Shuhei Imayama

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa.

We report here a novel type of ceramidase ofPseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides,i.e. it hydrolyzed ceramides containing C12:0–C18:0 fatty acids and 7-nitrobenz-2-oxa-1,3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from aPseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370–24374). This bacterial ceramidase had a pH optimum of 8.0–9.0, an apparent K m of 139 μm, and a V max of 5.3 μmol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.

Molecular Cloning, Sequencing, and Expression of the Gene Encoding Alkaline Ceramidase from Pseudomonas aeruginosa CLONING OF A CERAMIDASE HOMOLOGUE FROM MYCOBACTERIUM TUBERCULOSIS

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368–14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene inEscherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to thePseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.

Combination of patch test and IgE for dust mite antigens differentiates 130 patients with atopic dermatitis into four groups

BACKGROUND Patients with atopic dermatitis sometimes have positive responses to patch testing (PT) with dust mite antigens, which is believed to correlate with the elevated levels of specific IgE for those antigens. OBJECTIVE The purpose of this study is to identify the correlation between the PT and serum IgE concerning the mite antigens. METHODS We studied 130 patients with atopic dermatitis by the PT reaction and the serum level of specific IgE for Dermatophagoides pteronyssinus antigens. RESULTS Fifty-one of the 130 patients assessed as PT-positive had either high (32 of 130 patients; 24.6%) or low or no (19 of 130 patients; 14.6%) levels of mite-specific IgE; there was a significant difference between the groups with elevated and low IgE. Similarly, a total of 79 PT-negative patients also showed an elevated or low mite-specific IgE (42 of 130 patients [32.3%] or 37 of 130 patients [28.5%], respectively). It was noted that clinical morphologic findings were peculiar to three of the four groups; however, the patients who were PT-negative with a low IgE (37 of 130 patients) showed no particular clinical lesions. CONCLUSION Comparing the results from our 130 patients, there was no correlation between the serum IgE level and the PT reaction for dust mite antigens. Conversely, the results of PT and mite-specific IgE could be used to divide these patients into four distinct groups, each with its own particular clinical morphology, suggesting the heterogeneity of this disease.

Clinical, immunological and MRI features of myelitis with atopic dermatitis (atopic myelitis)

In order to clarify the characteristic features of myelitis with atopic dermatitis (AD), we compared the clinical, immunological and MRI findings between 14 myelitic patients with AD and 12 myelitic patients without AD. The myelitic patients with AD showed the following distinct features, compared with those without AD. (1) A preferential involvement of the cervical cord, as shown by neurologic as well as MRI examinations (14/14 vs. 5/12; P=0.0012), (2) paresthesia/dysesthesia as the predominant symptoms and a rare occurrence of definite muscle weakness (0/14 vs. 5/12; P=0.0120) and dysuria (1/14 vs. 8/12; P=0.0029), (3) a lower Expanded Disability Status Scale score (mean, 1.5 vs. 3.5; P=0.0018), (4) normal cerebrospinal fluid (CSF) findings including those for the IgG index and oligoclonal IgG bands and (5) a persistence of neurologic symptoms and MRI lesions during the follow-up periods (mean, 17 months). In addition, both the serum total IgE level and the frequency of specific IgE to Dermatophagoides farinae were significantly higher in the myelitic patients with AD (median IgE=1266 U/ml, specific IgE 14/14) than in those without AD (145 U/ml, P=0.0034 and 8/12, P=0.0331, respectively) and in 40 healthy controls (86 U/ml, P<0.0001 and 12/40, P<0.0001, respectively). Since myelitis with AD has distinct features and atopy to mite antigens appears to be the underlying cause of this condition, it may therefore be a distinct subtype of myelitis.

Response of naevus of Ota to Q-switched ruby laser treatment according to lesion colour

Lesions of naevus of Ota range in colour from light brown to blue, and even greenish‐black. To develop guidelines for optimal treatment, we evaluated the number of Q‐switched ruby laser treatments required to eliminate the pigmentation of such lesions classified by colour. Over a period of 6 years, we evaluated 151 Japanese patients with naevus of Ota who had been treated with the Q‐switched ruby laser at a low energy level (wavelength 694·3 nm; pulse duration 28 × 10−9 s; energy fluence 5 J/cm2; spot size 6·5 mm) every 2 months. Each lesion was classified by colour as brown (n = 22), brown–violet (n = 42), violet–blue (n = 81) and blue–green (n = 6). The 22 predominantly brown lesions attained an excellent (100–95%) or good (95–75%) cosmetic result following three laser treatments in all patients who received this number of treatments. In the 42 brown–violet lesions, 25 of the 29 good or excellent results were achieved after four treatments; the 13 less successful results were in patients who had one to three treatments. In the 81 violet–blue lesions, 54 of the 65 good or excellent results were achieved after four treatments and 64 of 65 after five treatments, whereas all 16 less good results were in patients who had only one to three treatments. However, in the six blue–green lesions, six or more treatments were required to achieve a similarly favourable result. At the end of treatment, the area was virtually free of scarring, and its texture resembled that of the surrounding normal skin. We have confirmed that the use of the Q‐switched ruby laser at a low energy level can eliminate the pigmentation of naevus of Ota. While the desired improvement can be obtained within 1 year, the number of treatments appears to depend on the predominant colour of the lesion.

