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Biomedical subjects

J C Kvedar

Publications and source records attributed to J C Kvedar.

At least 19 recordsLinked to original sources
PubMed1992

Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine.

A method providing more sensitive detection of transglutaminase substrates was developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when [14C]putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r2 = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.

Animals↗
PubMed1992

Keratinocytes stimulate prostaglandin I2 synthesis by 3T3 cells and exhibit enhanced cornification when exposed to prostaglandin I2 analogues.

The predominant cyclooxygenase products of keratinocytes are prostaglandin (PG)E2 and PGF2 alpha with only trace amounts of PGI2 synthesis detected. When normal or immortal (NM1) keratinocytes were co-cultured with mitomycin C-treated 3T3 cells, increased synthesis of PGI2 was noted compared to mitomycin C-treated 3T3 cells alone. The PGI2 level in co-cultures was maximum within the first week and diminished rapidly thereafter. These results suggested keratinocytes enhance the production of PGI2 by 3T3 cells. Keratinocyte cultures incubated with Iloprost and Piriprost, stable PGI2 analogues, showed evidence of increased cornification as demonstrated by staining with rhodanile blue, decreased shedding of cells into the culture medium, and more cornified material adhering to the culture surface. The cultures appeared to be responsive between the first and second weeks after plating and the inhibition of shedding could not be reversed by changing to drug-free medium. Control and treated cultures showed identical electrophoretic protein patterns. Immunoblots showed involucrin unchanged in extracts of control and treated cultures while the 22 kd pancornulin was absent in treated cultures. The findings that keratinocytes enhance the production of PGI2 by 3T3 cells and that PGI2 analogues enhance cornification of confluent keratinocytes raise the possibility that eicosanoids may serve as autoregulatory signals together with other factors.

3T3 Cells↗
PubMed1992

Characterization of sciellin, a precursor to the cornified envelope of human keratinocytes.

The cornified envelope, located beneath the plasma membrane of terminally differentiated keratinocytes, is formed as protein precursors are cross-linked by a membrane associated transglutaminase. This report characterizes a new precursor to the cornified envelope. A monoclonal antibody derived from mice immunized with cornified envelopes of human cultured keratinocytes stained the periphery of more differentiated cells in epidermis and other stratified squamous epithelia including hair and nails. The epitope was widely conserved among mammals as determined by immunohistochemical and Western analysis. Immunoelectron microscopy localized the epitope to the cell periphery in the upper stratum spinosum and granulosum of epidermis. In the hair follicle, the epitope was present in the internal root sheath and in the infundibulum, the innermost aspect of the external root sheath. The antibody recognized a protein of relative mobility (M(r)) 82,000, pI 7.8. The protein was a transglutaminase substrate as shown by a dansylcadaverine incorporation assay. Purified cornified envelopes absorbed the reactivity of the antibody to the partially purified protein and cleavage of envelopes by cyanogen bromide resulted in release of immunoreactive fragments. The protein was soluble only in denaturing buffers such as 8 M urea or 2% sodium dodecyl-sulfate (SDS). Partial solubility could be achieved in 50 mM TRIS pH 8.3 plus 0.3 M NaCl (high salt buffer); the presence of a reducing agent did not affect solubility. Extraction of cultured keratinocytes in 8 M urea and subsequent dialysis against 50 mM TRIS pH 8.3 buffer resulted in precipitation of the protein with the keratin filaments. Dialysis against high salt buffer prevented precipitation of the protein. The unique solubility properties of this protein suggest that it aggregates with itself and/or with keratin filaments. The possible role of the protein in cornified envelope assembly is discussed. We have named this protein Sciellin (from the old english "sciell" for shell).

Animals↗
PubMed1992

Loose anagen hair as a cause of hereditary hair loss in children.

