were affected; the second was carried to term. Epidermal extracts were prepared from blisters of the newborn for analysis of keratin and filaggrin proteins. Abnormal clumps of keratin filaments were present in all layers of the prekeratinized fetal epidermis except the periderm and stratum germinativum. A significant population of amniotic fluid cells also contained the filament aggregations. Prenatal diagnosis of the disease should be possible using cells obtained at amniocentesis, thus avoiding fetal skin biopsy. Biochemical studies showed abnormalities in keratin and filaggrin proteins. The structural alterations in the tissue might be a consequence of altered interaction between these two abnormal epidermal proteins.

Amniotic Fluid↗
PubMed1983

Identification of filaggrin in cultured mouse keratinocytes and its regulation by calcium.

Filaggrin is a histidine-rich cationic protein present in cells of the stratum corneum in vivo and derived from a precursor in keratohyalin granules. Biochemical and immunologic methods were used to determine the presence of filaggrin in keratinocytes cultured in vitro and induced to differentiate by increasing the extracellular calcium concentration from 0.07 to 1.2 mM. Indirect immunofluorescence using antibody to rat filaggrin was negative in cells cultured in low-calcium medium but positive in cells switched to high-calcium medium. Large immunofluorescent granules were identified in a perinuclear distribution starting at 6 hours after the shift in calcium concentration, coinciding with the time of appearance of phase-dense cytoplasmic granules. Radiolabeled histidine was preferentially incorporated into proteins of 95, 37, and 27 K. The 37 and 27 K bands were not adsorbed by DE52 cellulose and therefore are cationic. A 27 K cationic, histidine-labeled protein was readily extracted from frozen pellets of cells cultured in high-calcium medium. It comigrates with purified mouse filaggrin (27 K) and reacts with antibody to rat filaggrin on immunoautoradiography. Only trace amounts of this protein could be detected in cells cultured in low-calcium medium. Our observation of filaggrin-immunoreactive granules confirms the previous ultrastructural identification of keratohyalin granules after the shift to high-calcium medium. The results suggest that filaggrin synthesis is stimulated in keratinocytes induced to differentiate by the shift to high extracellular calcium concentration.

Animals↗
PubMed1983

Hyperthyroidism due to a thyrotrophin-secreting microadenoma.

We report a 63-year-old female with a TSH-secreting pituitary adenoma causing hyperthyroidism. This case is apparently unique, and challenges the present concept of dividing patients with inappropriate TSH secretion into tumour and non-tumour groups on the basis of conventional pituitary fossa radiology.

Adenoma↗
PubMed1982

Characterization of a phosphorylated form of the intermediate filament-aggregating protein filaggrin.

Filaggrin and a phosphorylated form of filaggrin, which has been shown by pulse-chase studies to be a precursor form of the protein [Dale, B. A., & Ling, S. Y. (1979) Biochemistry 18, 3539-3546], were compared for functional, biochemical, and physical properties. Filaggrin reacts with keratin filaments to form visible macrofibrils, unlike the precursor which does not. Biochemical and peptide-mapping studies suggest that the two proteins have similar, perhaps identical, amino acid sequences. The major differences between the two proteins are in molecular weight (precursor, 44 200 g/mol; filaggrin, 38 400 g/mol), the existence of oligomeric forms of the precursor, and the presence of phosphate in the precursor (15-20 mol/mol of protein). Phosphoserine was identified in the precursor, but neither phosphothreonine nor phosphotyrosine was observed. The results of proteolytic digests of [32P]phosphate-radiolabeled precursor show that the phosphate is unevenly distributed throughout the molecule and may be localized in approximately 30% of the precursor. A discrete localization of the phosphate in the precursor may block a specific keratin filament combining site and so prevent premature aggregation of these filaments during epidermal differentiation. It is suggested that a specific phosphatase is involved in the dephosphorylation, because several phosphatases of general specificity, including rat epidermal lysosomal acid phosphatase, did not catalyze this conversion.

Amino Acids↗