The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes.

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.

Ultraviolet B radiation downregulates inducible nitric oxide synthase expression induced by interferon-gamma or tumor necrosis factor-alpha in murine keratinocyte Pam 212 cells.

Ultraviolet radiation causes inflammation characterized by erythema and swelling, but also exhibits antiinflammatory effects which have led to the use of ultraviolet B radiation (UVBR) and psoralen plus ultraviolet A (PUVA) in the treatment of psoriasis, chronic severe atopic dermatitis and uremic pruritus. In inflammatory dermatoses, a pathogenic role of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) has been suggested. To elucidate how UVBR regulates iNOS expression in skin under inflammatory conditions, we investigated the effect of UVBR on NO production and iNOS expression in cultured murine keratinocyte Pam 212 cells stimulated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha). Low doses of UVBR significantly suppressed IFN-gamma- or TNF-alpha-induced NO production. UVBR also downregulated IFN-gamma- or TNF-alpha-induced iNOS expression at both the mRNA level and the protein level. These findings suggest the possibility that the down-regulatory effect of UVBR on IFN-gamma- or TNF-alpha-induced iNOS expression may, in part, explain the antiinflammatory and therapeutic properties of UVBR in inflammatory dermatoses.

Suppressive effect of zinc ion on iNOS expression induced by interferon-gamma or tumor necrosis factor-alpha in murine keratinocytes.

Zinc, an essential metal, is a critical component of zinc binding proteins such as zinc fingers, zinc enzymes and metallothioneins. Recently, evidence for its anti-inflammatory property in skin has been accumulating, as shown in the treatment of acne, alopecia and zinc deficiency. In cutaneous inflammations, a large amount of nitric oxide (NO) is produced through induction of inducible nitric oxide synthase (iNOS) under the influence of proinflammatory cytokines, resulting in tissue damages in skin, as clarified in other organs. Therefore, we asked if the effect of zinc on NO production and/or on iNOS expression in keratinocytes may explain the anti-inflammatory property of zinc in skin. Accordingly, we sought to determine in this study whether zinc ion may have effect on IFN-gamma or TNF-alpha induced NO production and iNOS expression in cultured murine keratinocytes. Ten microM of zinc ion remarkably suppressed cytokine-induced NO production in keratinocytes. Furthermore, zinc ion also suppressed cytokine-induced iNOS expression in the protein level as well as in the messenger RNA level. These results suggest the possibility that the suppressive effect of zinc ion on cytokine-induced NO production in keratinocytes may be in part implicated in the anti-inflammatory property of zinc in some of skin disorders.

Mutational analysis of superantigen activity responsible for the induction of skin erythema by streptococcal pyrogenic exotoxin C.

Streptococcal pyrogenic exotoxin C (SPEC), when injected intradermally, induces erythema in unsensitized rabbits. In the present study, we examined whether this erythema induction is due to the T-cell stimulatory activity of SPEC as a superantigen. Analysis by using single-residue mutant SPECs indicated that mutant SPECs Y15I, A16E, and Y17I, in which tyrosine 15, alanine 16, and tyrosine 17 were replaced with isoleucine, glutamic acid, and isoleucine, respectively, exhibited significantly reduced mitogenic activity for Vbeta2(+) human T cells in vitro, and Y15I showed as much as a 1, 000-fold reduction. Y15I mutant SPEC, however, retained the ability to bind to major histocompatibility complex class II antigen and to form a homodimer, implying that residue 15 is critically important for the interaction of SPEC with T-cell antigen receptor beta chains. When injected intradermally into normal rabbits, wild-type SPEC induced a characteristic erythema after 3 h in a dose-dependent fashion, which was associated with polymorphonuclear and mononuclear cell infiltration. This erythema formation was found to be severely suppressed by systemic pretreatment with cyclosporin A, suggesting the involvement of host T cells. Y15I mutant SPEC exhibited nearly 1, 000-fold less erythema induction in vivo than wild-type SPEC. Altogether, the present results strongly suggest that erythema induction in rabbits by SPEC is attributable mostly to its T-cell stimulatory activity as a superantigen.

Analysis of mechanisms of epidermal proliferation induced by intracutaneous injection of cholera toxin by the use of site-specifically mutated cholera toxins.

Intracutaneous injection of cholera toxin (CT) into rabbits increases vascular permeability and induces epidermal proliferation. To understand the mechanisms of these effects on the skin, we evaluated the involvement of the ADP-ribosyltransferase activity of the A subunit of CT and receptor-binding interactions between GM1-ganglioside and the B subunit of CT. We constructed two mutant CTs, E112K and W88K, by site-directed mutagenesis. Mutant CT-E112K, in which glutamic acid at position 112 (E112) of the A subunit of CT was replaced by lysine, has been shown to have lost its biological activity on Chinese hamster ovary (CHO) cells because of its abolished ADP-ribosyltransferase activity. Mutant CT-W88K, in which tryptophan at position 88 (W88) of the B subunit of CT was replaced by lysine, has been shown to have lost its binding ability to GM1-ganglioside. Intracutaneous injection of these mutant CTs evoked less vascular permeability and less epidermal proliferation than recombinant wild-type CT. These results suggest that: (1) the ADP-ribosyltransferase activity carried by E112 of the A subunit of CT; and (2) the binding ability to GM1-ganglioside via W88 of the B subunit of CT are essential for these effects of CT on the skin.

Loss of biological activity due to Glu-->Arg mutation at residue 11 of the B subunit of cholera toxin.

Since it has been reported that a single amino acid mutation of Gly-->Arg in the CAGYC region of the beta chain of human thyroid stimulating hormone (hTSH) was responsible for congenital isolated TSH deficiency, and that the same amino acid substitution in this site of hTSH and human chorionic gonadotropin (hCG) introduced by site-directed mutagenesis resulted in loss of activity, the authors studied the role of glutamic acid at position 11 (Glu-11) from the N-terminus of the B subunit of cholera toxin (CT), which corresponds to the glycine in the CAGYC region of the beta chain of hTSH and hCG. A mutant CT constructed by site-directed mutagenesis in which Glu-11 was replaced by Arg (CT-E11R) did not induce either morphological changes or accumulation of cytosolic cyclic AMP in Chinese hamster ovary cells, although it formed the holotoxin AB5, retained the ability to bind to GM1-ganglioside and showed ADP-ribosyltransferase activity. Weak assembly of the B subunits in mutant CT-E11R demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-heating conditions might explain the loss of biological activity.

Anomaly of the ulnar artery arising from the brachial artery.

A high origin of the ulnar artery was encountered in the body of an 87-year-old Japanese man in the course of normal anatomic dissection at Nara Medical University. The left ulnar artery originated from the brachial artery at the height of the left inferior angle of the left scapula and passed medially to the brachial artery and the median nerve. In the forearm, the ulnar artery passed deep to the flexor carpi ulnaris muscle. Thereafter, the two branches from the ulnar artery contributed to form the superficial and deep palmar arterial arches. The left brachial artery was divided into two terminal branches of the radial and common interosseous arteries.

Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose.

A simple method for purification of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and for purification of cholera toxin using immobilized D-galactose column is described. A single run of column chromatography yielded homogeneous toxin as demonstrated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis.