Psoriasis and the arachidonic acid cascade.

Arachidonic acid (5.8,11,14-eicosatetraenoic acid C20:4, n-6) is released from the cell membrane by the action of phospholipases on membrane phospholipids. Metabolites of arachidonic acid, which are generically termed eicosanoids, including prostaglandins, thromboxane, leukotrienes and hydroxyeicosatetraenoic acids, have been implicated as mediators or modulators of a number of physiological functions and pathological conditions in both normal and diseased human skin. Particularly, eicosanoids have been suspected to play an important role in the pathogenesis of psoriasis, because a number of phenomena observed in psoriasis can be explained, at least in part, by the action of eicosanoids. This review will focus on recent progress regarding the significance of eicosanoids in the pathogenesis of psoriasis. Recent developments in the molecular biology in the eicosanoids have renewed interest in the role of eicosanoids in psoriasis. New understanding of the etiology of psoriasis and advances in its treatment due to recent progress in eicosanoid biology will also be presented.

Cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from patients with atopic dermatitis: correlation with respiratory atopy.

We determined the cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in peripheral blood mononuclear leucocytes from 100 patients with atopic dermatitis (AD) aged 13-57 years (mean +/- SD, 29.8 +/- 17.7 years). The correlation between cAMP-PDE activity and clinical parameters such as the severity of eczema and a personal or family predisposition to atopic respiratory diseases (ARD) (asthma or allergic rhinitis) was examined. Although the enzymic activity varied from normal to very high in the AD patients, cAMP-PDE activity was significantly (P < 0.005) elevated in AD patients (42.1 +/- 22.0 units) as compared with the normal controls (12.4 +/- 5.6) and clinical control subjects (13.4 +/- 9.5). In contrast, we found no correlation between cAMP-PDE activity and the severity of eczema when AD patients were classified into four categories (remission, mild, moderate and severe) according to the extent of their skin involvement. Furthermore, we found that systemic corticosteroid therapy in severe AD patients did not alter the cAMP-PDE activity. cAMP-PDE activity was significantly (P < 0.01) higher in those AD patients who had a personal history of ARD (47.2 +/- 11.2) than in AD patients with a family history of ARD (37.2 +/- 17.4) and those without a personal or family history ('pure' AD) (34.4 +/- 19.8). Nevertheless, the cAMP-PDE activity was significantly higher even in 'pure' AD patients than in the controls. These results suggest that an elevation of cAMP-PDE activity is closely related to a predisposition to respiratory atopy, and does not follow inflammation in AD patients.

Induction of apoptosis, p53 and heme oxygenase-1 by cytotoxic prostaglandin delta12-PGJ2 in transformed endothelial cells.

Delta12-prostaglandin (PG)J2, which has been reported to have potent growth inhibitory activity in various tumor cells, induced apoptosis at 5 microg/ml culture medium in transformed mouse endothelial (F2) cells. Immunoblot analysis using anti-p53 or anti-WAF1 antibodies demonstrated that these two proteins had increased following delta12-PGJ2 treatment in F2 cells. Western blotting analysis using anti-heme oxygenase-1 (heat shock protein (HSP)32) antibody also revealed that delta12-PGJ2 induced HSP32 formation in F2 cells. HSP32 was also induced by heat shock treatment at 43 degrees C for 90 min. In contrast, HSP72 was not induced by heat shock or by delta12-PGJ2 treatment. In agreement with these findings, HSP32 immunofluorescence in the cytoplasm of F2 cells was intensified by delta12-PGJ2 treatment. More intense HSP32 immunoreactivity was similarly observed after heat shock treatment. These results suggest that delta12-PGJ2 caused the apoptotic cell death of F2 cells, which involved a certain process required for p53 or HSP32 induction.

Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans.

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.

Human melanoma cells generate leukotrienes B4 and C4 from leukotriene A4.

