Neuroprotective effects of creatine in a transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a progressive neurodegenerative illness for which there is no effective therapy. We examined whether creatine, which may exert neuroprotective effects by increasing phosphocreatine levels or by stabilizing the mitochondrial permeability transition, has beneficial effects in a transgenic mouse model of HD (line 6/2). Dietary creatine supplementation significantly improved survival, slowed the development of brain atrophy, and delayed atrophy of striatal neurons and the formation of huntingtin-positive aggregates in R6/2 mice. Body weight and motor performance on the rotarod test were significantly improved in creatine-supplemented R6/2 mice, whereas the onset of diabetes was markedly delayed. Nuclear magnetic resonance spectroscopy showed that creatine supplementation significantly increased brain creatine concentrations and delayed decreases in N-acetylaspartate concentrations. These results support a role of metabolic dysfunction in a transgenic mouse model of HD and suggest a novel therapeutic strategy to slow the pathological process.

Reconstituted 3-dimensional human skin as a novel in vitro model for studies of carcinogenesis.

EpiDerm (MatTek Co., MA) is a reconstituted human skin equivalent which exhibits morphological and growth characteristics similar to human skin. This model has previously been utilized to evaluate the cytotoxicity and irritant potential of various cosmetic and household products. In this study, we show for the first time that EpiDerm can be used successfully to evaluate the genotoxicity of different types of known carcinogenic agents such as benzo[a]pyrene (BaP), ultraviolet B radiation (UVB), ultraviolet A radiation (UVA), and psoralen-ultraviolet A radiation (PUVA) at the molecular level. The topical application of 50 microg/cm2 BaP to EpiDerm resulted in the accumulation of BaP-DNA adducts and c-fos and p53 proteins as evidenced by immunohistochemical localization. Similarly, exposure to UVB (50 mJ/cm2) and UVA (2.5 J/cm2) enhanced the epidermal expression of c-fos and p53 proteins in the human skin equivalent. PUVA treatment of EpiDerm, however, resulted in the formation of both DNA-8-MOP adducts and augmented expression of c-fos and p53 proteins. Most of these changes reached a peak 8 h after the treatments except in the case of UVA where maximum changes in the expression of c-fos and p53 proteins were observed 24 h after treatment. These results are similar to those previously reported in human and murine skin following exposure to BaP, UVB, UVA, or PUVA indicating that human skin equivalents can be used as a convenient and cost-effective alternative to animal testing for assessing the genotoxicity and mechanism of action of mutagens/carcinogens in human skin.

New epidermal model for dermal irritancy testing.

An interlaboratory comparison of a new model of the human epidermis (EpiDerm) was conducted using a range of anionic and non-ionic surfactants and surfactant-containing final formulations. The toxicity of the materials was estimated by MTT conversion, using both concentration (EC(50)) and time (ET(50)) protocols. A range of 16 compounds was tested on different production lots of EpiDerm following storage periods of 1 and 2 days (after shipping) at MatTek and at two independent testing laboratories, Microbiological Associates (MA), USA and Scotland, UK. The EC(50) and ET(50) values were compared and the least squares fit lines with resulting correlation coefficients (r) calculated. Correlation of in vitro results to human clinical chamber irritation and repeat handwash testing gave r values ranging from 0.977 to 0.993 and comparison of the results obtained in the independent laboratories with the site of manufacture was good (MA, USA, r = 0.84; MA, UK, r = 0.74). The model appears to have utility in predicting clinically observed dermal irritation in vitro which is reproducible in different laboratories and after transatlantic shipping, such that it is worthy of further investigation.

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.

The pancornulins: a group of basic low molecular weight proteins in mammalian epidermis and epithelium that may function as cornified envelope precursors.

1. A monoclonal antibody (HCE-2) to human epidermal and epithelial cornified envelopes identified a group of soluble basic protein precursors. 2. Using HCE-2, envelope-like staining was observed in the epidermis and stratified squamous epithelium of a number of mammalian species. 3. Basic polypeptides reactive to HCE-2 varied in size and number among the different animals. 4. In those species studied, HCE-2-reactive peptides were substrates for transglutaminase and protease treatment of cornified envelopes released HCE-2-reactive degradation products. 5. These results suggest a new family of proteins in mammalian epidermis that may function as cornified envelope precursors.

