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We report a 37-year-old female of intractable rheumatoid arthritis (RA) complicated by systemic lupus erythematosus (SLE), who was successfully treated with a combination of tocilizumab (TCZ) and tacrolimus. She was diagnosed with RA when she was 21 years old, and was administered oral prednisolone, injectable gold and salazosulfapyridine, but deformity of her hands gradually developed. She developed high fever and thrombocytopenia when she was 35 years old. Renal involvement, pericarditis, positive antinuclear antibody and high level of anti-double-stranded DNA antibody were found and the patient was diagnosed with SLE. Polyarthritis and immunological abnormalities developed despite aggressive immunosuppressive therapy including high-dose corticosteroids and intravenously administered cyclophosphamide. Tacrolimus (TAC) therapy gave only partial improvement of joint symptoms. After the initiation of combination therapy with TCZ, not only was a complete remission of RA obtained, but also the serum levels of SLE markers dramatically decreased. Our report suggests the possibility that this combination therapy is effective in treating SLE as well as RA.

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A 12-residue marinostatin [MST(1-12): (1)FATMRYPSDSDE(12)] which contains two ester linkages of Thr(3)-Asp(9) and Ser(8)-Asp(11) strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K(i) value of 1SS(C(3)-C(9)) ((1)FACMRYPSCSDE(12)), which has a single disulfide linkage of Cys(3)-Cys(9) was comparable with those of MST(1-12) and MST-2SS ((1)FACMRYPCCSCE(12)), a doubly linked analog of Cys(3)-Cys(9) and Cys(8)-Cys(11). However, 1SS(C(3)-C(9)) and MST-2SS showed temporary inhibition, but not MST(1-12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. (1)H NMR study showed that 1SS(C(3)-C(9)) has two conformations, which contain a cis- (70%) or trans- (30%) Pro residue, while MST-2SS as well as MST(1-12) takes a single conformation containing only a cis-Pro residue. Hydrogen-deuterium exchange rate of the Arg(5) (P1') NH proton of the MST analogs was about 100 times faster than that of MST(1-12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis-conformation of the Pro(7) residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease-inhibitor complex.

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AIMS: To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T. halophilus. METHOD AND RESULTS: The growth, lactic acid production and pH reduction ability of five strains of T. muriaticus and T. halophilus in MRS broth medium under various culture conditions such as salt concentration (3, 7, 15 and 23% NaCl), temperature (20, 30 and 40 degrees C), and initial medium pH (5.8, 6.5 and 7.5) were investigated. Those of T. halophilus were seriously affected by a high salinity (23% NaCl); in contrast, those of T. muriaticus were affected by a low initial pH (5.8). CONCLUSIONS: The results indicate that high saline concentrations and low pH values have significant impact on the growth, lactic acid production and pH reduction ability of T. halophilus and T. muriaticus, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: This study appears to be important in biopreservation during the manufacture of fermented food products. Both T. muriaticus and T. halophilus may support each other in reducing pH in hypersaline or low pH environment. To our knowledge, this is the first report on the fermentation ability of T. muriaticus.

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A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior. The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However, these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or became small in Ca2+-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward current under conditions where K+ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Sr2+, Mg2+, or Mn2+, and was lost upon replacing with equimolar concentration of Ni2+. On the other hand, the cold-induced inward current was not affected significantly by Ba2+, or Sr2+, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations of Mg2+, Mn2+, or Ni2+. These results indicate that Paramecium cells have heat-activated Ca2+ channels and cold-activated Ca2+ channels and that the cold-activated Ca2+ channel is different from the heat-activated Ca2+ channel in the ion selectivity and the calcium-dependent inactivation.

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IC202A, a new immunosuppressive compound, was isolated from the culture filtrate of Streptoalloteichus sp. 1454-19. It showed a suppressive effect on mixed lymphocyte culture reaction with an IC50 value of 3.6 microg/ml and mitogen induced lymphocyte blastogenesis in vitro.

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The protoplasts of two strains of Micromonospora which were sensitive to kanamycin (KM(s)) and utilized raffinose (Raf+), and one strain of Streptomyces griseus which was resistant to KM (KM(r)) and did not utilize raffinose (Raf-), were prepared, mixed in the presence of polyethylene glycol (PEG) and incubated on regeneration agar plates. Recombinant colonies showing KM(r)*Raf+ were obtained at a frequency of 2 x 10(-6). Their recombinants displayed a significant exchange of taxonomic characteristics between the two genera, although the majority appeared similar to the parent Micromonospora in their morphology as well as growth at 40 degrees C. Their patterns of utilization of carbohydrates, amino acids and diammonium hydrogenphosphate were different from those of the Micromonospora. Intermediate or novel types which different from their parents in their tolerance to NaCl and sensitivity to aminoglycoside antibiotics were also observed. Out of the 31 fusants obtained, two showed antimicrobial activity against Bacillus subtilis PCI 219, without any activity against Escherichia coli K-12 or Candida albicans 3147. The active substance may be a newly formed antibiotic, different from streptomycin in S. griseus.

