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1.
Eur J Pain ; 21(10): 1723-1731, 2017 11.
Article in English | MEDLINE | ID: mdl-28722339

ABSTRACT

BACKGROUND: The goal was to test the effectiveness of a structured pain management programme after invasive electrophysiological interventions in cardiology including ablation of atrial fibrillation (AF) or ventricular tachycardia (VT) and implantation, or explantation, of pacemakers or implantable cardioverter defibrillators. METHODS: This was a prospective study with a pre-/post-design where a post-intervention group (116 consecutive patients) was compared to a pre-intervention group (102 consecutive patients) after implementation of a structured pain-management programme using the numeric rating scale (NRS 0-10) and classified as moderate-to-severe if NRS > 3. Measurements were recorded every two hours during the first 24 h post-operatively. The location of the pain and the amount of analgesic used were also recorded. RESULTS: The proportion of patients who experienced moderate-to-severe pain after the procedure decreased after initiation of the pain-management program: 47% versus 61%; p = 0.048. This difference was driven primarily by reduced pain late (8-24 h) after the procedure; 16% versus 39%; p < 0.001. The risk to develop late (8-24 h) post-procedural pain was reduced approximately three-fold after implementation of the pain-management programme (OR = 0.32, 95% CI 0.16-0.64, p = 0.001). Multivariate analysis indicated chronic pain, early pain (0-6 h), and type of intervention were associated with late post-interventional pain. In contrast, age, diabetes mellitus, BMI, gender and procedure time were not related. CONCLUSION: The findings illustrate the potential value of a structured pain-management programme. The proportion of patients who experienced moderate-to-severe pain after these electrophysiological procedures decreased significantly. SIGNIFICANCE: This is the first exploratory study that evaluates the impact of a multidisciplinary pain-management programme after cardiac electrophysiological interventions. It demonstrates that significant quality improvement is achievable following simple rules together with patient and staff education. The programme reduces the proportion of patients with moderate-to-severe pain after electrophysiological procedures significantly.


Subject(s)
Cardiac Surgical Procedures/adverse effects , Catheter Ablation/adverse effects , Pain Management , Pain, Postoperative/therapy , Aged , Aged, 80 and over , Analgesics/therapeutic use , Controlled Before-After Studies , Defibrillators, Implantable , Female , Humans , Male , Middle Aged , Pain, Postoperative/etiology , Prospective Studies , Treatment Outcome
2.
Pathologe ; 38(2): 112-116, 2017 Mar.
Article in German | MEDLINE | ID: mdl-28120026

ABSTRACT

We report a case of ALK1-positive anaplastic large cell lymphoma with expression of placental alkaline phosphatase (PLAP) in many tumor cells. Initially, due to the positivity of tumor cells for CD30 and PLAP, lymph node metastasis of a germ cell neoplasm was discussed. Anaplastic large cell lymphomas of T­cell lineage form a group of rare non-Hodgkin lymphomas with heterogeneous morphological and immunohistochemical appearance. They may imitate other neoplasms, such as large cell B­cell lymphomas, metastasis of a carcinoma, melanoma, embryonal carcinoma or seminoma, rhabdomyosarcoma and inflammatory myofibroblastic tumor. Only an extended immunohistochemistry panel leads to an accurate diagnosis.


Subject(s)
Alkaline Phosphatase/metabolism , Isoenzymes/metabolism , Lymphatic Metastasis/diagnosis , Lymphoma, Large-Cell, Anaplastic/diagnosis , Activin Receptors, Type II/genetics , Adult , Biomarkers, Tumor , GPI-Linked Proteins/metabolism , Humans , Immunohistochemistry , Lymph Nodes/pathology , Lymphoma, Large-Cell, Anaplastic/genetics , Male , Neck , Neoplasms, Germ Cell and Embryonal/pathology
3.
J Atr Fibrillation ; 6(3): 934, 2013.
Article in English | MEDLINE | ID: mdl-28496900

ABSTRACT

Since the pulmonary veins (PVs) were identified as a major source of AF triggers, ablation strategies targeting the PVs have evolved from focal ablation inside the PVs to wide area circumferential PV isolation (PVI) which at this juncture is the standard approach. Despite the widespread popularity of PVI, a universal definition is lacking. While "entrance block" is a generally accepted endpoint for PVI, the role of "exit block" has yet to be determined. Inexcitability of the circular ablation line has been introduced as a promising additional endpoint for PVI and was associated with an improved clinical outcome in a randomized trial. Correct interpretation of PV electrograms during an ablation procedure is critical in terms of efficacy and safety. A variety of electrophysiological techniques help to correctly differentiate components of complex PV electrograms. Resumption of PV conduction after initially successful PVI leading to AF recurrence remains a major problem and confirmation of bi-directional conduction block does not exclude reversible tissue damage along the ablation line. Prolongation of post-PVI monitoring and application of provocative procedures such as the administration of adenosine after initial PVI to unmask dormant PV conduction may improve clinical outcome although there is lack of valid data supporting these strategies. This article aims on clarifying the electrophysiological criteria for complete pulmonary vein isolation and the explain the importance of this cornerstone in almost all atrial fibrillation ablation procedures.