BACKGROUND AND DESIGN: The loose anagen hair syndrome is a recently described disorder with enhanced hair loss in which anagen hairs can be painlessly pulled from the scalp. RESULTS: In three families, we have observed an autosomal dominant form of inheritance. We have confirmed that the extracted bulbs consist only of cortical cells and that the hair shafts have an abnormal shape. Using routine light and electron microscopy, we observed disadhesion between all layers of the follicle and within some layers and premature keratinization of the inner root sheath of the follicle. In addition, abnormal maturation of the various layers was observed at the electron microscopic level, including regions of the cortex that contained decreased numbers of filaments. The disorder appears to be cyclic, since relatively normal and markedly affected regions of the shaft can be observed within a single follicle. CONCLUSIONS: Possible mechanisms responsible for the disease include abnormal intercellular signaling and disturbances of desmosomal components and/or cadherins.

Alopecia↗
PubMed1991

Dietary management reverses grooving and abnormal polarization of hair shafts in argininosuccinase deficiency.

We have observed that the fragile hair of two untreated patients with argininosuccinic aciduria showed abnormal alternating zones of bright and dark banding by polarizing microscopy. Scanning electron microscopy documented discontinuous grooves with a 50 to 100 microns periodicity. Results of amino acid analysis of the hair were essentially normal. After the patients were treated with a low-protein, arginine-supplemented diet, the hair assumed a normal appearance. Five patients already treated with diet showed no hair abnormalities. The pathogenesis of the hair changes in unknown, but our findings suggest that products generated in the disease can adversely affect metabolically active tissue such as hair.

Amino Acid Metabolism, Inborn Errors↗
PubMed1991

Nail changes in cutaneous disease.

Nail involvement in cutaneous disease provides the clinician with diagnostic clues, and may be of great cosmetic significance to patients. Nail manifestations of the most common and/or important cutaneous diseases are discussed. Photographic illustrations of these changes are also presented.

Humans↗
PubMed1990

Involucrin-like proteins in non-primates.

A monoclonal and two polyclonal antibodies to human involucrin were used to look for involucrin epitopes in other species. All antibodies react strongly with the same proteins of monkey, and both polyclonal antibodies react with specific proteins of cow and dog. One of the polyclonal antibodies also reacts with proteins of sheep, guinea pig, rat, and finback whale. The immunoreactive proteins from cow and dog could be purified using a procedure developed for human involucrin. The reaction with the purified dog protein could be blocked by purified human involucrin. The results suggest that involucrin-like proteins have a wider species distribution than originally appreciated.

Animals↗
PubMed1988

Selective inhibition by minoxidil of prostacyclin production by cells in culture.

The effect of minoxidil on arachidonic acid metabolism by cells in culture was studied. In bovine aorta endothelial cells, treatment with minoxidil in the presence of various stimulators of arachidonic acid metabolism was accompanied by a dose-dependent inhibition of prostacyclin production (measured as 6-keto-prostaglandin F1 alpha). Synthesis of the other cyclooxygenase products (prostaglandins E2, F2 alpha and thromboxane) was not inhibited. When the bovine aorta endothelial cells were stimulated by the Ca2+ ionophore A-23187, the inhibition was seen as early as 2 min. Minoxidil also inhibited prostacyclin production by a second cell line of bovine aorta endothelial cells (the established CPAE cell line), bovine aorta smooth muscle cells, porcine aorta endothelial cells, and rat liver cells (the C-9 cell line)--the latter, less effectively. Again, formation of all the other cyclooxygenase products studied was not inhibited. Minoxidil did not affect significantly prostaglandin E2 and F2 alpha production by newborn rat keratinocytes (the NBR cell line)--a cell that does not produce PGI2. The clinical, biochemical, and pharmacologic implications are discussed.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

A new class of soluble basic protein precursors of the cornified envelope of mammalian epidermis.