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Amino acid analogs (AAA) were used as selective pressures for isolation of marine bacteria with novel physiological properties and as effecters for antibiotic production. Relatively small numbers of isolates were obtained from a minimal medium containing aminoethylcysteine (AC), 3,4-dehydroproline (DP), 5-methyltryptophan (MT), and selenomethionine (SM). These bacteria exhibited a high probability (68%) of antibiotic production in the presence of AAA, which was 10-fold higher than that (7%) in the absence of AAA. Among them, strain 14, obtained as the only SM-resistant and SM-dependent antibiotic (selenohomocystine) producer, was characterized for microbiological properties. It showed taxonomic properties falling into those of the genus Bacillus, required seawater for growth, and exhibited a high level (0.5 mM) of resistance to all the AAAs tested. Neither known Bacillus spp. nor other marine isolates showed such properties. Therefore, the strain 14 appears to be the first marine Bacillus strain with unique AAA resistance and AAA-dependent antibiotic productivity. The AAA-resistance-based strategy was thus demonstrated to be effective for isolation of novel bacteria as well as for screening for antibiotic production.

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New inhibitors of dipeptidyl peptidaseIII (EC 3.4.14.4) from human placenta, designated as fluostatins A and B, were discovered in the fermentation broth of a strain isolated in our institute. The strain has been identified as Streptomyces sp. TA-3391 on the basis of taxonomic studies. Fluostatins A and B were purified by Diaion HP-20 chromatography, ethyl acetate extraction, silica gel chromatography and reverse phase preparative HPLC. With the synthetic substrate, arginyl-arginine-2-naphthylamide, the IC50 values of fluostatins A and B were 0.44 and 24.0 micrograms/ml, respectively. Fluostatins A and B were slightly inhibitory against other dipeptidyl peptidases. Fluostatin A showed mixed-type (competitive and noncompeptitive) inhibition with human leucine-enkephalin as a substrate, and the inhibition constant (Ki) was 14.2 microM.

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One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

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The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.

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Changes of intracellular calcium concentration ([Ca2+]i) induced by the extracellular application of ATP and bradykinin in mouse mammary tumour cells (MMT060562) were investigated by image analysis of fluo-3 fluorescence at 24 degrees C and 35 degrees C. ATP (0.1-100 microM) and bradykinin (0.1 nM-1 microM) induced the increase of [Ca2+]i at both temperatures and Ca(2+)-depletion did not affect these [Ca2+]i responses. Both [Ca2+]i responses became more sensitive at 35 degrees C than at 24 degrees C. A clear latency of [Ca2+]i increase after the application of the agonists was observed, and it changed with the concentration of the agonist. As concentrations of ATP or bradykinin became lower, the latency and rise time became longer. At higher concentrations, the latency and rise time approached a constant value. The latency shortened remarkably at 35 degrees C. These results suggested the involvement of a regenerative or threshold process in the [Ca2+]i responses in mammary tumour cells.

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The gene (aprI) encoding alkaline serine protease (AprI; subtilase) from Alteromonas sp. strain O-7 was cloned and sequenced. The nucleotide sequence of aprI has been identified. The deduced amino acid sequence indicated that aprI codes for a precursor of 715 amino acids and the precursor is composed of four regions including a signal peptide, an N-terminal pro-region, a mature protease region and a C-terminal extension region of 215 amino acids as previously described for aprII [H. Tsujibo et al., Gene, 136, 247-251 (1993)]. The amino acid sequence of the mature AprI (AprI-M) showed high sequence homology with those of other class I subtilases. The C-terminal region was characterized by a repeat of 94 amino acids residues, which showed about 50% similarity with those of the C-terminal pro-region of several known proteases from Gram-negative bacteria.

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The effects of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on the endothelial nitric oxide (NO) pathway were studied in vitro. Vanadate caused endothelium-dependent relaxations in isolated porcine coronary arteries, which were abolished by N omega-nitro-L-arginine methyl ester. The relaxations were also abolished by pertussis toxin, an inhibitor of certain G proteins. Tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenyl-methylcinnamamide (ST-638), significantly attenuated the vanadate-induced relaxations. Vanadate also caused pertussis toxin-sensitive, endothelium-dependent relaxations in isolated porcine renal and femoral arteries and jugular veins. Immunoblots, using an antibody to phosphotyrosines and to c-Src in native porcine aortic endothelial cells, respectively, showed that vanadate induced an elevation of phosphotyrosine proteins and a decrease in the amount of the active form of c-Src family kinases; both changes were markedly suppressed by cotreatment with ST-638. These results indicate that in porcine blood vessels, vanadate causes a synthesis of endothelium-derived NO for which endothelial tyrosine kinases and pertussis toxin-sensitive G protein are considered to be closely involved.

