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Transcriptome Analysis of the Rhizosphere Bacterium Azospirillum brasilense Reveals an Extensive Auxin Response

MICROBIAL ECOLOGY 2011年第4期61卷 723-728页

作者： Van Puyvelde, Sandra Cloots, Lore Engelen, Kristof Das, Frederik Marchal, Kathleen Vanderleyden, Jos Spaepen, Stijn Katholieke Univ Leuven Ctr Microbial & Plant Genet B-3001 Heverlee Belgium

The rhizosphere bacterium Azospirillum brasilense produces the auxin indole-3-acetic acid (IAA) through the indole-3-pyruvate pathway. As we previously demonstrated that transcription of the indole-3-pyruvate decarboxylase (ipdC) gene is positively regulated by IAA, produced by A. brasilense itself or added exogenously, we performed a microarray analysis to study the overall effects of IAA on the transcriptome of A. brasilense. The transcriptomes of A. brasilense wild-type and the ipdC knockout mutant, both cultured in the absence and presence of exogenously added IAA, were compared. Interfering with the IAA biosynthesis/homeostasis in A. brasilense through inactivation of the ipdC gene or IAA addition results in much broader transcriptional changes than anticipated. Based on the multitude of changes observed by comparing the different transcriptomes, we can conclude that IAA is a signaling molecule in A. brasilense. It appears that the bacterium, when exposed to IAA, adapts itself to the plant rhizosphere, by changing its arsenal of transport proteins and cell surface proteins. A striking example of adaptation to IAA exposure, as happens in the rhizosphere, is the upregulation of a type VI secretion system (T6SS) in the presence of IAA. The T6SS is described as specifically involved in bacterium-eukaryotic host interactions. Additionally, many transcription factors show an altered regulation as well, indicating that the regulatory machinery of the bacterium is changing.

关键词： RHIZOBACTERIA AZOSPIRILLUM AUXIN INDOLEACETIC acid GENETIC transcription microarray technology BIOSYNTHESIS CARRIER proteins

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Rituximab Treatment Induces the Expression of Genes Involved in Healing Processes in the Rheumatoid Arthritis Synovium

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ARTHRITIS AND RHEUMATISM 2011年第5期63卷 1246-1254页

作者： Gutierrez-Roelens, I. Galant, C. Theate, I. Lories, R. J. Durez, P. Nzeusseu-Toukap, A. Van den Eynde, B. Houssiau, F. A. Lauwerys, B. R. Clin Univ St Luc Dept Rheumatol RUMA 5390 B-1200 Brussels Belgium Catholic Univ Louvain B-1200 Brussels Belgium Katholieke Univ Leuven Louvain Belgium Ludwig Inst Canc Res Brussels Branch Brussels Belgium Duve Inst Brussels Belgium

Objective. Rituximab displays therapeutic benefits in the treatment of patients with rheumatoid arthritis (RA) resistant to tumor necrosis factor (TNF) blockade. However, the precise role of B cells in the pathogenesis of RA is still unknown. We undertook this study to investigate the global molecular effects of rituximab in synovial biopsy samples obtained from anti-TNF-resistant RA patients before and after administration of the drug. Methods. Paired synovial biopsy samples were obtained from the affected knee of anti-TNF-resistant RA patients before (time 0) and 12 weeks after (time 12) initiation of rituximab therapy. Total RNA was extracted, labeled according to standard Affymetrix procedures, and hybridized on GeneChip HGU133 Plus 2.0 slides. Immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction experiments were performed to confirm the differential expression of selected transcripts. Results. According to Student's paired t-tests, 549 of 54,675 investigated probe sets were differentially expressed between time 0 and time 12. Pathway analysis revealed that genes down-regulated between time 0 and time 12 were significantly enriched in immunoglobulin genes and genes involved in chemotaxis, leukocyte activation, and immune responses (Gene Ontology annotations). In contrast, genes up-regulated between time 0 and time 12 were significantly enriched in transcripts involved in cell development (Gene Ontology annotation) and wound healing (Gene Set Enrichment Analysis). At baseline, higher synovial expression of immunoglobulin genes was associated with response to therapy. Conclusion. Rituximab displays unique effects on global gene expression profiles in the synovial tissue of RA patients. These observations open new perspectives in the understanding of the biologic effects of the drug and in the selection of patients likely to benefit from this therapy.

