A gene, cots, encoding a spore coat polypeptide of Bacillus subtilis, was isolated from an EcoRl fragment (54 kb) of the chromosome by using synthetic oligonucleotide probes corresponding to the NH,-terminal amino acid sequence of Cot40-2 previously purified from the spore coat of B. subtilis. The nucleotide sequence (2603 bp) was determined and sequence analysis suggested the presence of two contiguous ORFs, ORF X and cots, followed by the 5'-region of an additional ORF, ORF Y, downstream of cots. The cots gene is 1053 nucleotides long and encodes a polypeptide of 351 amino acids with a predicted molecular mass of 41 083 Da. The predicted amino acid sequence was in complete agreement with the NH,-terminal amino acid sequence of Cot40-2. The orfx gene is 1131 nucleotides long and encodes a polypeptide of 377 amino acids with a predicted molecular mass of 42911 Da. The gene product of cots was confirmed to be identical to Cot40-2 by SDS-PAGE and immunoblotting from Escherichia coli transformed with a plasmid containing the cots region. Northern hybridization analysis indicated that a transcript of cots and orfx appeared at about 5 h after the onset of sporulation. The transcriptional start point determined by primer extension analysis suggested that -10 and -35 regions are present upstream of o m and are very similar to the consensus sequence for the $<-dependent promoter. Terminator-like sequences were not found in the DNA fragment (2603 bp) sequenced in this paper, which suggested that the cots locus may be part of a multicistronic operon. The cots gene is located between dnaB and degQ a t about 27&275" on the genetic map. Insertional mutagenesis of the cots gene by introducing an integrative plasmid resulted in no alteration of growth or sporulation, and had no effect on germination or resistance to chloroform.

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Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-beta-D-galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of epsilon N-2,4-dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound beta-D-galactosidase activity by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers.

The novel immunomodulator (immunosuppressant) FTY720 1,2) is a synthetic structural analogue of myriocin (ISP-I), a metabolite of Isaria cinclairii.3) FTY720 was discovered by Tetsuro Fujita, in collaboration with Taito Co., Ltd., Japan and Yoshitomi Pharmaceutical Industries, Ltd., Japan. FTY is an abbreviation of Fujita, Taito and Yoshitomi. The efficacy of FTY720 has been well established in preclinical transplantation models (rat heart, liver, skin, small intestine, dog kidney and monkey kidney), 4) and also recently in renal transplantation in humans. [5][6][7][8] The mechanism of action of FTY720 differs from that of established immunosuppressants (cyclosporin and tacrolimus hydrate). FTY720 is phosphorylated by sphingosine kinase, and the product, FTY720 monophosphate, is the active form of the drug. FTY720 monophosphate acts as a potent agonist at four sphingosine 1-phosphate receptors (S1P), especially S1P 4 (endothelial differentiation gene (edg) 6) and S1P 5 (edg-8), and modulates chemotactic responses of lymphocytes and lymphocyte trafficking. 9,10) As a result, FTY720 suppresses immune response by sequestering lymphocytes from blood and peripheral tissues to the secondary lymphoid tissues. 11,12) However, Sugito and Fukuzawa reported that administration of FTY720 to mice without lymph nodes, Payer's patches and spleen still resulted in peripheral lymphopenia, prevented the infiltration of CD4 ϩ T cells into skin allografts, and prolonged skin allograft survival.13) Even though the mechanisms of pharmacological action of FTY720 remain to be fully clarified, 14) it is clear that FTY720 has no inhibitory effect on cytokine production, in contrast to the established immunosuppressants. 12)NC/Nga mice have been used as an animal model for human atopic dermatitis.15) The mice raised in conventional (nonsterile) circumstances spontaneously develop human atopic dermatitis-like skin lesions with hyper IgE production, while those raised in a specific pathogen-free environment show neither dermatitis nor hyper IgE production. 15) In this study, we examined the efficacy of FTY720 for preventing the development of dermatitis in this animal model (NC/Nga mice) of human atopic dermatitis. MATERIALS AND METHODSAnimals NC/Nga mice (4-week-old males) bred under specific pathogen-free (SPF) conditions were purchased from Japan SLC Inc., Shizuoka, Japan. The mice were given gray-irradiated food (CRF-1, Oriental Bio Co., Kyoto, Japan) and distilled water for injection (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).FTY720 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720) was given kindly from Yoshitomi Pharmaceutical Industries, Ltd., Japan.Study Protocol Twelve NC/Nga mice were divided into three groups (nϭ4/group), of which two (treated and untreated groups) were kept under conventional conditions, and one (control group) was kept in an SPF environment. From 5 weeks of age (when the mice had no skin lesions), the treated group was orally given FTY720 in water (0.1 mg/kg) once a week. The untrea...