We examined the synthesis of leukotrienes (LTs) in human melanoma cells in order to assess the function of LTs in human melanocytes. LTA4 hydrolase, which catalyzes the conversion of LTA4 to LTB4, was detected in the supernatant of cultured human melanoma (MeWo) cells and melanoma cells obtained from patients. Immunoblotting analysis using an antihuman LTA4 hydrolase antibody showed LTA4 hydrolase to be a 70-kDa protein in both MeWo and melanoma cells. Considerable activity of LTC4 synthase, which catalyzes the conversion of LTA4 to LTC4, was detected in the microsomal fraction of both MeWo and melanoma cells. The HPLC profile of the LTC4 synthase reaction products revealed that LTC4 was the main product. LTD4 was not detected under these conditions, indicating that the microsomal fraction of human melanoma cells lacks the membrane-bound gamma-glutamyl transferase that converts LTC4 to LTD4. LTC4 synthase activity was inhibited by the addition of MK-886, and was not altered by treatment with N-ethylmaleimide or 1-chloro-2,4-dinitrobenzene. These results indicate that the enzyme responsible for the conversion of LTA4 to LTC4 in human melanoma cells is LTC4 synthase rather than a nonspecific or microsomal glutathione-S-transferase. These results also suggest that human melanoma cells can generate LTB4 and LTC4 from LTA4, and that this process is catalyzed by two enzymes: LTA4 hydrolase and LTC4 synthase.

Werner's syndrome--chromosome analyses of cultured fibroblasts and mitogen-stimulated lymphocytes.

Two cases of Werner's syndrome are reported. Fibroblasts derived from both patients revealed reduced population doubling numbers. Chromosomal analyses for fibroblasts from both patients and lymphocytes from one patient revealed that chromosomal aberrations occur frequently and randomly. Although some of the chromosomal aberrations involved sites where tumour suppressor genes have been mapped, neither of our patients demonstrated malignancy. Chromosomal aberration at one critical site may not be sufficient to induce cancer or additional factors may be necessary.

An acquired bullous dermatosis due to an autoimmune reaction against uncein.

A 59-year-old male showed acquired, mechanically induced, scarring blisters on the fingers, toes, scalp and abdomen, as well as in the oral cavity. Ultrastructural and immunohistochemical examination of the bullae revealed junctional epidermal-dermal separation and IgG deposits in the lamina lucida of the basement membrane zone (BMZ), where the reactivity of the 19-DEJ-1 monoclonal antibody was decreased. Anti-BMZ autoantibodies detected in his serum were reactive to the lower lamina lucida region of normal human skin. SDS-PAGE of affinity purified antigens from human keratinocytes with IgG from the patient's serum revealed three polypeptide bands at 165, 135 and 100 kDa, in reduced condition. The indirect immunofluorescence test of his serum was negative on skin cryosections from patients with lethal junctional epidermolysis bullosa. Pretreatment of normal human skin sections with the patient's serum, blocked the binding of 19-DEJ-1 monoclonal antibody but not that of the GB3 monoclonal antibody. This case is considered to be an acquired autoimmune bullous dermatosis due to an autoantibody reaction against uncein (19-DEJ-1 antigen), a component of anchoring filaments.

Leukotriene A4 hydrolase in peripheral leukocytes of patients with atopic dermatitis.

We examined the enzymatic activity of leukotriene (LT) A4 hydrolase, which catalyzes the conversion of LTA4 to LTB4, in peripheral leukocytes of patients with atopic dermatitis. The patients were divided into three categories (severe, moderate and mild) on the basis of clinical severity. The LTA4 hydrolase activities in the supernatant fraction of peripheral blood polymorphonuclear leukocytes (PMN) were significantly higher in preparations of cells from severe atopic dermatitis patients (123.94 +/- 16.61 pmol/10(6) cells per min) than in those from moderate (49.03 +/- 9.43 pmol/ 10(6) cells per min; P < 0.01) and mild (28.75 +/- 11.42 pmol/10(6) cells per min; P < 0.01) atopic dermatitis patients and normal controls (15.14 +/- 1.74 pmol/10(6) cells per min; P < 0.01). LTA4 hydrolase activities were also higher in peripheral blood mononuclear cells (PBMC) from severe atopic dermatitis patients (27.81 +/- 8.28 pmol/10(6) cells per min) than in those from moderate (11.31 +/- 2.11 pmol/10(6) cells per min; P < 0.05) and mild (6.16 +/- 2.62 pmol/10(6) cells per min; P < 0.05) atopic dermatitis patients and normal controls (11.17 +/- 0.83 pmol/10(6) cells per min; P < 0.05). LTA4 hydrolase activities in PMN were reduced after improvement of the disease in eight patients with severe or moderate atopic dermatitis. These results suggest that LTA4 hydrolase, which synthesizes LTB4, plays a significant role in the pathogenesis and development of atopic dermatitis.

