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1.
FEMS Microbiol Lett ; 364(7)2017 04 01.
Article in English | MEDLINE | ID: mdl-28333255

ABSTRACT

The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides.


Subject(s)
Bacterial Proteins/metabolism , Endopeptidases/metabolism , Pseudomonas aeruginosa/enzymology , Pyocyanine/biosynthesis , Animals , Bacterial Proteins/genetics , Caseins/metabolism , Caseins/pharmacology , Culture Media/chemistry , Electrophoresis, Polyacrylamide Gel , Endopeptidases/genetics , Gelatin/metabolism , Genetic Complementation Test , Milk/metabolism , Proteolysis , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/pathogenicity , Virulence Factors/genetics
2.
Plasmid ; 84-85: 44-50, 2016.
Article in English | MEDLINE | ID: mdl-26997534

ABSTRACT

Pseudomonas aeruginosa PAO1 organisms harbouring different plasmids were cultured in broths containing appropriate antibiotic(s). Extracellular proteins were more abundant in the presence of tetracycline or kanamycin than in the presence of other antibiotics. Zymography revealed that alkaline protease (AprA) production was interfered by these antibiotics. Extracellular proteins were not observed at the same level when AprA-deficient EG03 strains were cultured in the presence of different antibiotics. The extracellular protein levels were dependent on the antibiotics and plasmid derivative groups. Levels of extracellular protein were not significantly different between PAO1 (pBBR1MCS-5) and EG03 (pAprcomp-MCS5), and profiles of the extracellular proteome were comparable. In contrast, the level of EG03 (pBBR1MCS-MCS5) extracellular protein was higher than those observed in the other two strains. These results suggested that although AprA partially contributes to the alteration of extracellular protein level, the effect is limited.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Endopeptidases/genetics , Endopeptidases/metabolism , Plasmids/genetics , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , Bacterial Proteins/biosynthesis , Endopeptidases/biosynthesis , Kanamycin/pharmacology , Tetracycline/pharmacology
3.
Arthritis Rheum ; 62(12): 3751-9, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20722021

ABSTRACT

OBJECTIVE: To investigate whether fasciitis is histopathologically demonstrable in patients with dermatomyositis (DM), and to analyze the process of inflammatory progression in myopathy accompanying DM. METHODS: STIR or fat-suppressed T2-weighted magnetic resonance imaging (MRI) and en bloc biopsy were performed in 14 patients with newly diagnosed adult-onset DM. The severity of inflammatory cell infiltration around the fascial and intramuscular small blood vessels was evaluated using the total vascular inflammation score (TVIS). RESULTS: In all patients, MRI revealed abnormal hyperintensity in the fascia and in marginal sites of the muscle, predominantly over central sites. En bloc biopsy revealed the presence of fasciitis in most of the patients, as shown by inflammatory infiltrates around the fascial small blood vessels. In those patients who underwent en bloc biopsy earlier than 2 months after the appearance of muscle symptoms, the TVIS of the fascia was significantly higher than the TVIS of the muscle. In contrast, in those patients who underwent en bloc biopsy >2 months after muscle symptom onset, the TVIS of the fascia did not differ significantly from the TVIS of the muscle. CONCLUSION: Fasciitis was histopathologically demonstrated in patients with newly diagnosed adult-onset DM as early as 2 months after the onset of muscle symptoms. These results indicate that fasciitis is a common lesion of DM and suggest that the fascial microvasculature is the primary site of inflammatory cell infiltration in DM. Fasciitis may contribute to muscle symptoms in patients with DM without myositis.


Subject(s)
Dermatomyositis/pathology , Fasciitis/pathology , Muscle, Skeletal/pathology , Adult , Aged , Biopsy/methods , Disease Progression , Female , Humans , Magnetic Resonance Imaging/methods , Male , Middle Aged , Retrospective Studies , Severity of Illness Index , Time Factors
4.
J Rheumatol ; 37(6): 1121-8, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20436077

ABSTRACT

OBJECTIVE: To evaluate the clinical significance of serum levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2) in patients with rheumatoid arthritis (RA). METHODS: The subjects were 70 patients with RA. Serum VEGF, Ang-1, and Ang-2 levels were determined by ELISA. As indices of disease activity, serum levels of C-reactive protein (CRP) and matrix metalloprotease (MMP)-3 were examined, and the 28-joint count Disease Activity Score (DAS28)-CRP was calculated. Power Doppler ultrasonography was performed in the bilateral wrists, elbows, shoulders, knees and ankles. The synovial blood flow signals were scored using a 3-grade scale (0-2), and the total of the scores in the 10 joints was regarded as the total signal score (TSS). RESULTS: Serum VEGF level showed significant correlations with serum CRP and MMP-3 levels, DAS28-CRP, and TSS. Serum Ang-1 level showed significant correlations with serum MMP-3 level and DAS28-CRP. Serum Ang-2 level showed significant correlations with serum CRP level and TSS. CONCLUSION: The serum VEGF level is important as an index of the activity of RA based on angiogenesis and a prognostic factor regarding joint destruction. Serum Ang-1 level may be useful as an index of sustained arthritis based on the maintenance of newly formed vessels. Serum Ang-2 level may reflect a state of marked angiogenesis.