FTY720(Fingolimod) is known to have a significant therapeutic effect on experimental autoimmune encephalomyelitis (EAE). Here, we used an EAE mouse model, which had been established by immunizing C57BL/6J mice with a partial peptide of myelin oligodendrocyte glycoprotein (MOG 35-55 ), to examine the relapse of EAE upon discontinuation of treatment with FTY720 alone or in combination with MOG 35-55 . Relapse was confirmed to occur in all animals (n‫)6؍‬ within one week after discontinuation of FTY720, with increase in the number of lymphocytes infiltrating the spinal cord and demyelination. However, in the case of combination therapy with FTY720 and MOG 35-55 , relapse following discontinuation of treatment was completely suppressed. The autoantigenic peptide might serve to suppress the clonal selection of relapse-associated autoantigen-specific T cells.

It has been well established that myasthenia gravis (MG) is caused by autoimmune responses against nicotinic acetylcholine receptor (AChR) on postsynaptic membrane in neuromuscular junctions.1) Experimental autoimmune myasthenia gravis (EAMG) has been used as an animal model for human MG. This model is prepared by immunizing mice or rats with AChR from Torpedo californica; the animals develop MG-like symptoms, including muscle weakness, with production of autoantibody against AChR.2)The novel immunomodulator (immunosuppressant) FTY720, which was discovered by Fujita et al. 3,4) of our group, is a synthetic structural analogue of myriocin (ISP-I), a metabolite of Isaria cinclairii.5) The efficacy of FTY720 has been well established in preclinical transplantation models, 6) and also recently in renal transplantation in humans. [7][8][9][10] Although the mechanisms of pharmacological action of FTY720 remain to be fully clarified, 11,12) it has been proposed that FTY720 is phosphorylated by sphingosine kinase, and the product, FTY720 monophosphate, suppresses immune response by sequestering lymphocytes from blood and peripheral tissues to the secondary lymphoid tissues.13-16) FTY720 has no inhibitory effect on cytokine production in vitro, in contrast to established immunosuppressants (cyclosporin and tacrolimus hydrate).16) Its mechanism of action is unique and differs from that of the established immunosuppressants. The development of FTY720 was described in our previous report. 17)In this study, we examined the efficacy of FTY720 for preventing the development of EAMG, an animal model of human MG. MATERIALS AND METHODSAnimals Specific pathogen-free (SPF) C57BL/6 mice (7 weeks of age, females) were purchased from Japan SLC Inc., Shizuoka, Japan. The mice were given g-ray-irradiated food (CRF-1, Oriental Bio Co., Kyoto, Japan) and distilled water for injection (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).FTY720 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride (FTY720) was kindly provided by Yoshitomi Pharmaceutical Industries, Ltd., Japan.Acetylcholine Receptor from Torpedo californica Electric organ of Torpedo californica was purchased from Aquatic Research Consultants (San Pedro, CA, U.S.A.), and acetylcholine receptor (tAChR) was purified by the method of Froehner and Rafto using cobrotoxin-coupled Sepharose.18) AChR concentration was quantified by the Lowry method.Evaluation of Clinical Symptoms of EAMG Muscle strength was evaluated by the inverted hanging technique as described by Karachunski et al. 19,20) Mice with a holding time of 10 min or more were considered normal, and those with a holding time of less than 10 min were considered as having EAMG.Study Protocol Twelve C57BL/6 mice were immunized intracutaneously on the back with tAChR (5 mg) in the presence of Freund's complete adjuvant at 8, 10 and 11 weeks of age. Six mice (treated group, Nos. 1-6) out of the twelve were orally given FTY720 in water (1.0 mg/kg) three times a week from the day before the first immunization. The other...

A novel enzyme immunoassay of anti-insulin IgG in human serum is described. A serum sample containing anti-insulin IgG was treated with dextran-charcoal at pH 6.0 to remove endogenous insulin and subsequently incubated with dinitrophenyl biotinyl nonspecific rabbit IgG-insulin conjugate. The reaction mixture was further incubated with a rabbit (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene ball to trap the complex formed between anti-insulin IgG and the conjugate. After washing to eliminate nonspecific IgG in the test serum, the polystyrene ball was incubated with dinitrophenyl-L-lysine to elute the complex. The eluate was incubated with an avidin-coated polystyrene ball. Finally, the amount of human antiinsulin IgG in the complex trapped onto the avidin-coated polystyrene ball was measured by incubation with rabbit (antihuman IgG (?-chain)) Fab'-peroxidase conjugate. This enzyme immunoassay was 1,000-fold more sensitive than the conventional enzyme immunoassay, in which an insulinbovine serum albumin-coated polystyrene ball was incubated with a serum sample containing anti-insulin IgG and subsequently with rabbit (antihuman IgG (ychain)) Fab'-peroxidase conjugate. The principle of the novel enzyme immunoassay can be used to more sensitively measure antibodies for most kinds of haptens and antigens than the conventional enzyme immunoassay.