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Biomedical subjects

P Fleckman

Publications and source records attributed to P Fleckman.

52 records · Page 3Linked to original sources
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↗
PubMed1990

The distinctive pattern of proteoglycan and glycosaminoglycan free chain synthesis by cultured human epidermal keratinocytes.

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Cells, Cultured↗
PubMed1988

Glycosaminoglycan synthesis by human keratinocytes: cell growth and medium calcium effects.

The influences of cell density, differentiation, and medium calcium levels on glycosaminoglycan biosynthesis were evaluated in cultured human epidermal keratinocytes. Following metabolic labeling with [35S]-sulfate and [3H]-glucosamine under steady state conditions in "high" medium calcium (greater than 1.0 mMol), the majority of sulfated glycosaminoglycans remained associated with the cell layers, whereas hyaluronic acid, which was present in smaller amounts than the sulfated products, was about equally distributed between the medium and the cell layers. Of the sulfated glycosaminoglycans, heparan sulfate and chondroitin 4/6-sulfate were the major species and were present in roughly comparable amounts, whereas dermatan sulfate was quantitatively the lesser of the products. The effects of "low" medium calcium (0.3 and 0.025 mM) were complex, although a consistent decrease in the incorporation of the [3H]-glucosamine precursor was found at high cell density, probably reflecting a decrease in its intracellular specific activity. In "high" calcium cultures, there was a strong inverse correlation (r = -0.92) between keratinocyte cell number and cellular production of sulfated glycosaminoglycans, whereas no such relationship was evident in cultures grown in "low" calcium medium at comparable cell density. Because keratinocyte differentiation is inhibited in the low calcium conditions, the results suggest that the decrease in production of sulfated glycosaminoglycans by confluent keratinocytes may actually correlate with differentiation rather than with cell number.

Calcium↗
PubMed1987

Characterization of two monoclonal antibodies to human epidermal keratohyalin: reactivity with filaggrin and related proteins.

Two monoclonal antibodies (AKH1 and AKH2) were elicited with partially purified human filaggrin and characterized by immunohistochemistry on normal and abnormal skin biopsies, immunoblotting techniques, and antigen purification. Both antibodies react strongly with the granular cell layer consistent with the distribution of keratohyalin and show a more diffuse reaction with the stratum corneum in normal skin biopsies. Reaction in cultured human keratinocytes is limited to immunofluorescent granules in flattened, well-differentiated cells in confluent cultures, in which we have previously demonstrated keratohyalin. On immunoblots AKH1 reacts with filaggrin (37 kD) and profilaggrin (400 kD), while AKH2 primarily stains bands of 150 and 300 kD. The AKH2 antigens were identified in the cationic protein fraction used for immunization and were purified by gel permeation and high-performance liquid chromatography. Amino acid composition of these proteins differs only slightly from filaggrin. Immunohistochemical staining patterns of the two antibodies are very similar in the genetic disorders of keratinization tested, except for ichthyosis vulgaris, and reflect the presence and distribution of keratohyalin. In ichthyosis vulgaris, AKH1 staining is weak, consistent with the morphology and with biochemical absence of profilaggrin/filaggrin; however, AKH2 staining is positive, although weaker than normal, suggesting the presence of the AKH2 antigens even when keratohyalin is absent or abnormal. Antibodies AKH1 and AKH2 may be useful as differentiation markers for keratinization in tissues and for cells in culture. Antibody AKH1 can be used specifically for detection of profilaggrin/filaggrin in tissues, cultured keratinocytes, and extracts.

Antibodies, Monoclonal↗
PubMed1987

Keratinocytes cultured from subjects with ichthyosis vulgaris are phenotypically abnormal.

Ichthyosis vulgaris (IV) is an autosomal dominant, scaling disorder in which keratohyaline granules and filaggrin are reduced in or absent from the epidermis of affected individuals. Morphologic and biochemical markers of epidermal differentiation were studied in keratinocytes cultured from clinically unaffected skin of patients with IV, from clinically unaffected skin of an obligate gene carrier, and from normal skin of unaffected family members and an adult volunteer. Cultured keratinocytes from affected subjects formed thickened layers of scaly cells that failed to react with monoclonal antibody to filaggrin. In contrast, normal cells contained many large, immunoreactive granules. Electron microscopy confirmed the absence of keratohyaline granules in affected cells and the presence of large keratohyaline granules in normal cells. Immunoblot analysis of keratinocyte extracts from subjects with ichthyosis showed that profilaggrin was absent, but no differences in keratins were detected between affected and control cells. For all parameters, findings in cells of the clinically unaffected obligate gene carrier were intermediate between those from affected patients and controls. We conclude that keratinocytes cultured from patients with IV maintain structural and biochemical phenotypic characteristics of the disease in vitro.