Subject(s)
Angiopoietin-1/blood , Angiopoietin-2/blood , Arthritis, Rheumatoid/blood , Vascular Endothelial Growth Factor A/blood , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/diagnostic imaging , Arthritis, Rheumatoid/physiopathology , Biomarkers/blood , Blood Flow Velocity , C-Reactive Protein/analysis , Enzyme-Linked Immunosorbent Assay , Female , Humans , Joints/diagnostic imaging , Joints/physiopathology , Male , Middle Aged , Severity of Illness Index , Synovial Membrane/blood supply , Ultrasonography, Doppler/methods , Young Adult
5.
Article in Japanese | MEDLINE | ID: mdl-20190509

ABSTRACT

Treatment with corticosteroid and etanercept was started for a 69-year-old woman with rheumatoid arthritis. Eight weeks later, she developed fever and anorexia, and was admitted to our hospital with a poor general condition. With the suspicion of bacterial infection, antibiotic treatment was started on the first hospital day, but was ineffective. From the fourth hospital day, multiple generalized, partially confluent, erythematous papules appeared. To exclude viral infection, we measured antibody titers against various viruses, and found that the levels of anti-measles IgG and IgM antibodies were elevated, which led to a diagnosis of modified measles. During the course of measles, she developed severe leukopenia, and bone marrow aspiration revealed bone marrow suppression. This was considered to be due to measles infection, leading to a diagnosis of severe modified measles. This is the first case report of modified measles which developed during the use of an anti-TNF-alpha preparation, and suggests that immunosuppression induced by the preparation may play a role in measles reinfection and its aggravation.


Subject(s)
Antirheumatic Agents/adverse effects , Arthritis, Rheumatoid/drug therapy , Immunoglobulin G/adverse effects , Immunosuppressive Agents/adverse effects , Measles/etiology , Aged , Etanercept , Female , Humans , Receptors, Tumor Necrosis Factor
6.
BMC Musculoskelet Disord ; 10: 45, 2009 Apr 30.
Article in English | MEDLINE | ID: mdl-19405944

ABSTRACT

BACKGROUND: Bombina variegate peptide 8 (Bv8) is a small protein secreted by frog skin. Recently it has been shown to contribute to tumor angiogenesis in mouse model. The purpose of this study was to investigate Bv8 in mice with type II collagen-induced arthritis (CIA). METHODS: We induced CIA in male DBA/1J mice. The severity of arthritis was evaluated based on an arthritis score. RNA was extracted from the joint, and examined for Bv8 mRNA expression by RT-PCR and real-time RT-PCR. Synovial tissue and bone marrow were immunohistochemically examined using anti-Bv8 antibody. RESULTS: The level of Bv8 mRNA expression in the joint was below the detection limit in the control group, but was elevated in the CIA group, and was correlated with the arthritis score. In addition, an increase in Bv8-positive cells was observed in the synovium and bone marrow in the CIA group. CONCLUSION: Bv8 was elevated in the synovium and bone marrow of CIA mice, suggesting that Bv8 plays an important role in the pathogenesis of arthritis.


Subject(s)
Arthritis, Experimental/metabolism , Gastrointestinal Hormones/genetics , Joints/metabolism , Neuropeptides/genetics , Synovial Membrane/metabolism , Up-Regulation/physiology , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/physiopathology , Biomarkers/analysis , Biomarkers/metabolism , Bone Marrow/metabolism , Bone Marrow/pathology , Bone Marrow/physiopathology , Collagen/toxicity , Disease Models, Animal , Joints/pathology , Joints/physiopathology , Male , Mice , Mice, Inbred DBA , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Synovial Membrane/pathology , Synovial Membrane/physiopathology
7.
Mod Rheumatol ; 19(2): 187-91, 2009.
Article in English | MEDLINE | ID: mdl-19219643

ABSTRACT

The objective of the study is to examine the relationship between synovial blood flow signals and vascular endothelial growth factor (VEGF) involved in angiogenesis by Doppler ultrasound. Twenty-one patients meeting the diagnostic criteria of the American College of Rheumatology (ACR) were enrolled in this study. Doppler ultrasound signals of blood flow in the wrist synovial membrane were measured and classified into three grades: grade 1 = no flow; grade 2 = mild flow; grade 3 = intense flow. A significant correlation was observed between blood flow signals in the wrist synovial membrane and serum VEGF levels (r = 0.5681, P = 0.0072). These results suggest that the measurement of Doppler ultrasound signals of blood flow in the wrist synovial membrane is useful in the evaluation of angiogenesis.