The cornified envelope has been shown to be formed beneath the plasma membrane as a result of the cross-linking of soluble and membrane-associated precursor proteins by transglutaminase. We have obtained a monoclonal antibody which reacts with the periphery of cells in the upper layers of human epidermis by indirect immunofluorescence (IIF) following immunization of mice with cornified envelopes of cultured human keratinocytes. The antibody also stained the cell peripheries of bovine, rat and mouse epidermis as well as stratified epithelium. Neutral buffer extracts of human cultured keratinocytes and epidermis examined under denaturing conditions contained polypeptides of molecular weight 14,900 and 16,800 which reacted with the antibody, and an additional component of molecular weight 24,800 was found in cultured cells. The polypeptides were shown to have a pI of about 9.0. Under non-denaturing conditions the two lower-molecular-weight polypeptides had an apparent molecular weight of 30,000, while the 24,800 protein had one of 60,000. Incubation of the polypeptides under conditions that activate transglutaminase resulted in a disappearance of the polypeptides or the formation of cross-linked products. Basic polypeptides with somewhat different pI values and molecular weights were identified in neutral buffer extracts of bovine and rat epidermis. The HCE-2 antibody appears to identify a new class of basic protein precursors of mammalian cornified envelope.

Animals↗
PubMed1987

Topical minoxidil in the treatment of male pattern alopecia.

Male pattern hair loss (androgenetic alopecia) is a common problem. In fact, it affects nearly all males to some degree. Expression of the disorder is variable, and while it is never life-threatening, it often becomes a major source of consternation. The biology of the process is poorly understood, and no current therapy can halt or reverse the process. Only cosmetic surgery, which is painful, time consuming, and expensive, has been effective. In the past 7 years, since it was noted that a patient taking minoxidil for hypertension had reversal of male pattern hair loss, awareness of a possible therapeutic role for topical minoxidil in the management of this disorder has grown among physicians, scientists, and the general public. It can be concluded from available data that topical application of minoxidil is effective in providing cosmetically satisfying thickening of hair in a select group of individuals with male pattern hair loss. The drug's mechanism of action remains obscure. No serious side effects have been demonstrated with its use, however, and it is therefore advised in selected patients.

Administration, Topical↗
PubMed1987

Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM 1).

The long-lived keratinocyte line, NM 1, was isolated from the epidermis of a pool of foreskins obtained from apparently normal neonates at the time of circumcision. Cultures were initiated in Dulbecco's minimal essential medium containing 20% fetal bovine serum, 0.4 micrograms/ml hydrocortisone, 10(-9) M cholera toxin, and 10 ng/ml epidermal growth factor using mitomycin C-treated 3T3 cells as a feeder layer. Unlike normal keratinocytes which survive for only 150 generations these cells have been in culture for more than a year and have been carried for more than 400 doublings. The cells seem to follow a pathway of growth and differentiation that is very similar to normal keratinocytes. Cytokeratin fibrils, intercellular attachments, and cornified envelopes were observed. The keratin polypeptides isolated from the NM 1 cells were similar to those previously described in normal cultured cells; the presence of profilaggrin and involucrin was demonstrated by sodium dodecyl sulfate electrophoresis and immunoblotting with monoclonal antibodies specific to these proteins. The NM 1 cells showed a reduced dependency on 3T3 feeder cells but did not form tumors when placed into athymic nude mice. Screening of the cells for SV40, BK, HPV 16, and HPV 18 viruses was negative. The NM 1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of keratinocytes.

Animals↗
PubMed1987

Effect of minoxidil on pre- and postconfluent keratinocytes.

Studies in our laboratory have shown that minoxidil prolongs the life of keratinocytes in culture and extends the time after confluence that cells can be subcultured. These data suggest that the drug reduces the rate at which cells are lost from the germinative pool and hence slows senescence. In a dose-response study with minoxidil, the maximal effect of the drug was seen at doses from 6 to 12 micrograms/ml; however, activity could be detected at doses below 1 microgram/ml. Cells subcultured during log growth failed to demonstrate that minoxidil increased the total number of generations attainable under these conditions, although as expected, epidermal growth factor extended the life span of cells. When the experiments were repeated in a keratinocyte cell line that does not require a fibroblast feeder layer, the same results were obtained, indicating that the difference observed between log phase and postconfluence growth cannot be explained by the presence of fibroblasts. Minoxidil's effect on postconfluent cells was blunted by the addition of cholera toxin to the medium, suggesting that elevation of cyclic adenosine monophosphate cannot be a mechanism, although reduction of cyclic adenosine monophosphate is a possibility. Finally, maintaining keratinocytes at a 20 to 40 mM calcium concentration greatly reduced the ability of postconfluent cells to be subcultured, in comparison with the normal calcium concentration of 2 mM. That minoxidil almost completely reversed this inhibitory effect suggests it may work by preventing cross-linking by transglutaminase, which is activated by elevated calcium concentrations.