A case of autoimmune bullous dermatosis with features of pemphigus vulgaris and bullous pemphigoid.

Pleomorphic blisters, including tense bullae and annularly arranged vesicles around the erythema as well as erosive eruptions in the oral cavity, appeared on a 61-year-old woman 5 years after surgery for cholangiocellular carcinoma. A biopsy specimen from the oral cavity showed intraepidermal blisters, and those from skin lesions showed subepidermal blisters with infiltrates of eosinophils and neutrophils. The early-stage vesicles showed infiltrates along the epidermal-dermal junction, where electron microscopy disclosed disruption of the lamina densa, basal cells remaining on the dermis, and acantholytic keratinocytes among the infiltrates, but there was no cleavage of the epidermal-dermal junction at the lamina lucida. Direct immunofluorescence studies showed immune deposition at the intercellular space (ICS) and along the basement membrane zone (BMZ). Indirect immunofluorescence studies confirmed coexistence of IgG class anti-ICS and anti-BMZ antibodies. Although this case showed immunohistochemical features of bullous pemphigoid, the presence of suprabasal cleavage in the oral mucosa, acantholytic cells in the blister cavity, the deposition of IgG at the ICS of the perilesional epidermis, and circulating anti-ICS antibodies strongly suggested that this case was primarily pemphigus. The strong inflammation along the epidermal-dermal junction due to unknown factors may have modified the clinical appearance and the histopathology.

Bilateral inguinal scrofuloderma during steroid therapy in a patient with bullous pemphigoid.

This report described a case of scrofuloderma that developed in the bilateral inguinal regions during treatment of bullous pemphigoid with systemic corticosteroid. Analysis of the literature on scrofuloderma between 1978-1993 disclosed that the number of cases with extracervical involvement are increasing. Immunosuppression could disseminate tuberculous focuses, resulting in extracervical involvement of SD connected with the underlying extrapulmonary tuberculous lesions.

Effect of cytotoxic prostaglandin, delta 12-prostaglandin J2 on E-cadherin expression in transformed epidermal cells in culture.

The cyclopentenone prostaglandins (PGs), such as delta 12-PGJ2 and PGA1, are potent inhibitors of growth in a variety of cultured cells, including human epidermal cells. To clarify the mechanism of the cytotoxicity of these PGs, we examined the effects of delta 12-PGJ2 on the function and expression of E-cadherin, which plays a major role in the maintenance of intercellular adhesion, in transformed human epidermal cells in culture (HSC-1). A 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 did not affect the cell-binding activity of E-cadherin expressed in HSC-1 cells. Immunoblot analysis using a monoclonal antibody specific to human E-cadherin revealed that a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2 induced E-cadherin expression in HSC-1 cells. Immunofluorescence using a monoclonal antibody against human E-cadherin demonstrated that E-cadherin was localized to the cell-cell contact regions in HSC-1 cells. Following a 12-h incubation with 5 micrograms/ml of delta 12-PGJ2, E-cadherin was also detected in a uniform pattern along cell junctions, although cell morphology was changed by the presence of cytotoxic PGs. These results suggest that the cytotoxicity of cyclopentenone PGs is related, at least in part, to E-cadherin expression in transformed human epidermal cells.

Elevated cyclic adenosine monophosphate phosphodiesterase activity in peripheral blood mononuclear leucocytes from children with atopic dermatitis.

We examined peripheral blood mononuclear leucocyte cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) activity in 80 children (aged 2-12 years) with atopic dermatitis. The enzyme activity (35.1 +/- 18.6 U) in children with atopic dermatitis was significantly higher than that (19.1 +/- 12.6 U) in age-matched non-atopic controls. There was no significant difference in the cAMP-PDE activity between children with mild atopic dermatitis and children with severe atopic dermatitis. These findings support the view that elevation of peripheral mononuclear leucocyte cAMP-PDE activity in patients with atopic dermatitis is a gene-associated abnormality.

Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: a possible role for rho protein in epidermal cells.