Subject(s)
Arthritis, Rheumatoid/diagnostic imaging , Synovial Membrane/blood supply , Vascular Endothelial Growth Factor A/blood , Adult , Aged , Arthritis, Rheumatoid/blood , Cell Movement , Female , Humans , Male , Middle Aged , Neovascularization, Pathologic/diagnostic imaging , Synovial Fluid/metabolism , Synovial Membrane/diagnostic imaging , Ultrasonography, Doppler , Wrist Joint/blood supply , Wrist Joint/diagnostic imaging
8.
Cancer Sci ; 94(7): 606-11, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12841869

ABSTRACT

The expression levels of the Wilms' tumor gene WT1 were examined in 34 primary thyroid cancers (24 papillary, 5 follicular, 1 anaplastic, and 4 medullary carcinomas), 17 thyroid follicular adenomas, and 6 normal-appearing thyroid tissues using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In 33 of 34 thyroid cancers, the WT1 mRNA was expressed at levels ranging from 5.0 x 10 (-5) to 8.3 x 10 (-2) levels (WT1 expression level in K562 leukemic cells was defined as 1.0). The WT1 mRNA expression levels were significantly higher than those in either thyroid follicular adenomas (P < 0.001) or normal-appearing thyroid tissues (P < 0.01). Immunohistochemical analysis confirmed the expression of WT1 protein in 20 of 21 thyroid cancers with WT1 mRNA expression. WT1 protein was also detected in 6 of 7 follicular adenomas with WT1 mRNA expression. However, the intensity of staining of WT1 protein in adenoma cells was weaker than that in cancer cells and its expression was restricted to approximately 30-80% of adenoma cells in the tumors examined. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of the 10 exons of the WT1 gene in all of the 9 different thyroid cancers. These findings indicate an important role of the wild-type WT1 gene in the tumorigenesis of primary thyroid cancer.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Genes, Wilms Tumor , Thyroid Neoplasms/genetics , WT1 Proteins/genetics , Adenocarcinoma, Follicular/genetics , Adenocarcinoma, Follicular/pathology , Adenoma/genetics , Adenoma/pathology , Adult , Aged , Base Sequence , Carcinoma, Medullary/genetics , Carcinoma, Papillary/genetics , DNA Primers , Female , Humans , Kidney Neoplasms/genetics , Male , Middle Aged , Polymerase Chain Reaction , Thyroid Neoplasms/pathology , Wilms Tumor/genetics
9.
Cancer Sci ; 94(3): 271-6, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12824921

ABSTRACT

The expression levels of the Wilms' tumor gene WT1 were examined in 36 cases of various types of human bone and soft-tissue sarcomas using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). They included 12 malignant fibrous histiocytomas (MFH), 3 malignant peripheral nerve sheath tumors (MPNST), 6 synovial sarcomas (SyS), 4 myxoid liposarcomas (MyLS), one angiosarcoma (AGS), one clear cell sarcoma (CCS), and 9 osteosarcomas (OS). Eleven (92%) of 12 MFH, 2 (67%) of 3 MPNST, all (100%) of 6 SyS, 2 (50%) of 4 MyLS, one AGS, one CCS, and 5 (56%) of 9 OS cases overexpressed WT1 in the range of 1.4 x 10(-3)-3.9 x 10(-1) levels (WT1 expression level in K562 leukemic cells was defined as 1.0). Thus, 28 (78%) out of 36 various types of human bone and soft-tissue sarcomas overexpressed the WT1 gene. Immunohistochemical analysis showed positive staining for WT1 protein in all of 4 cases (one case each of MFH, MyLS, AGS and OS) with WT1 gene overexpression detected by RT-PCR analysis, demonstrating clearly that WT1 was expressed at the protein level in various types of human bone and soft-tissue sarcomas. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of 10 exons of the WT1 gene in 8 different sarcoma samples (3 MFH, one SyS, one MyLS, one AGS, and 2 OS). The present study demonstrates that various types of human bone and soft-tissue sarcomas frequently overexpress the wild-type WT1 gene, suggesting an important role of the wild-type WT1 gene in tumorigenesis of various types of human bone and soft-tissue sarcomas.


Subject(s)
Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, Wilms Tumor , Sarcoma/genetics , WT1 Proteins/genetics , Adult , Aged , Base Sequence , Bone Neoplasms/classification , Bone Neoplasms/pathology , DNA Primers , Female , Humans , Immunohistochemistry , Kidney Neoplasms/genetics , Male , Middle Aged , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma/classification , Sarcoma/pathology , WT1 Proteins/analysis , Wilms Tumor/genetics
10.
Int J Cancer ; 100(3): 297-303, 2002 Jul 20.
Article in English | MEDLINE | ID: mdl-12115544

ABSTRACT

Expression of the Wilms' tumor gene WT1 in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WT1 gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WT1 protein with an anti-WT1 antibody. Overexpression of the WT1 gene was also demonstrated in 5/6 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WT1 gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the nonmutated, wild-type WT1 gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WT1 gene and its products.


Subject(s)
Lung Neoplasms/genetics , WT1 Proteins/genetics , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Small Cell/genetics , Carcinoma, Small Cell/pathology , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Mutation , Neoplasm Staging , RNA Splicing
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