Identification of a novel carotenoid, 2'-isopentenylsaproxanthin, by Jejuia pallidilutea strain 11shimoA1 and its increased production under alkaline condition.

Carotenoids are a class of naturally occurring pigment, carrying out important biological functions in photosynthesis and involved in environmental responses including nutrition in organisms. Saproxanthin and myxol, which have monocyclic carotenoids with a γ-carotene skeleton, have been reported to show a stronger antioxidant activity than those with ß-carotene and zeaxanthin. In this research, a yellow-orange bacterium of strain 11shimoA1 (JCM19538) was isolated from a seaweed collected at Nabeta Bay (Shizuoka, Japan). The 16S rRNA gene sequence of strain 11shimoA1 revealed more than 99.99 % similarity with those of Jejuia pallidilutea strains in the family Flavobacteriaceae. Strain 11shimoA1 synthesized two types of carotenoids. One of them was (3R, 3'R)-zeaxanthin with dicyclic structure and another was identified as (3R, 2'S)-2'-isopentenylsaproxanthin, a novel monocyclic carotenoid with pentenyl residue at C-2' position of saproxanthin, using FAB-MS, (1)H NMR, and CD analyses. Culturing strain 11shimoA1 in an alkaline medium at pH 9.2 resulted in a markedly increased in production of 2'-isopentenylsaproxanthin per dry cell weight, but a decreased in zeaxanthin production as compared to their respective production levels in medium with pH 7.0. These carotenoids are likely to play some roles in the adaptation of the bacterium to the environmental conditions.

Simple conjugation and outgrowth procedures for tagging vibrios with GFP, and factors affecting the stable expression of the gfp tag.

AIM: Our goal was to develop a simple system for tagging wild-type marine bacteria with gfp. METHODS AND RESULTS: Escherichia coli strain CC118lambdapir carrying the conjugative helper plasmid pEVS104 and the gfp-containing plasmid pKV111 was used to transfer gfp to Vibrio recipients. Four different media were tested for their ability to support the growth of recipients, but not the E. coli donor, to allow powerful enrichment of gfp-tagged wild-type vibrios from mating mixes. Forty-three vibrio strains, representing 39 different species, were successfully tagged with gfp using the conjugative transfer from E. coli followed by selective outgrowth at 15 degrees C on ZoBell 2216E agar containing 0.5% sodium alginate. Using this outgrowth medium, colonies of GFP-expressing vibrio clones were detectable within 4 days. The percentage of visibly fluorescent cells in three representative GFP-tagged vibrios was higher at 15 degrees C than at 20 or 25 degrees C (c. 50% vs. 45% or 40%, respectively), and was also higher during the aerobic rather than the anaerobic culturing (c. 50% vs. 35%, respectively). CONCLUSIONS: We found a simple selective outgrowth technique that enabled us to isolate a wide variety of GFP-tagged marine vibrios following the conjugative transfer of gfp from E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Tagging cells with GFP and related fluorescent proteins is a powerful approach for investigating the bacteria in situ, particularly during the colonization of hosts. The simple and cost-effective outgrowth condition described in this study could be applied to construct a wide variety gfp-tagged marine bacteria.

Thalassomonas loyana sp. nov., a causative agent of the white plague-like disease of corals on the Eilat coral reef.

The taxonomic position of the coral pathogen strain CBMAI 722T was determined on the basis of molecular and phenotypic data. We clearly show that the novel isolate CBMAI 722 T is a member of the family Colwelliaceae, with Thalassomonas ganghwensis as the nearest neighbour (95 % 16S rRNA gene sequence similarity). CBMAI 722T can be differentiated from its nearest neighbour on the basis of phenotypic and chemotaxonomic features, including the utilization of cellobiose and L-arginine, the production of alginase and amylase, but not oxidase, and the presence of the fatty acids 12:0 3-OH and 14:0, but not 10:0 or 15:0. The DNA G+C content of CBMAI 722T is 39.3 mol%. We conclude that this strain represents a novel species for which we propose the name Thalassomonas loyana sp. nov., with the type strain CBMAI 722T (=LMG 22536T). This is the first report of the involvement of a member of the family Colwelliaceae in coral white plague-like disease.