Calcium↗
PubMed1987

NM1 keratinocyte line is cytogenetically and biologically stable and exhibits a unique structural protein.

We have previously described a human keratinocyte line, NM1, which had been carried for more than 400 doublings, was trisomic for chromosome 8, and appeared to make a number of structural proteins characteristic of keratinocytes. This line has now been carried for more than 800 doublings and grows with the same vigor. It reaches confluence in 7 to 10 days and can be grown without a feeder layer for more than 15 passages. Its karyotype has remained 47,XY, +8. The current NM1 cells make readily detectable amounts of 67 kd and 48 kd keratins, and it has been established that the previously poorly resolved 58 kd band actually consists of 58 kd and 59 kd bands. We have also found that the apparent 56 kd band consists of the 56 kd and 56.5 kd bands. A unique basic polypeptide precursor of the cornified envelope has been discovered in the NM1 line. Although similar in charge to one in normal cells it is lower in molecular weight.

Carcinogens↗
PubMed1986

Cytokeratins of the bovine hoof: classification and studies on expression.

The bovine hoof has been examined as a model for the study of keratinized skin appendages. We characterized the keratin polypeptides of hoof bed and matrix and compared them to epidermis using two-dimensional electrophoresis and immunoblot techniques. Both hoof tissues express keratins 6 and 16 (as described by Franke et al. (1981) J. Mol. Biol. 153, 933-959) and b2 and a1-4 which are previously undescribed proteins unique to the bovine hoof. Keratins of hoof matrix and bed share one or more common antigenic components as defined by immunoblot analysis. Hoof matrix expresses keratins 7 and 14, which are absent in hoof bed, and also expresses a greater number of isoelectric variants of keratin 6. Biopsies of hoof bed and matrix transplanted onto athymic mice both made hard hoof and underwent active keratin synthesis as evidenced by incorporation of [3H]leucine. Indirect immunofluorescence studies of the grafts showed that they had the histology and immunoreactivity previously noted for hoof bed and matrix. The two-dimensional gel electrophoretic patterns of both grafts were similar and expressed keratins b2 and a1-4. We conclude that a unique group of keratins exists in hoof. Furthermore, while hoof matrix is the major contributor to hard hoof, hoof bed epidermis maintains the capacity to make hard hoof and may contribute to the synthesis of the hoof plate in vivo. The ability to graft hoofs onto athymic mice provides an opportunity for the study of a number of aspects of hoof formation.

Animals↗
PubMed1986

Immunologic detection of markers of keratinocyte differentiation. Its use in neoplastic and preneoplastic lesions of skin.

We examined seven invasive squamous cell carcinomas, five squamous cell carcinomas in situ, four keratoacanthomas, two actinic keratoses, and two seborrheic keratoses by indirect immunofluorescence. We used a panel of three antibodies: one directed against filaggrin, one against involucrin, and one against peptidylarginine deiminase. Anti-involucrin stained all the lesions studied, but the pattern within a given category of lesions was variable and consistent differences between the categories were not observed. Similarly, the antibodies against peptidylarginine deiminase and filaggrin were not able to distinguish differences between the various types of tumors. We conclude that in tumors of epidermis, benign or malignant, products of differentiation are expressed independently of histologic atypia or clinical aggressiveness. Therefore, markers of differentiation do not appear to be reliable indexes for distinguishing benign from malignant lesions.

Carcinoma, Squamous Cell↗