Howard P. Baden

Exposing the human nude phenotype.

The recent discovery of the human counterpart of the hairless mouse phenotype has helped our understanding of the molecular genetics of hair growth. But there are no reports of a defect in the human homologue of the best known of the ‘bald’ mouse phenotypes, the nude mouse. This may be because affected individuals are so gravely ill from the accompanying immunodeficiency that their baldness goes unnoticed. We have carried out a genetic analysis that reveals a human homologue of the nude mouse.

DNA synthesis in normal and xeroderma pigmentosum fibroblasts following treatment with 8-methoxypsoralen and long wave ultraviolet light.

Abstract Furocoumarins are known to photosensitize biological systems to 360 nm irradiation and an important mechanism is coupling of the compounds to thymine of DNA. In order to study the effect on DNA synthesis, incorporation of [ 3 H]thymidine into DNA of cultured fibroblasts was studied following 8-methoxypsoralen treatment and 360 nm irradiation. Normal and xeroderma pigmentosum cells showed identical inhibition of scheduled DNA synthesis but the latter were more sensitive to killing than the former. Repair synthesis of 8-methoxypsoralen and ultraviolet-damaged DNA could be domonstrated in normal cells but not in those from xeroderma pigmentosum. The amount of repair DNA synthesis relative to the degree of inhibition of scheduled DNA synthesis was much less for 8-methoxypsoralen and long wave ultraviolet-treated cells then those exposed to 254 nm radiation. Studies of guinea pig epidermal DNA following treatment of the skin with [ 3 H]trimethylpsoralen and long wave ultraviolet light indicated there is a progressive elimination of the photoadduct.

A comparative study of the physicochemical properties of human keratinized tissues

Abstract Stratum corneum, hair and nail are all derived from ectodermal cells but show significant structural differences in their fully differentiated form. However, X-ray diffraction studies indicate that they all contain an α-fibrous protein with the same molecular dimensions. The greater tensile strength and stability to heating of hair and nail compared to stratum corneum can be explained by their higher content of half cystine. Studies of the isolated structural proteins indicate that the appendages contain a non-helical matrix component very rich in cystine while stratum corneum does not. Furthermore, stratum corneum has an α-fibrous protein with physicochemical properties quite different from those of hair and nail, which are very similar to one another. Despite different morphological characteristics, it appears that hair and nail have differentiated along very similar lines. However, subtle differences in the relative proportion and composition of the structural proteins can be detected. A distinguishing feature of stratum corneum is its large content of lipids which make it an effective barrier to the diffusion of water.

Tyrosinemia with plantar and palmar keratosis and keratitis

An 111/2-year-old boy, born of a consanguineous marriage, had mental retardation, painful plantar and palmar keratosis, and dendritic keratitis associated with tyrosinemia, p-hydroxyphenylpyruvicaciduria, p-hydroxylaceticaciduria, and p-hydroxyphenylaceticaciduria. Liver and renal functions were within normal ranges, and vitamin C loading did not correct the metabolic abnormalities. Maintenance on a low-tyrosine, low-phenylalanine diet was associated with resolution of the epithelial lesions and a decrease in the metabolic abnormalities. Four other patients with similar metabolic abnormalitiers may have had this clinical syndrome.

Purification and properties of a brain enzyme which deiminates proteins

The deimination of guanidyl groups of peptides, proteins and other arginine-containing compounds is catalyzed by enzymes found in mammalian brain and epidermis. In cow, the brain and epidermal enzymes differ kinetically and physically, but both may be quantitated by measuring the production of benzoyl citrulline ethyl ester from benzoyl-arginine ethyl ester. The brain enzyme has been purified to apparent homogeneity, as judged by the presence of only one 85,000 dalton band in purified preparations when examined by SDS-polyacrylamide gel electrophoresis. An antibody raised to this band precipitates pure and partially purified brain enzyme but not partially purified epidermal enzyme, using the Ouchterlony technique. The antibody bound to an insoluble matrix removes brain enzyme activity from solution but not epidermal enzyme activity. The Km of the brain enzyme for benzoyl-arginine ethyl ester is about 0.33 mM.

Isolation and characterization of a spontaneously arising long-lived line of human keratinocytes (NM1)

SummaryThe long-lived keratinocyte line, NM1, was isolated from the epidermis of a pool of foreskins obtained from apprently, normal neonates at the time of circumcision. Cultures were initiated in Dulbecco’s minimal essential medium containing 20% fetal bovine serum, 0.4 μg/ml hydrocortisone, 10−9M 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 NM1 cells showed trisomy of chromosome 8. The long-lived nature of these cells makes them a valuable model for studying growth and differentiation of kerationocytes.

The ichthyoses--a review.

The ichthyoses are a group of hereditary disorders of keratinization which share in common the accumulation of large amounts of scale. The four major types, as well as the rarer forms, are reviewed, and current therapy is discussed.

Partial purification and specificity of an arginine-converting enzyme from bovine epidermis

An enzyme which catalyzes the conversion of intraprotein arginine residue to intraprotein citrulline residue is present in bovine snout epidermis. This arginine-converting enzyme has been partially purified by (NH4)2SO4 preciptation chromatography on DEAE-cellulose and chromatography on Sephadex G-200. The enzyme is active at neutral pH, requires Ca2+ and a reducing agent and has an apparent molecular weight of 69 000. Its substrates include histone, polyarginine, S-carboxymethyl cysteine-hair keratin, S-carboxymethyl cysteine epidermal keratin and prekeratin and S-carboxymethyl cysteine-trichohyalin. A large number of proteins, synthetic and naturally occurring peptides, and other guanidine-containing compounds were substrates of the arginine-converting enzyme.

Epidermal proteins of cultured human and bovine keratinocytes

Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.

The polypeptide composition of epidermal prekeratin.

An alpha-fibrous protein, prekeratin, has been isolated from cow snout epidermis with citrate buffer, pH 2.65. Using acrylamide electrophoresis with 0.1% sodium dodecyl sulfate, prekeratin can be shown to contain three polypeptide chains of different molecular weights. The two faster migrating components are very similar with a mol. wt of about 47,000 while the slower one has a mol. wt of about 58,000. Chromatography on a number of molecular sieve and exchange resins does not separate the components, but use of Sepharose 2B with 0.1 M Tris, pH 9.0, containing 10% propanol gives two peaks of protein. The first and major peak contains all three components while the second has only the two with the faster mobility. The two more rapidly migrating components and the slower one were isolated by acrylamide electrophoresis, and the latter has an amino acid composition more compatible with a non-helical protein. Enzymatic digestion with tosyl-L-phenylalanine chloromethylketone-treated (TPCK-)trypsin shows that the component of mol. wt 58,000 is more susceptible to hydrolysis than the other two. These data suggest that prekeratin is not homogenous in composition and consists of several interacting polypeptide chains. One of these components would appear to be non-helical in structure.