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample.

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.

Aberrant HS1 molecule in a patient with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of autoreactive B lymphocytes, which are supposed to carry aberrant signal transduction after the stimulation of B-cell receptor (BCR). To investigate abnormalities in BCR-mediated signaling pathway in lupus B lymphocytes, we analyzed HS1, a molecule downstream of BCR, in 80 Japanese SLE patients. We identified 37 amino acid deletion of HS1 in a 25-year-old female patient, and the aberrant HS1 lacked a part of a functional motif. Analysis of genomic DNA revealed that the aberrant HS1 was caused by exon skipping. Family study showed that the patient as well as her father and sister are heterozygous for the abnormality. WEHI-231 cell, a mouse B cell line, transfected with the aberrant HS1 displayed a significantly increased cell death upon cross-linking of BCR. Additionally, peripheral B lymphocytes from the patient exerted increased apoptosis after BCR stimulation compared to those from control SLE patients. These data suggest that the aberrant HS1 molecule may transmit an accelerated signal after BCR stimulation and may play a role in the activation of autoreactive B lymphocytes.

Oligonucleotide probe for detecting Enterobacteriaceae by in situ hybridization.

AIMS: To develop oligonucleotide probes for visualizing bacteria belonging to Enterobacteriaceae. METHODS AND RESULTS: 24-mer oligonucleotide probe (probe D) was designed by comparison of 16S rDNA sequences of 35 species of Enterobacteriaceae, eight species of Vibrionaceae and six species of Pasteurellaceae. The sequence of the probe corresponding to the complementary sequence of a position 1251-1274 of Escherichia coli 16S rRNA was found to be a highly conserved region of 16S rDNA sequence in Enterobacteriaceae different from that of Vibrionaceae and Pasteurellaceae. The fluorescent dye-labelled probe was tested for the specificity by in situ hybridization and epifluorescence microscopy. Seventy-six out of 78 strains belonging to Enterobacteriaceae were visualized in an optimal hybridization condition. No bacterial strains belonging to Vibrionaceae (31 strains) and Gram-positive bacteria (three strains) were visualized. CONCLUSIONS: In situ hybridization using probe D allows the detection of bacterial cells belonging to Enterobacteriaceae without false positive reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: In situ hybridization techniques using the probe D are potential tools for detecting Enterobacteriaceae in food and water samples.

Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens.

The heterotrophic microbial enrichment community established during degradation of brown algae Fucus evanescens was characterized. A two-species bacterial community of marine culturable gamma-proteobacteria consisted of Pseudoalteromonas and Halomonas. The first member of the community, Pseudoalteromonas sp., was highly metabolically active, had bacteriolytic and hemolytic activities, produced proteinases (gelatinase and caseinase), lipases, DNases, and fucoidanhydrolases, laminaranases, alginases, pustulanases, beta-glucosidases, beta-galactosidases, beta-N-acetylglucosaminidases, and beta-xylosidases. The second member of the community, Halomonas marina, produced only caseinase and DNase, and it did not hydrolyze algal polysaccharides. Both members of the studied bacterial community utilized a range of easily assimilable monosaccharides and other low molecular weight organic substances. The results provide an evidence of the complex metabolic interrelations between two members of this culturable community. One of them Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation; the other one, H. marina, resistant to the bacteriolytic activity of the former, is able to utilize the products of degradation of polysaccharides.

Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase.

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity.

[A case of chronic hypersensitivity pneumonitis due to wild pigeons].