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Extracellular chitinase from marine Alteromonas sp. strain O-7 is unique because of the activation by four major cations contained in sea water, such as Na+, K+, Mg2+, and Ca2+. The positions of S-S bonds of Alteromonas chitinase were identified. Alteromonas chitinase was fragmented by TPCK-trypsin and Staphylococcus aureus V8 protease. The amino acid and sequence analyses of three peptides showed that the positions of disulfide bonds are Cys(94)-Cys(99), Cys(174)-Cys(196), and Cys(386)-Cys(395).

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beta-N-Acetylglucosaminidase (EC 3.2.1.30) was purified from the outer membrane of a marine bacterium, Alteromonas sp. strain O-7. The enzyme (GlcNAcase A) was purified by successive column chromatographies. The purified enzyme was found to be homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass and pI of GlcNAcase A were 92kDa and 4.9, respectively. The optimum pH and temperature were 6.0-7.0 and 45 degrees C, respectively. GlcNAcase A was stable up to 40 degrees C at pH 7.0, and hydrolyzed N-acetylchitooligosaccharides from dimer to hexamer. The amino-terminal 16 amino acid residues of GlcNAcase A were sequenced.

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The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.

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Pyrostatins A and B, new inhibitors of N-acetyl-beta-D-glucosaminidase (GlcNAc-ase), have been purified from the culture broth of Streptomyces sp. SA-3501 isolated from a marine environment. They were purified by chromatography on Dowex 50W, silica gel and Capcell Pak C18 (HPLC) followed treatment with active carbon and then isolated as white powders. The structures of pyrostatins A and B were determined by NMR studies to be 4-hydroxy-2-imino-1-methylpyrrolidine-5-carboxylic acid and 2-imino-1-methylpyrrolidine-5-carboxylic acid, respectively. They were competitive with the substrate, and the inhibition constants (Ki) of pyrostatins A and B were 1.7 x 10(-6) M and 2.0 x 10(-6) M respectively.

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The gene (cht60) encoding N-acetyl-beta-glucosaminidase (Cht; EC 3.2.1.30) from the marine bacterium Alteromonas sp. strain O-7 was cloned into pUC18 in Escherichia coli JM109. The nucleotide (nt) sequence of cht60 was determined. A 1797-bp open reading frame encoded a polypeptide of 598 amino acids (aa) (M(r) 64,535). The aa sequence of the cloned enzyme (Cht60) deduced from the nt sequence showed no significant sequence homologies with available aa sequences from databases. Cht60 was purified from the periplasmic fraction of E. coli cells carrying pCHT982. The enzyme was most active towards p-nitrophenyl-N-acetyl-beta-D-glucosaminide(PNP-beta-GlcNAc) and diacetylchitobiose. The optimum pH and temperature of the enzyme were pH 7.5 and 37 degrees C, respectively. The N-terminal 11 aa residues of Cht60 were sequenced, and the location of the signal peptide cleavage site was clarified.

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The gene (aprII) encoding alkaline serine protease (AprII; subtilase) from Alteromonas sp. strain O-7 was cloned in plasmid pUC19 and transformed into Escherichia coli JM109. The nucleotide (nt) sequence of aprII has been determined. A single open reading frame (ORF) encoded a protein consisting of 621 amino acids (aa) with a M(r) of 63,958. The results of aa sequence analysis indicated that AprII is produced as a large precursor consisting of four domains: the signal sequence, the N-terminal pro-region (AprII-N), the mature AprII (AprII-M) and the C-terminal pro-region (AprII-C). The aa sequence of AprII-M shows high sequence homology with those of class-II subtilases. Two conserved sequences were found in AprII-N which might play a critical role in the maintenance of chaperone-like activity. Repeated aa sequences were observed in AprII-C (AprII-C1 and AprII-C2). The aa sequences of AprII-C1 and AprII-C2 show high sequence homology with those of the C-terminal pro-region of the other known proteases.

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Chitinase (Chi85) from Alteromonas sp. strain O-7 contains the two conserved regions common to microbial and plant chitinases. We did site-directed mutagenesis of Chi85 to investigate the effects of the conserved amino acid residues on chitinase activity. We suggest that Asp-290 and Glu-292 of Chi85 may be the essential amino acid residues for the cleavage of beta-glycosidic linkage of chitin.

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