关键词： RITUXIMAB ANALYSIS of variance BIOPSY IMMUNOHISTOCHEMISTRY LONGITUDINAL method POLYMERASE chain reaction FINANCING of research RHEUMATOID arthritis T-test (Statistics) GENOMICS EQUIPMENT & supplies REVERSE transcriptase polymerase chain reaction microarray technology THERAPEUTIC use

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The Antiproliferative Effect of Dietary Fiber Phenolic Compounds Ferulic Acid and p-Coumaric Acid on the Cell Cycle of Caco-2 Cells

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NUTRITION AND CANCER-AN INTERNATIONAL JOURNAL 2011年第4期63卷 611-622页

作者： Janicke, Birgit Hegardt, Cecilia Krogh, Morten Onning, Gunilla Akesson, Bjorn Cirenajwis, Helena M. Oredsson, Stina M. Lund Univ Ctr Chem & Chem Engn Lund Inst Technol SE-22100 Lund Sweden Univ Lund Hosp Dept Clin Sci Oncol S-22185 Lund Sweden Lund Univ Dept Theoret Phys SE-22100 Lund Sweden Univ Lund Hosp Dept Clin Nutr S-22185 Lund Sweden Lund Univ Dept Cell & Organism Biol SE-22100 Lund Sweden

Epidemiological and animal studies have shown that dietary fiber is protective against the development of colon cancer. Dietary fiber is a rich source of the hydroxycinnamic acids ferulic acid (FA) and p-coumaric acid (p-CA), which both may contribute to the protective effect. We have investigated the effects of FA and p-CA treatment on global gene expression in Caco-2 colon cancer cells. The Caco-2 cells were treated with 150 M FA or p-CA for 24 h, and gene expression was analyzed with cDNA microarray technique. A total of 517 genes were significantly affected by FA and 901 by p-CA. As we previously have found that FA or p-CA treatment delayed cell cycle progression, we focused on genes involved in proliferation and cell cycle regulation. The expressions of a number of genes involved in centrosome assembly, such as RABGAP1 and CEP2, were upregulated by FA treatment as well as the gene for the S phase checkpoint protein SMC1L1. p-CA treatment upregulated CDKN1A expression and downregulated CCNA2, CCNB1, MYC, and ODC1. Some proteins corresponding to the affected genes were also studied. Taken together, the changes found can partly explain the effects of FA or p-CA treatment on cell cycle progression, specifically in the S phase by FA and G2/M phase by p-CA treatment.

关键词： DNA PHYSIOLOGY RNA -- Physiology PHENOLS PHYTOCHEMICALS COLON tumors PREVENTION CELL culture CELL cycle CELL physiology DIET FIBER in human nutrition FLOW cytometry GENES NUTRITION PROFESSIONAL associations WESTERN immunoblotting LIFESTYLES microarray technology THERAPEUTIC use

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Increased immunoreactivity of membrane type-1 matrix metalloproteinase (MT1-MMP) and β-catenin in high-risk basal cell carcinoma

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BRITISH JOURNAL OF DERMATOLOGY 2011年第6期165卷 1197-1204页

作者： Oh, S. T. Kim, H. S. Yoo, N. J. Lee, W. S. Cho, B. K. Reichrath, J. Catholic Univ Korea Dept Dermatol Seoul South Korea Catholic Univ Korea Dept Pathol Seoul South Korea Saarland Univ Hosp Dept Dermatol D-66421 Homburg Germany