A 61-year-old woman was admitted to the Oita Medical University Hospital because of a nonproductive cough and exertional dyspnea. Interstitial changes had been seen on her chest radiograph 5 years previously, but no respiratory symptoms were identified at that time. On admission, chest radiography revealed linear and ground-glass opacities in the middle and lower lung fields. Computed tomography provided evidence of bronchiectasis and micro-honeycombing of the lungs, while lymphocyte and neutrophil counts in the bronchoalveolar lavage fluid were increased. Transbronchial lung biopsy demonstrated alveolitis and Masson's bodies. The patient was not a pigeon breeder, but she could have been exposed to pigeons at her workplace. Indeed, she had specific antibodies against pigeon serum and droppings, and her peripheral lymphocytes showed proliferation in response to pigeon serum. A positive provocation test involving inhalation of pigeon serum confirmed that she had chronic hypersensitivity pneumonitis caused by allergy to pigeons. This is a rare case of chronic hypersensitivity pneumonitis associated with wild pigeons, that progressed to pulmonary fibrosis. Antigen provocation testing proved to be of great value.

Shewanella japonica sp. nov.

Two strains of agar-digesting bacteria, KMM 3299T and KMM 3300, respectively isolated from sea water and the mussel Protothaca jedoensis, have been characterized. Based on sequencing of the 16S rRNA gene, KMM 3299T showed the highest similarity (93-95%) to members of the genus Shewanella. The G+C contents of the DNAs of these strains were 43-44 mol%. The level of DNA homology between the two strains was conspecific (95%), indicating that they represent a distinct genospecies. These organisms were non-pigmented, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic, neutrophilic and able to degrade a wide range of high molecular mass polymers, including alginate, carrageenan, laminaran and agar. The novel organisms were susceptible to gentamycin, carbenicillin, lincomycin and oleandomycin. The predominant cellular fatty acids were i-15:0, 16:0, 16:1(n-7), 18:1(n-7). Eicosapentaenoic acid, 20:5(n-3), was detected in the two isolates at levels of 1-8%, depending on the temperature of cultivation. Phylogenetic evidence, together with phenotypic characteristics, showed that the two isolates studied constitute a novel species of the genus Shewanella. The name Shewanella japonica is proposed; the type strain is KMM 3299T(= LMG 19691T = CIP 106860T).

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Defect of lck in a patient with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a congenital immunological disorder characterized by defective antibody production with normal count of peripheral B lymphocytes. The basic immunologic defects that leads to CVID are still unknown, however, a proportion of CVID is suggested to be caused by decreased CD4+ helper T cell activity. In addition, recent reports indicate that a defect of T cell receptor (TCR)-associated signaling molecules results in congenital immune deficiency in human. In the present study, we investigated lck, a signaling molecule downstream of TCR, in a patient with CVID plus CD4 lymphopenia, and found an aberrantly spliced lck transcript lacking the entire exon 7 associated with the decrease in the expression of lck protein. An identical splicing abnormality has been previously demonstrated in a case of severe combined immunodeficiency with selective CD4 lymphopenia, although the case showed almost complete loss of the expression of lck protein. Considering these findings, the aberrant splicing of lck gene is suggested to be correlated, at least with a subset of congenital immunodeficiency plus CD4 lymphopenia.

Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells.

The membrane TNF-alpha is known to serve as a precursor of the soluble form of TNF-alpha. Although it has been reported the biological functions of the membrane TNF-alpha as a ligand, the outside-to-inside (reverse) signal transmitted through membrane TNF-alpha is poorly understood. Here we report a novel function mediated by outside-to-inside signal via membrane TNF-alpha into the cells expressing membrane TNF-alpha. Activation by anti-TNF-alpha Ab against membrane TNF-alpha on human T cell leukemia virus (HTLV) I-infected T cell line, MT-2, or PHA-activated normal human CD4(+) T cells resulted in the induction of an adhesion molecule, E-selectin (CD62E), on the cells with the peak of 12-24 h, which completely disappeared by 48 h. When wild-type or mutant membrane TNF-alpha (R78T/S79T) resistant to proteolytic cleavage was introduced into Jurkat or HeLa cells, E-selectin was induced by the treatment with anti-TNF-alpha Ab with the similar kinetics. Membrane TNF-alpha-expressing Jurkat cells also up-regulated E-selectin when brought into cell-to-cell contact with TNF receptor-expressing HeLa cells. Northern blot analysis and RT-PCR analysis showed that the membrane TNF-alpha-mediated E-selectin expression was up-regulated at the level of transcription. These results not only confirmed our previous findings of reverse signaling through membrane TNF-alpha, but also presented evidence that E-selectin was inducible in cell types different from endothelial cells. It is strongly suggested that membrane TNF-alpha is a novel proinflammatory cell surface molecule that transmits bipolar signals in local inflammation.