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.

Characterization of monoclonal antibodies generated to the cornified envelope of human cultured keratinocytes.

The cornified envelope of keratinocytes is an insoluble structure formed beneath the plasma membrane at the base of the stratum corneum. It is made by cross-linking precursor proteins by a membrane-associated transglutaminase. We have prepared monoclonal antibodies to the cornified envelope of cultured human keratinocytes and used these to identify precursor proteins using Western blotting. We have uncovered a number of precursors including involucrin and a 195 kD membrane-associated protein, which had previously been reported. Antibodies to these precursors, with the exception of the one to involucrin, reacted with the epidermis of other mammalian species, suggesting structural conservation in at least some envelope components.

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.

Identification of new components of the cornified envelope of human and bovine epidermis.

Antibodies raised in rabbits to purified cornified envelopes (CEs) of cultured human keratinocytes reacted in a peripheral fashion with the granular and spinous layers of human, cow, rat, and mouse epidermis. This reaction could not be abolished by absorption of the antibody with purified human involucrin to which the antibody reacted by immunoblot, thus indicating the presence of an additional antigenic determinant(s). Antibodies raised to CEs of human epidermis stained the cytoplasm of epidermal cells and gave a strong reaction to cytokeratins and a weak one to involucrin, indicating that in tissue the keratins are also cross-linked. The antibody prepared to bovine CEs reacted with keratins, but when absorbed with prekeratin it gave a peripheral staining pattern with epidermis and reacted strongly with a 126 kD component of the neutral buffer extract of cow snout epidermis and weakly with 205 kD and 85 kD ones. This antibody reacted with human involucrin by immunoblot while an antibody to involucrin stained 143 kD, 119 kD, 113 kD, and 107 kD polypeptides in the bovine extract. These latter 3 bands were shown to be substrates of transglutaminase. Further, a monoclonal antibody to bovine CEs reacted with the 119 kD and 113 kD bands and gave a peripheral staining pattern in the epidermis. Proof that the 126 kD protein was a precursor of the envelope was obtained by preparing an antibody to it and demonstrating peripheral staining of epidermal cells. These results point out the value of preparing antibodies to CE as an additional approach to studying the composition of CEs and demonstrate previously undescribed components in human and bovine tissue.

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.

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.

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.

The occurrence of profilaggrin and its processing in cultured keratinocytes.

An affinity-purified antibody to rat filaggrin detects filaggrin and profilaggrin in extracts of newborn rat epidermis, and a monoclonal antibody to human filaggrin, HF-1, detects the two proteins in extracts of human epidermis. Immunohistologic studies show that HF-1 reacts with keratohyaline granules of human epidermis and those seen in cultured human keratinocytes. Immunoblotting studies have demonstrated that profilaggrin is synthesized in both cultured human keratinocytes and in a long-lived line of cultured rat keratinocytes, but only in the latter is the protein processed to a product of the molecular weight of filaggrin.

The occurrence and immunology of peptidylarginine deiminase and its preparation from bovine epidermis by an improved method.

Peptidylarginine deiminase activity has been found in some tissues from at least one representative of each vertebrate class, suggesting that the occurrence of the enzyme throughout the vertebrates is widespread. Using a three-step procedure including affinity chromatography on arginine agarose, a greatly improved purification of bovine epidermal peptidylarginine deiminase is presented. The purified enzyme preparation contains a 70-75 kD band and several minor components when examined by sodium dodecyl sulfate electrophoresis. A polyclonal antibody raised to the enzyme of bovine brain cross-reacts with human and newborn rat epidermis by indirect immunofluorescence. This cross-reactivity is markedly diminished by absorption of the antibody to the purified brain enzyme.

A comparative study of the immunologic properties of hoof and nail fibrous proteins.