Background Although various immunohistological markers have been investigated to assess the aggressive characteristics of basal cell carcinoma (BCC), the role of membrane type-1 matrix metalloproteinase (MT1-MMP) has not been well established. Objectives To clarify the precise role of MT1-MMP in BCC, MT1-MMP expression was studied in various histological subtypes of BCC. Materials and methods High-risk subtypes of BCC were compared by assessing the expression of beta-catenin and MT1-MMP. The tissue microarray technique was used for immunohistochemical staining. Fifty-eight samples were divided into six subtypes (10 nodular, 12 mixed, nine infiltrative, eight morphoeiform, 10 micronodular and nine basosquamous). Overall, the 10 nodular BCC samples were classified as low-risk BCC and the remaining 48 samples were classified as high-risk BCCs. Results beta-Catenin immunoreactivity was increased in the high-risk BCCs compared with the low-risk (nodular) BCC (P < 0 001). Nuclear beta-catenin immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesion (P < 0 01). For the mixed BCC (P < 0 01), infiltrative BCC (P < 0 001), morphoeiform BCC (P < 0 001), micronodular BCC (P < 0 001) and basosquamous (P < 0 001) carcinoma, beta-catenin immunoreactivity was increased at the invading front compared with nodular BCC. MT1MMP immunoreactivity was increased in the high-risk BCCs compared with the low-risk (nodular) BCC (P < 0 01). The membranous MT1-MMP immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesions (P < 0 01). For the mixed (P < 0 01), infiltrative (P < 0 05), morphoeiform (P < 0 05), micronodular (P > 0 05) and basosquamous (P < 0 05) BCC, MT1-MMP immunoreactivity was also increased at the invading front compared with nodular BCC. Conclusions The results of this study suggest that MT1-MMP might be a novel marker for high-risk BCC. In

关键词： METALLOPROTEINASES BASAL cell carcinoma microarray technology PATHOLOGICAL histology TUMOR suppressor proteins

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Expression of coordinately regulated defence response genes and analysis of their role in disease resistance in Medicago truncatula

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MOLECULAR PLANT PATHOLOGY 2011年第8期12卷 786-798页

作者： Samac, Deborah A. Penuela, Silvia Schnurr, Judy A. Hunt, E. Nicole Foster-Hartnett, Dawn Vandenbosch, Kathryn A. Gantt, J. Stephen ARS USDA Plant Sci Res Unit St Paul MN 55108 USA Univ Minnesota Dept Plant Pathol St Paul MN 55108 USA Ft Valley State Univ Ft Valley GA 31030 USA Univ Minnesota Dept Plant Biol Ctr Biol Sci 250 St Paul MN 55108 USA

microarray technology was used to identify the genes associated with disease defence responses in the model legume Medicago truncatula. Transcript profiles from M. truncatula cv. Jemalong genotype A17 leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify the genes expressed in response to all three pathogens and genes unique to an interaction. The A17 genotype is resistant to C. trifolii and E. pisi, exhibiting a hypersensitive response after inoculation, and is moderately susceptible to P. medicaginis. Among the most strongly up-regulated genes in all three interactions were those encoding a hevein-like protein, thaumatin-like protein (TLP) and members of the pathogenesis response (PR) 10 family. Transcripts of genes for enzymes in the phenylpropanoid pathway leading to the production of isoflavonoid phytoalexins increased dramatically in response to inoculation with the foliar pathogens. In P. medicaginis-inoculated roots, transcripts of genes in the phenylpropanoid pathway peaked at 5 days post-inoculation, when symptoms became visible. Transcript accumulation of three PR10 family members, a TLP and chalcone synthase (CHS) was assessed in M. truncatula genotype R108 plants. The R108 plants are resistant to C. trifolii and moderately susceptible to E. pisi and P. medicaginis. Transcript accumulation paralleled the stages of pathogen development. To evaluate the role of a TLP, a PR10 family member and CHS in disease resistance, transgenic R108 plants containing interfering RNA (RNAi) constructs were produced. Reduced expression of PR10 and TLP had no effect on the disease phenotype, whereas reduced expression of CHS resulted in increased susceptibility to necrotrophic pathogens.