We examined the role of rho gene products (rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC-1), using recombinant botulinum C3 exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with a molecular weight of 23,000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 exoenzyme at 1, 3, 10, 30 and 60 micrograms/ml culture medium resulted in 9.0 +/- 1.8%, 20 +/- 2.9%, 26 +/- 2.3%, 50 +/- 1.4% and 40 +/- 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells.

Leukotriene A4 hydrolase in human skin.

The biochemical properties and immunohistochemical localization of leukotriene (LT) A4 hydrolase were investigated in human skin. The activity of LTA4 hydrolase, which catalyzes the hydrolysis of LTA4 to LTB4, the most chemotactic compound known, was detected in the 100,000 x g supernatant of homogenates of human epidermis and a transformed epidermal cell line (HSC-1). No significant LTA4 hydrolase activity was detected in human whole skin or dermis. The enzymatic properties of LTA4 hydrolase isolated from human keratinocytes and peripheral leukocytes were similar. Their activities were inhibited by bestatin and captopril, and they were completely absorbed by anti-human LTA4 hydrolase antibody. By immunoblotting analysis using this antibody, LTA4 hydrolase was detected as a 70-kDa protein in human epidermis and HSC-1 and was found to be similar to the enzyme detected in peripheral mononuclear leukocytes. In human dermis, LTA4 hydrolase was barely detected by Western blotting. On the other hand, LTA4 hydrolase was demonstrated in the cytoplasm of keratinocytes in the epidermis, and in fibroblasts, infiltrating and endothelial cells in the dermis of normal human skin by immunohistochemical analysis using the immunoperoxidase method. These results suggest that LTB4 can be generated from LTA4 by LTA4 hydrolase in keratinocytes as well as fibroblasts, infiltrating and endothelial cells in the dermis of human skin.

Isolation, structures, and antifungal activities of new aureobasidins.

Aureobasidins are a group of cyclic depsipeptides with antifungal activity and are produced by Aureobasidium pullulans. Aureobasidins are composed of eight amino acids and one hydroxy acid such as 2-hydroxy-3-methylpentanoic acid (Hmp), and highly lipophilic. Five new aureobasidins, S1, S2a, S2b, S3 and S4, which have higher hydrophilicity in reversed phase HPLC than the known aureobasidins A-R, were discovered in a fermentation broth of A. pullulans R106 by means of on-line liquid chromatography/mass spectrometry with electrospray ionization. We identified the structures of the compounds and studied their antifungal activities. Three of the new aureobasidins, S2b, S3 and S4, which have hydroxylated Hmp as the hydroxy acid, were highly active against Candida spp. and Cryptococcus neoformans.

Relationship between heat shock protein induction and the binding of antibodies to the extractable nuclear antigens on cultured human keratinocytes.

The importance of environmental factors such as ultraviolet light and temperature in the pathogenesis of cutaneous lupus erythematosus is well recognized. Recent evidence suggests the presence of autoantibodies to heat shock proteins (HSP) in the sera and enhanced expression of the HSP70 gene in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. We designed experiments to determine how HSP or stress protein inducers affect the cell surface binding of IgG antibodies from sera containing anti-SS-A/Ro and anti-ribonuclear protein (RNP) antibodies to keratinocytes because these antibodies are considered to be one of the immunologic triggers of cutaneous lupus erythematosus. Immunofluorescence and immunoblot analysis using a monoclonal antibody to the 72 kDa of HSP revealed that an 18-h incubation with 10 micrograms/ml of delta 12-PGJ2, one of cytotoxic prostaglandins, induced HSP72 formation in cultured human keratinocytes. delta 12-PGJ2 augmented the binding of IgG antibodies from sera containing anti-U1RNP and anti-SS-A/Ro antibodies to cultured keratinocytes, but produced no enhancement of the binding of IgG antibodies from sera containing anti-Sm or anti-DNA antibodies. Similar results were also obtained by using flow cytometry analysis. HSP was also induced by ultraviolet B irradiation. These results suggest that exposure of keratinocytes to stressors such as delta 12-PGJ2 and ultraviolet light increases the binding sites for U1RNP,SS-A/Ro, and SS-B/La antibodies. The association between HSP induction and the appearance of extractable nuclear antigens may provide a better understanding of why environmental stimuli can promote the development of erythematous lesions in the skin.

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.