We have studied the localization of epidermal (soft) and nail and hoof (hard) fibrous keratins in the various anatomic regions of bovine hoof and human nail. Indirect immunofluorescence was performed on frozen sections of various parts of hoof and nail with antibodies demonstrated to be specific for hard and soft fibrous keratins by the Ouchterlony technique. The antibody to hard fibrous keratin reacted with the upper region of hoof bed and matrix tissue but not perihoof epidermis. The antibody to soft fibrous keratin reacted with hoof bed, matrix tissue, and perihoof epidermis. Electrophoretic analysis of the fibrous proteins of hoof bed indicated they contained both soft and hard fibrous keratins while matrix tissue contained only hard keratins. Immunoblot analysis of matrix fibrous proteins indicated that most of the polypeptides reacted with both antibodies. These results indicate that the antibody to soft fibrous keratin cross-reacts with hard fibrous keratin but the antibody to hard fibrous keratin appears to be specific. Immunologic studies, therefore, must be correlated with electrophoretic studies in order to define the localization of the various types of fibrous keratins. Similar results were obtained with human nail.

Isopeptide bond formation in epidermis.

Proteins which are major substrates of epidermal transglutaminases can be identified in cultured keratinocytes of human, cow, and new-born rat. Cow and human keratinocytes both contain substrate proteins which are 30 000 to 50 000 daltons in size but dissociable in SDS to 12 000 daltons or less. In both species these proteins correspond to in vivo synthesized proteins which are probable precursors of cornified envelope. Human keratinocytes synthesize a 125 000 dalton protein which is also a precursor of cornified envelope both in cells and tissue. By SDS electrophoresis two 100 000 dalton substrate proteins are seen in cow keratinocyte extracts and a 23 000 dalton substrate protein is seen in rat keratinocyte extracts. Minor substrates of transglutaminase are seen in human keratinocytes, and one has been isolated by preparative electrophoresis. Major structural proteins of epidermis which are in vitro substrates of epidermal transglutaminase include the keratins and the stratum corneum basic protein.

Effect of minoxidil on cultured keratinocytes.

Minoxidil has been shown to stimulate hair growth and these studies were undertaken to determine whether the drug had a direct effect on keratinocytes. Cultures of human epidermal cells were treated with minoxidil and it was found that they survived longer than control cultures. In addition, minoxidil prolonged the time that cells could be passed after reaching confluence. The results suggest that minoxidil slows the senescence of keratinocytes, which is similar to what has been found with epidermal growth factor.

Fibrous proteins of bovine hoof.

The matrix region of calf hoof was identified as the living precursor layer of the hardened hoof plate. Fibrous protein was isolated from the matrix with citrate buffer, pH. 2.65, while Tris buffer, pH. 9.5, with 8 M urea and a reducing agent was required to dissolve the cornified hoof. The purified matrix protein had a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern similar to hoof plate and different from bovine epidermis. When the s-carboxymethyl derivatives of matrix, hoof plate, and hair proteins was compared by urea-polyacrylamide gel electrophoresis, they were identical, and different from that of epidermal protein. The matrix protein reacted with an antibody to hair fibrous protein. Cultured matrix keratinocytes appeared to be identical to cultured epidermal cells, pointing to the importance of the dermis in epidermal cell differentiation.

Modulation of differentiation by retinoids.

At high levels of vitamin A and other retinoids (3 X 10(-6) M) attachment of human keratinocytes to 3T3-coated plastic dishes is mildly inhibited. Retinoids at this concentration in culture media seem to have an antikeratinizing effect in that cells appear to be less differentiated. Retinoid-treated cultures are less stratified, having fewer cell layers, and display larger intercellular spaces and rounder, less flattened cells. Treated cultures also contain higher percentages of saline-soluble proteins and lower percentages of proteins requiring reduction and/or denaturants for solubility. This suggests that in treated cultures, the most keratinized cells are absent. Growth curves show that those most keratinized cells are sloughed from the dish and appear in the media. Thus at 3 X 10(-6) M, the major retinoid effect is to promote desquamation. At higher concentrations, retinoids are toxic to the keratinocyte, but at lower concentrations, they may be stimulative.