关键词： GENE expression MEDICAGO microarray technology COLLETOTRICHUM ERYSIPHE PHYTOPATHOGENIC microorganisms PLANT proteins PHYTOALEXINS

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Identification of cell surface-specific markers to target human nucleus pulposus cells: Expression of carbonic anhydrase XII varies with age and degeneration

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ARTHRITIS AND RHEUMATISM 2011年第12期63卷 3876-3886页

作者： Power, Karen A. Grad, Sibylle Rutges, Joost P. H. J. Creemers, Laura B. van Rijen, Mattie H. P. O'Gaora, Peadar Wall, J. Gerard Alini, Mauro Pandit, Abhay Gallagher, William M. AO Res Inst Davos CH-7270 Davos Switzerland Univ Coll Dublin Dublin 2 Ireland Natl Univ Ireland Network Excellence Funct Biomat Galway Ireland Univ Med Ctr Utrecht Netherlands

Objective Back pain is a major cause of disability, affecting millions of people worldwide. One cause of axial back pain is degeneration of the nucleus pulposus (NP) of the intervertebral disc. This study was undertaken to investigate associations of NP cells with cell surfacespecific proteins that differ from proteins in closely related cell types, i.e., intervertebral disc anulus fibrosus (AF) cells and articular cartilage (AC) chondrocytes, in order to identify potential surface molecules for directed delivery of therapeutic agents. Methods. We conducted a complementary DNA microarray analysis of 16 human samples from 6 donors, followed by gene list reduction using a systematic approach. Genes that were more highly expressed in NP than AC cells, contained transmembrane domains, and appeared attractive for targeting were assessed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). As a viable candidate, carbonic anhydrase XII (CAXII) was analyzed at the protein level by immunohistochemistry and functional study. Results. microarray results demonstrated a clear divide between the AC and AF and between the AC and NP samples. However, the transcriptomic profile of AF and NP samples displayed a greater intersubject similarity. Of the 552 genes with up-regulated expression in NP cells, 90 contained transmembrane domains, and 28 were quantified by RT-PCR. Most intense CAXII labeling was observed in the NP of discs from young subjects and in degenerative tissue. Conclusion. CAXII may be considered for detection or targeting of degenerating disc cells. Furthermore, CAXII may be involved in pH regulation of NP cells. Its potential for directed delivery of regenerative factors and its functional role in NP cell homeostasis warrant further investigation.

关键词： INTERVERTEBRAL disk PHYSIOLOGY AGING BACKACHE GENES IMMUNOHISTOCHEMISTRY POLYMERASE chain reaction FINANCING of research EQUIPMENT & supplies REVERSE transcriptase polymerase chain reaction microarray technology

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Affinity profiling using the peptide microarray technology: A case study

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ANALYTICAL BIOCHEMISTRY 2007年第1期363卷 108-118页

作者： Tapia, Victor Bongartz, Juliane Schutkowski, Mike Bruni, Nicole Weiser, Armin Ay, Bernhard Volkmer, Rudolf Or-Guil, Michal Charite Univ Med Berlin Inst Med Immunol Mol Lib & Recognit Grp D-10115 Berlin Germany Humboldt Univ Inst Theoret Biol D-10115 Berlin Germany JPT Peptide Technol GmbH D-12489 Berlin Germany

Little about the reliability of measurements obtained using synthetic peptide microarrays is known. We report results from a study on the quantitative reliability of microarrays manufactured by robot-supported immobilization of presynthesized peptides for different microarray platforms. Technological precision is assessed for inter- and intra-device readout comparisons. Correlations between measured signals and known dissociation constants using a phenomenological model derived from the mass action law are discussed. Special emphasis is on discussing the pitfalls of high-throughput affinity measurements. We show that the quantitative determination of binding affinities is prone to be biased toward a mean affinity of around 10(-7) M, while the classification of peptides into either "binders" or "nonbinders" provides very high prediction accuracy. The experimental requirements needed to obtain reliable binding affinity predictions are discussed. (c) 2007 Elsevier Inc. All rights reserved.