Biopsy↗
PubMed1987

Glycosaminoglycan synthesis by proliferating and differentiated human keratinocytes in culture.

Glycosaminoglycan (GAG) synthesis and compartmentalization were studied in populations of human neonatal keratinocytes under conditions of proliferation and terminal differentiation in vitro. Following isotopic labeling with the precursors [3H]glucosamine and [35S]sulfate, GAGs were extracted from the keratinocytes into several operationally created compartments associated with the cells and extracellular matrix. Chondroitin sulfate and heparan sulfate accounted for the majority of the incorporated label in all preparations. Although total sulfated GAGs per culture increased from proliferative to differentiated conditions, GAG content normalized to the DNA content of the cultures demonstrated the reverse trend. This was particularly evident for the chondroitin sulfates, which declined 60-70% in the differentiated cultures. Furthermore, label incorporation into chondroitin and heparan sulfates revealed a relative compartmental shift to a trypsin-accessible site upon keratinocyte differentiation. An analysis of heparan sulfate structure by characterization of the oligosaccharide products resulting from low pH nitrous acid deaminative degradation provided evidence that the parent material from differentiated keratinocytes contains a larger region of N-sulfated glucosamine residues unassociated with ester sulfate groups. The correlation of variations in GAG content and compartmentalization with the growth condition of human keratinocytes constitutes evidence that this heterogeneous group of cell surface-associated carbohydrates is involved in some aspect of cell function associated with growth control or differentiation. Furthermore, the apparent differences in heparan sulfate primary structure indicate that there is structure-function specificity to this association.

Cell Compartmentation↗
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↗
PubMed1985

Anatomy and physiology of the nail.

The nail plate lies under the proximal nail fold and over the matrix, nail bed, and hyponychium. The nail plate is made by the matrix. Although the few cells the nail bed contributes to the nail plate do not increase the bulk of the plate, they are important in attaching the plate to the bed. The interrelationships of the components of the nail and knowledge about their normal function are reviewed.

Female↗
PubMed1984

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells.

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.

Animals↗
PubMed1984

Epidermal growth factor stimulates ornithine decarboxylase activity in cultured mammalian keratinocytes.

The influence of epidermal growth factor (EGF) on ornithine decarboxylase has been examined in cultured bovine keratinocytes. Keratinocyte ornithine decarboxylase activity was maximal at pH 6.3 in MES buffer in the presence of dithiothreitol and EDTA. When cultured cells, deprived of serum, were exposed to EGF, the activity of ornithine decarboxylase was stimulated severalfold. Enzyme activity increased in a dose-dependent manner with EGF. The time course of this stimulation is unlike any previously reported in cultured cells. The increase in activity was maximal by 8 h. A small dip in activity was seen between 8 and 12 h. Increased activity was sustained for as long as 24 h after exposure to EGF. The prolonged increased in enzyme activity was reduced by actinomycin D. When cycloheximide was added 1 h before EGF, ornithine decarboxylase activity was obliterated. This is the first demonstration of ornithine decarboxylase stimulation following exposure to EGF in cultured keratinocytes. The prolonged duration of ornithine decarboxylase stimulation is unexplained but may be related to processing of EGF by the keratinocytes.

Animals↗
PubMed1983

Classification and therapy of atrophie blanche.

Atrophie blanche usually appears as painful purpuric papules that evolve into ulcerations and, finally, angular scars on the lower extremities. The literature on this subject presents a confusing picture of its causes, pathogenesis, and treatment. From our review of the literature and our experience in evaluating and treating cases of atrophie blanche, we conclude that it is best categorized as a clinicopathologic entity with multiple causes. Its characteristic histopathologic features and clinical evolution indicate that the common pathologic event is occlusion of vessels in the middle and deep dermis. No single form of therapy has been consistently effective for the treatment of atrophie blanche, but drugs that inhibit platelet thrombus formation or stimulate endogenous fibrinolytic activity arrest the disease in most patients.

Adult↗
PubMedDate not supplied

Current and future nail research - areas ripe for study.

This discussion examines five common topics that affect nails adversely, onychomycosis, brittle nails, developmental nail disorders, chronic paronychia and onycholysis. What is known about these processes, what areas of research, old and new, might lead to improved understanding of the underlying basis of the problems and what prospects the future might hold are considered.

Cytoskeleton↗