Association of tumor necrosis factor receptor type II polymorphism 196R with Systemic lupus erythematosus in the Japanese: molecular and functional analysis.

OBJECTIVE: To investigate whether a polymorphism(s) or mutation(s) in the tumor necrosis factor receptor II (TNFRII) gene is involved in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: All 10 exons of the TNFRII gene were analyzed by exon-specific polymerase chain reaction-single-strand conformation polymorphism, followed by nucleotide sequencing of exons that displayed aberrant bands. To analyze the function of the TNFRII polymorphisms, the full-length TNFRII complementary DNA of each allele was transfected in HeLa cells and then studied for specific binding of 125I-TNFalpha, as well as interleukin-6 (IL-6) production and cytotoxic activity after treatment with recombinant human TNFalpha. RESULTS: We identified 4 polymorphisms, at codons 56, 181, 196, and 232. The latter 2 had amino acid substitutions M196R and E232K, respectively. Only the 196R allele was significantly associated with SLE in our 105 Japanese SLE patients, with an allele frequency of 20.5%, compared with 12.6% in 99 healthy controls (P = 0.0335). More importantly, using TNFRII-transfected HeLa cells, we demonstrated significantly increased IL-6 production by 196R TNFRII compared with 196M TNFRII. The cytotoxic activity induced by 196R TNFRII was also increased compared with that of 196M TNFRII. This increase was achieved without affecting the binding affinity of TNFalpha to TNF-RII, as demonstrated by the finding that specific TNFalpha binding to the HeLa transfectants of 196R and 196M TNFRII was similar, with Kd values of 3.12 x 10(-10)M and 4.34 x 10(-10)M, respectively. CONCLUSION: These results suggest that 196R TNFRII, which transduces the signals of TNFalpha more effectively than does 196M TNFRII, is involved in the pathogenesis of SLE.

Accumulation of common clonal T cells in multiple lesions of sarcoidosis.

BACKGROUND: T cells recognizing as yet unknown antigens (Ags) are considered to play an important role in the development and perpetuation of the disease process of sarcoidosis. Several studies have shown that T cells that bear a limited T-cell receptor (TCR) repertoire may play an important role in this disorder. However, regarding variable (V) gene repertoire usage, the results differ among various reports. One reason for such inconsistency may be due to the materials used in these studies. Most studies analyzed the T-cell repertoire in the sarcoid lung. However, clonal expansion of pulmonary T cells, probably due to the activation by inhaled exogenous Ags, was observed and such expansion may seriously influence the repertoire analysis. MATERIALS AND METHODS: Reverse transcriptase-polymerase chain reaction and subsequent single-strand conformation polymorphism analysis were used for the analysis of TCR repertoire. To exclude unrelated T-cell clones, we used intramuscular sarcoid nodules and/or lymph node (LN) sarcoid lesions as our materials. We also analyzed sarcoid lesions from different organs and then compared the results. RESULTS: T cells of the same clonality were found to exist in widely separated sites in intramuscular and LN sarcoid lesions in almost all Vbeta subfamilies. Identical T-cell clones were present in the sarcoid lesions from different organs in several Vbeta subfamilies. CONCLUSIONS: Some of the common T-cell clones in separated sites in intramuscular and LN sarcoid lesions and in sarcoid samples from different organs may recognize Ags that are related to the pathogenesis of sarcoidosis.

[Misoprostol-induced pneumonitis].

