Makoto Negi

Keratinolytic proteinase produced by Candida albicans

Candida albicans was cultivated in various media that contained human stratum corneum, human scalp hair or keratin powder (cows hoof) as a nitrogen source. Production of a keratinolytic proteinase (KPase) was observed when C. albicans was incubated in the medium containing stratum corneum. However, there was no production of a KPase that could digest human stratum corneum in the medium containing hair or keratin powder. alpha-fibrous protein extracted from human stratum corneum was digested by the KPase. The pH optimum of the enzyme was 4.0 and enzyme activity was inhibited by pepstatin A and chymostatin. The KPase, a kind of carboxyl proteinase, may be important for C. albicans to enable it to play a pathogenic role in vivo.

Partial characterization of the extracellular keratinase from Microsporum canis

The extracellular keratinase of Microsporum canis released peptides from alpha-type fibrous protein and the membranous fraction isolated from human stratum corneum. Inhibition of the enzyme by phenylmethyl-sulfonylfluoride and its weak inhibition by N-ethylmaleimide and etheneglycol tetra-acetic acid indicated that it is probably a serine proteinase.

ISOLATION AND CHARACTERIZATION OF THE MEMBRANEOUS FRACTION IN HUMAN STRATUM CORNEUM

Membraneous fractions were isolated from human stratum corneum. The fractions obtained after frequent reextraction with urea and 2 ME at pH 9.0 (A), and further digestion of (A) by trypsin (B) were compared using electron microscopy and amino acid analysis. Low molecular weight proteins or amino peptides were solubilized from fraction (A) following digestion with trypsin, chymotrypsin and pronase P. The amounts of solubilized material showed a plateau after 30 minutes of digestion with trypsin. A small fraction of contaminants thought to be the fibrous and interfibrous materials were still observed in Fraction (A) by electron‐microscopy and SDS gel electrophoresis, but no remarkable contaminants were observed in Fraction (B), which was composed of trilaminar membraneous parts including the marginal band. Fraction (A) contained 43 half cystine residues per 1000 amino acid residues, whereas Fraction (B) contained 81 half cystine residues per 1000 amino acid residues.

Alteration of human epidermal transglutaminase during its activation

The effects of enhancement of enzymatic activity by heating at 56 degrees C or by limited treatment with dimethylsulfoxide, trypsin and cathepsin D on two forms (Mr = 50 kDa and 72 kDa) of human epidermal transglutaminase were studied by immunoblots using rabbit antihuman epidermal transglutaminase. Both 50 kDa and 72 kDa transglutaminase bands were detected without any alteration in the mobility of the transglutaminase bands during activation induced by heating at 56 degrees C or by pretreatment with dimethylsulfoxide. With a preincubation period longer than 60 min, the trypsin pretreated sample showed progressive disappearance of the 72 kDa transglutaminase band in conjunction with the loss of transglutaminase activity. On the other hand, samples preincubated with cathepsin D showed a complete disappearance of the 50 kDa band after 180 min. These studies suggest the different forms of human epidermal transglutaminase may regulate enzyme activity each other during normal epidermal differentiation.

FRACTIONATION AND CHARACTERIZATION OF THE HUMAN EPIDERMAL STRATUM CORNEUM IN KERATINIZATION DISORDERS

Stratum corneum obtained from persons with a variety of keratinization disorders was fractionated into keratin fibers, water soluble proteins and cell membranes. SDS gel electrophoresis was used to study the keratin fibrous and water soluble fractions, and amino‐acid analysis was employed for the membraneous fractions.

Biochemical changes after the oral administration of retinoid in the horny layer of patients with keratinization disorders.

Four cases with keratinization disorders were treated with aromatic retinoid (Ro 10‐9359), with resulting clinical improvement. Horny layers from these patients were obtained before and after treatment, and fractionated into keratin fibers, soluble proteins, and membraneous fractions. These were studied using SDS gel electrophoresis and amino‐acid analysis, and a comparison was made between the findings before and after retinoid administration. Membraneous fractions from diseased horny layers contained abnormal amino acid compositions, in particular, reduced half‐cystine and proline residues, which tended to become normalized after the treatment with retinoid. Normalization of keratin fibers and soluble fractions was not as obvious on SDS gel electrophoresis.

FRACTIONATION AND CHARACTERIZATION OF THE EPIDERMAL STRATUM CORNEUM IN BULLOUS CONGENITAL ICHTHYOSIFORM ERYTHRODERMA (BCIE)

The stratum corneum obtained from seven cases of bullous congenital ichthyosiform erythroderma (BCIE) and one case of Vörner type keratosis palmaris et plantaris (KPP) was fractionated into keratin fibers, soluble proteins, and cell membranes. To study the fractions, SDS polyacrylamide gel electrophoresis was used for the keratin fibrous and soluble fractions and amino acid analysis was employed for the membraneous fraction. In the electrophoresis of the keratin fibrous fractions, the 55,000 dalton band was either absent or greatly reduced in BCIE, whereas almost normal patterns were obtained in Vörner type KPP. The soluble fractions from BCIE demonstrated abnormal patterns in different cases on electrophoresis. Amino acid compositions of the keratin fibrous and membraneous fractions in BCIE were identical to those of the normal control. It is suggested that, as the 55,000 dalton constituent of keratin fibrous protein is absent or greatly reduced, this is a specific characteristic of BCIE, and that biochemical changes differ for BCIE and Vörner type KPP, which have been thought to be essentially identical due to their histological and electron‐microscopic similarity.

Localization and possible activation mechanisms of transglutaminase in the skin.

The activity of transglutaminase per unit of wet weight, dry weight, soluble protein, and DNA was determined in three fractions (stratum corneum, malpighii, and dermis) of cow snout skin. The stratum corneum showed the highest enzyme activity among these three fractions.

BIOCHEMICAL ANALYSES OF STRATUM CORNEUM IN HYPERKERATOSIS LENTICULARIS PERSTANS

The stratum corneum obtained from keratotic papules of hyperkeratosis lenticularis perstans was fractionated into keratin filamentous, water soluble, and membraneous fractions. SDS‐gel electrophoresis was used to examine the keratin filamentous and soluble fractions, and amino acid analysis was employed for all fractions.