Spindle cell haemangioendothelioma : probably a benign vascular lesion not a low-grade angiosarcoma : a clinicopathological, ultrastructural and immunohistochemical study

Ten cases of spindle cell haemangioendothelioma (SCH) were analysed clinicopathologically, including an immunohistochemical survey of seven cases and ultrastructural observations on one. There were seven females and three males, ranging from 16 to 76 years of age. All but one lesion developed on the extremities, predominantly on the hands and feet. Six of the ten patients presented multiple nodules or papules which gradually increased in size and number over a long duration. Among them, four patients had undergone operations twice or more, but no metastatic foci were recognized. Histologically, the lesions were composed of dilated vascular spaces and a proliferation of bland-appearing spindle cells and interspersed epithelioid endothelial cells. Ultrastructural and immunohistochemical studies demonstrated that the spindle cells were mainly made up of fibroblastic cells admixed with pericyte-like cells and macrophages. Smooth muscle cells and primitive mesenchymal cells were also present. The clinical and microscopic features suggest that SCH may be a benign vasoformative lesion of a heterochronological multicentric origin.

Sulfidoleukotriene Release Test (CAST) in Hypersensitivity to Nonsteroidal Anti-Inflammatory Drugs

There is a great need to develop a method for making an accurate and reliable in vitro diagnosis of adverse hypersensitivity reactions to drugs. We measured the amount of sulfidoleukotriene (sLT) released from the peripheral blood leukocytes obtained from 25 patients who developed hypersensitivity reactions following the administration of nonsteroidal anti-inflammatory drugs (NSAIDs); 12 patients demonstrated reactions to Voltaren, 8 patients to Bufferin, and 5 patients to Sedes G. The stimulation index, the ratio of the amounts of sLT (pg/ml) incubated with and without drugs, was considerably higher in the patients than in the controls, which consisted of 5 nonallergic healthy subjects. The sensitivity of the CAST (cellular antigen stimulation test) was evaluated to range from 62.5 to 80%, while the specificity was 70-100%. The CAST may thus be useful as a novel in vitro test system in order to screen for possible hypersensitive reactions to NSAIDs with both reliability and safety.

Ultraviolet-B irradiation deforms the configuration of elastic fibers during the induction of actinic elastosis in rats

We used scanning electron microscopy combined with perfusion fixation, resin injection and a selective digestion procedure to determine the effects of ultraviolet-B (UVB) radiation on the three-dimensional architecture of elastic fibers of rat skin. Chronic irradiation with a suberythematous dose of UVB (3 times/week for 12 weeks) produced a tortuous deformation of the superficial elastic fibers in the skin of the rat sole which normally are linearly arranged. Using computer analysis, we evaluated 40 individuals elastic fibers every 3 weeks for 12 weeks following the irradiation. This procedure confirmed the increasing tortuosity of the fibers, which was related to a decline in the elastic property of the skin in situ. Fine elastic branches developed among the deformed fibers and eventually anastomosed to produce an irregular network in the superficial dermal connective tissue, which may correspond to the development of actinic elastosis, that is, the UV-related accumulation of elastic fiber material.

Clinicopathologic Analysis of 22 Cases of Subcutaneous Panniculitis-Like CD56- or CD56+ Lymphoma and Review of 44 Other Reported Cases

In 22 histologic cases of subcutaneous panniculitis-like lymphoma, we studied the clinicopathologic differences between CD56- and CD56+ cases (11 each). CD56- cases had skin ulcers (1 [9%]); tumor invasion in the superficial dermis (1 [9%]); erythrophagocytosis (10 [91%]); and medium-sized (11 [100%]), CD8+ (10 [91%]), T-cell receptor (TcR)betaF1+ (10 [91%]), and CD95 (Fas)- tumor cells. CD56+ cases had skin ulcers (9 [82%]); tumor invasion in the superficial dermis (8 [73%]); erythrophagocytosis (1 [9%]); and pleomorphic large (10 [91%]), CD8+ (2/10 [20%]), TcRbetaF1 + (3/10 [30%]), and CD95 (Fas)+ (7/10 [70%]) tumor cells. These 7 factors were significantly different between groups (P < .01). Median survival rates were 96 and 12 months for the CD56- and CD56+ groups, respectively. Age younger than 40 years, no skin ulcers, no tumor invasion in the superficial dermis, and CD8+, TcRbetaF1 +, CD95 (Fas)-, and CD56- tumor cells were significantly better prognostic factors (P < .01). The CD56- and CD56+ groups showed different tumor cell characteristics, clinicopathologic findings, and prognosis. In the CD56+ group, 1 was gamma/delta T-cell phenotype, 3 were alpha/beta T-cell, and 6 were TcRbetaF1- and gamma/delta- NK/T-cell, and 3 NK/T-cell cases had nuclear signals of Epstein-Barr virus-encoded RNA. Cases of CD56+ T- and NK/T-cell lymphoma had similar clinicopathologic findings and prognosis.

Reduction of Environmental Mites Improved Atopic Dermatitis Patients with Positive Mite-patch Tests

During a series of studies on the involvement of house dust mite antigens in 183 cases of atopic dermatitis, we observed an improvement in two patients following the removal of mites from their environment by means of a thorough housecleaning and replacement of the mattress. Both patients manifested the typical clinical skin lesions of atopic dermatitis and had similar laboratory findings. Although the serum IgE concentrations and specific IgE to Dermatophagoides pteronyssinus and Dermatophagoides farinae were each relatively low, the results of patch tests with these antigens were positive. Thus, a regimen aimed at reducing the presence of house dust mites can produce clinical improvement in a subset of patients with atopic dermatitis who show contact hypersensitivity to mite antigens on skin testing, but negative results on IgE (RAST; radioallergosorbent technique) testing.