A 76-year-old woman presented with non-productive cough and progressive dyspnea, and was admitted to Oita Medical University Hospital. Arterial blood gas values obtained on admission indicated severe hypoxemia. Chest roentgenograms and computed tomography disclosed diffuse interstitial infiltrates in both lungs. Transbronchial lung biopsy specimens demonstrated thickened alveolar walls with lymphocyte infiltration and swollen type II pneumocyte proliferation. Eosinophils were observed mainly around bronchioles. For approximately 6 months prior to hospitalization, the patient had been given misoprostol, sodium aurothiomalate, prednisolone, and loxoprofen sodium for the treatment of rheumatoid arthritis. Based on the clinical history and findings, drug-induced interstitial pneumonia was suspected. All medications were discontinued, and the patient was then placed on corticosteroids. After treatment, arterial blood gas values improved and the findings on chest roentgenograms cleared up. Positive lymphocyte stimulation tests and positive dermal reaction patch tests implicated misoprostol as an etiologic factor in the patient's interstitial pneumonia. High serum levels of KL-6 and cytokeratin subunit 19 fragment had been detected on admission. These values returned to normal after the interstitial infiltrates had disappeared. To our knowledge, this is the first reported case of misoprostol-induced interstitial pneumonia.

Analysis of p53 tumour suppressor gene somatic mutations in rheumatoid arthritis synovium.

OBJECTIVE: In order to study the role of the p53 tumour suppressor gene in the proliferation of rheumatoid arthritis (RA) synovium, we analysed the mutation of p53 in the synovial fibroblast-like type B synoviocyte from RA patients. METHODS: Synovial fibroblast-like type B synoviocytes were prepared from the synovial tissues from nine Japanese patients with RA. The p53 cDNA region from exons 4-11 was screened for mutations by the streamlined mutation detection method in which polymerase chain reaction (PCR) products are post-labelled and are analysed by automated capillary electrophoresis using single-strand conformation polymorphism conditions, followed by direct sequencing of the subclones of the PCR products. RESULTS: p53 mutation with possible functional alteration was detected in four of the nine RA patients (44.4%). Of a total of 262 p53 cDNA subclones, 10 subclones were carrying 10 p53 mutations, eight of which were associated with amino acid alterations or protein truncation. Of the p53 functional mutations, a substitution of Gly at amino acid residue 245 to Asp (G245D) was identified in two patients in three subclones. G245D was the first mutation that was recurrently identified in different RA individuals. G245D is also one of the relatively common mutations in human cancers. CONCLUSIONS: In some patients with RA, dysfunction of p53 might play a role in the proliferation of the synovial tissue. G245D mutation might especially need further study as it is the first recurrently identified p53 mutation in RA and is also one of the frequently identified mutations in human cancers.

Assignment of Alteromonas elyakovii KMM 162T and five strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species.

A marine bacterium, Alteromonas elyakovii KMM 162T, which was described recently, and five strains isolated from spot-wounded fronds of Laminaria japonica have been subjected to phylogenetic analysis, and geno- and phenotypic characterization. The phenotypic features of Pseudoalteromonas elyakovii strains were closely related to that of Pseudoalteromonas espejiana IAM 12640T, but utilization of three carbon compounds (D-mannose, L-tyrosine and trehalose) distinguished both species. The G+C content of Pseudoalteromonas elyakovii was between 38.5 and 38.9 mol%. Pseudoalteromonas elyakovii KMM 162T and the five Laminaria isolates constitute a single species different from any other Alteromonas and Pseudoalteromonas species as revealed by DNA-DNA hybridization data, especially Pseudoalteromonas distincta KMM 638T (52.4%), Pseudoalteromonas citrea KMM 216 (49.5%), Pseudoalteromonas carrageenovora NCIMB 302T (46.9%) and Pseudoalteromonas espejiana IAM 12640T (29.9%). All the data indicated that Alteromonas elyakovii KMM 162T should be reclassified as Pseudoalteromonas elyakovii and five strains isolated from Laminaria japonica have to be included in the species. Pseudoalteromonas elyakovii comb. nov. (type strain, KMM 162T = ATCC 700519T) is proposed and a set of phenotypic features which differentiate the Pseudoalteromonas species is described.