关键词： microarray technology synthetic peptide arrays mass action law precision accuracy binding affinity

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Chitinases in the Salivary Glands and Circulation of Patients With Sjogren's Syndrome Macrophage Harbingers of Disease Severity

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ARTHRITIS AND RHEUMATISM 2011年第10期63卷 3103-3115页

作者： Greenwell-Wild, Teresa Moutsopoulos, Niki M. Gliozzi, Maria Kapsogeorgou, Efstathia Rangel, Zoila Munson, Peter J. Moutsopoulos, Haralampos M. Wahl, Sharon M. Natl Inst Dent & Craniofacial Res NIH Oral Infect & Immun Branch Bethesda MD 20892 USA Natl Univ Athens Sch Med Athens Greece NIH Ctr Informat Technol Bethesda MD 20892 USA

Objective. Sjogren's syndrome (SS) is a chronic autoimmune disease of unknown etiology that targets salivary and lacrimal glands and may be accompanied by multiorgan systemic manifestations. To further the understanding of immunopathology associated with SS and identify potential therapeutic targets, we undertook the present study comparing the gene expression profiles of salivary glands with severe inflammation versus those of salivary glands with mild or no disease. Methods. Using microarray profiling of salivary gland tissue from patients with SS and control subjects, we identified target genes, which were further characterized in tissue, serum, and cultured cell populations by real-time polymerase chain reaction and protein analysis. Results. Among the most highly expressed SS genes were those associated with myeloid cells, including members of the mammalian chitinase family, which had not previously been shown to be associated with exocrinopathies. Both chitinase 3-like protein 1 and chitinase 1, highly conserved chitinase-like glycoproteins (one with enzymatic activity and one lacking enzymatic activity), were evident at the transcriptome level and were detected within inflamed tissue. Chitinases were expressed during monocyte-to-macrophage differentiation and their levels augmented by stimulation with cytokines, including interferon-alpha (IFN alpha). Conclusion. Because elevated expression of these and other macrophage-derived molecules corresponded with more severe SS, the present observations suggest that macrophages have potential immunopathologic involvement in SS and that the tissue macrophage transcription profile reflects multiple genes induced by IFN alpha.

关键词： SJOGREN'S syndrome CYTOKINES IMMUNOHISTOCHEMISTRY POLYMERASE chain reaction FINANCING of research SALIVARY glands U-statistics EQUIPMENT & supplies SEVERITY of illness index CASE-control method REVERSE transcriptase polymerase chain reaction DATA analysis software microarray technology GENETIC aspects

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Presence of amplifiable mRNA in acellular hair shafts: utilization to analyze gene expression profiles of black and white hairs

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INTERNATIONAL JOURNAL OF DERMATOLOGY 2011年第5期50卷 530-534页

作者： Tochio, Takumi Tanaka, Hiroshi Nakata, Satoru Yoshizato, Katsutoshi Nippon Menard Cosmet Co Ltd Res Labs Nishi Ku Nagoya Aichi 4510071 Japan Osaka City Univ Grad Sch Med Dept Dermatol Osaka 558 Japan Phoenix Bio Co Ltd Higashihiroshima Japan

Background We postulated that hair shafts preserve amplifiable messenger RNAs (mRNAs) and that these mRNAs could be utilized to characterize phenotypic differences in hairs at the gene expression level. Objectives This study aimed to prove the presence of amplifiable mRNAs in the hair shaft and then to utilize them to characterize black and white hairs at the gene expression level. Methods RNAs were extracted from black and white scalp hair shafts and amplified by quantitative real-time polymerase chain reaction (qRT-PCR). microarray analysis was performed using T7 RNA polymerase-amplified mRNAs derived from the hair shaft to examine the gene expression profiles of black and white hairs. Results We showed that hair shaft RNAs contained trace amounts of mRNAs and concluded that the acellular hair shaft contained mRNAs usable for microarray analysis. We termed the mRNA "fossil mRNA" (fmRNA) in this study. Totals of 10 and 2% of the detected genes were expressed at levels more than two-fold higher in black and white hairs compared with white and black hairs, respectively. We selected five genes and examined their expression levels in five donors by qRT-PCR. Among them, only hypothetical protein (L1H3 region) human was found to be expressed at 2.69 +/- 1.81 (relative ratio) in black hairs. Conclusions We demonstrated that the scalp hair shaft conserves fmRNAs which are probably remnants that have been expressed during hair differentiation. Consequently, fmRNA is expected to be a useful source of information for the study of phenotypic changes in hairs at the molecular level.

关键词： HAIR RESEARCH MESSENGER RNA GENE expression POLYMERASE chain reaction microarray technology GENES SCALP

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Generation of Human Induced Pluripotent Stem Cells From Osteoarthritis Patient-Derived Synovial Cells

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ARTHRITIS AND RHEUMATISM 2011年第10期63卷 3010-3021页

作者： Kim, Min-Jeong Son, Myung Jin Son, Mi-Young Seol, Binna Kim, Janghwan Park, Jongjin Kim, Jung Hwa Kim, Yong-Hoon Park, Su A. Lee, Chul-Ho Lee, Kang-Sik Han, Yong-Mahn Chang, Jae-Suk Cho, Yee Sook Korea Res Inst Biosci & Biotechnol Taejon 305806 South Korea Univ Sci & Technol Taejon South Korea Univ Ulsan Coll Med Seoul South Korea Asan Med Ctr Seoul South Korea Korea Inst Machinery & Mat Taejon South Korea Korea Adv Inst Sci & Technol Taejon 305701 South Korea

Objective. This study was undertaken to generate and characterize human induced pluripotent stem cells (PSCs) from patients with osteoarthritis (OA) and to examine whether these cells can be developed into disease-relevant cell types for use in disease modeling and drug discovery. Methods. Human synovial cells isolated from two 71-year-old women with advanced OA were characterized and reprogrammed into induced PSCs by ectopic expression of 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc). The pluripotency status of each induced PSC line was validated by comparison with human embryonic stem cells (ESCs). Results. We found that OA patient-derived human synovial cells had human mesenchymal stem cell (MSC)-like characteristics, as indicated by the expression of specific markers, including CD14-, CD19-, CD34-, CD45-, CD44+, CD51+, CD90+, CD105+, and CD147+. microarray analysis of human MSCs and human synovial cells further determined their unique and overlapping gene expression patterns. The pluripotency of established human induced PSCs was confirmed by their human ESC-like morphology, expression of pluripotency markers, gene expression profiles, epigenetic status, normal karyotype, and in vitro and in vivo differentiation potential. The potential of human induced PSCs to differentiate into distinct mesenchymal cell lineages, such as osteoblasts, adipocytes, and chondrocytes, was further confirmed by positive expression of markers for respective cell types and positive staining with alizarin red S (osteoblasts), oil red O (adipocytes), or Alcian blue (chondrocytes). Functional chondrocyte differentiation of induced PSCs in pellet culture and 3-dimensional polycaprolactone scaffold culture was assessed by chondrocyte self-assembly and histology. Conclusion. Our findings indicate that patient-derived synovial cells are an attractive source of MSCs as well as induced PSCs and have the potential to advance cartilage tissue engineering and cell-based models of cartilag

关键词： CLINICAL medicine research IMMUNOHISTOCHEMISTRY KARYOTYPES OSTEOARTHRITIS POLYMERASE chain reaction FINANCING of research STEM cells GENOMICS EQUIPMENT & supplies REVERSE transcriptase polymerase chain reaction microarray technology

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