4.
Curr Cardiol Rev ; 8(4): 354-61, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22920479

ABSTRACT

Atrial fibrillation (AF) is the most prevalent sustained arrhythmia in clinical practice. It is associated with significant morbidity and mortality and has been identified as an independent risk factor for ischemic stroke and thromboembolic events. Catheter ablation has become an established rhythm control therapy in patients with highly symptomatic drug-refractory AF. The definition of ablation success remains controversial since current symptom-based or intermittent electrocardiogram monitoring strategies fail to sufficiently disclose rhythm outcome. This failure is mainly related to the high incidence of asymptomatic AF recurrences, the unpredictable nature of arrhythmia relapses, and the poor correlation of symptoms and AF episodes. There is a clear correlation between the intensity of the monitoring strategy and the sensitivity for it to detect arrhythmia recurrences. Furthermore, several clinical studies assessing the long-term efficacy of catheter ablation procedures have reported late AF recurrences in patients who were initially considered responders to catheter ablation. In certain subsets of patients, precise long-term monitoring may help to guide therapy, e.g. patients in whom withdrawal of antithrombotic therapy may be considered if they are free of arrhythmia recurrences. Recently, subcutaneous implantable cardiac monitors (ICM) have been introduced for prolonged and continuous rhythm monitoring. The performance of a leadless ICM equipped with a dedicated AF detection algorithm has recently been assessed in a clinical trial demonstrating a high sensitivity and overall accuracy for identifying patients with AF. The clinical impact of ICM-based follow-up strategies, however, has to be evaluated in prospective clinical trials.


Subject(s)
Atrial Fibrillation/surgery , Catheter Ablation/methods , Atrial Fibrillation/diagnosis , Catheter Ablation/trends , Defibrillators, Implantable , Electrocardiography , Forecasting , Humans , Postoperative Care/methods , Secondary Prevention , Treatment Outcome
6.
Colorectal Dis ; 14(10): 1276-86, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22309286

ABSTRACT

AIM: Immunosuppression and steroid medication have been identified as risk factors for complicated sigmoid diverticulitis. The underlying molecular mechanisms have not yet been elucidated. We hypothesized that glucocorticoid-induced tumour necrosis factor receptor (GITR) and matrix metalloproteinase-9 (MMP-9) might play a role. METHOD: GITR and MMP-9 were analysed at protein [immunohistochemistry/immunofluorescence (IF)] and messenger RNA level (real-time polymerase chain reaction) in surgical specimens with complicated and non-complicated diverticulitis (n=101). IF double staining and regression analysis were performed for both markers. GITR expression was correlated with clinical data and its usefulness as a diagnostic test was investigated. RESULTS: High GITR expression (≥41%) was observed in the inflammatory infiltrate in complicated diverticulitis, in contrast to non-complicated diverticulitis where GITR expression was low (P<0.001). High GITR expression was significantly associated with steroid use and pulmonary diseases (both P<0.001). MMP-9 expression correlated with GITR expression (R(2) =0.7268, P<0.0001, r=0.85) as demonstrated with IF double-staining experiments. Co-labelling of GITR with CD68, but not CD15, suggested that GITR-expressing cells in diverticulitis are macrophages. GITR expression was superior to C-reactive protein (CRP), white cell count and temperature in distinguishing complicated and non-complicated diverticulitis. CONCLUSIONS: Our results suggest that GITR expression in inflammatory cells might potentially indicate a molecular link between steroid use and complicated forms of acute sigmoid diverticulitis. Increased MMP-9 expression by GITR signalling might explain the morphological changes in the colonic wall of perforated and phlegmonous diverticulitis. Analysis of soluble GITR might be a promising strategy for future research.


Subject(s)
Diverticulitis, Colonic/metabolism , Glucocorticoid-Induced TNFR-Related Protein/metabolism , Immunosuppressive Agents/adverse effects , Matrix Metalloproteinase 9/metabolism , Sigmoid Diseases/metabolism , Steroids/adverse effects , Aged , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Biomarkers/metabolism , Case-Control Studies , Diverticulitis, Colonic/chemically induced , Diverticulitis, Colonic/complications , Diverticulitis, Colonic/diagnosis , Female , Fucosyltransferases/metabolism , Humans , Immunohistochemistry , Lewis X Antigen/metabolism , Macrophages/metabolism , Male , Middle Aged , Odds Ratio , Prospective Studies , ROC Curve , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sigmoid Diseases/chemically induced , Sigmoid Diseases/complications , Sigmoid Diseases/diagnosis
7.
Cell Oncol (Dordr) ; 34(4): 315-26, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21573932

ABSTRACT

INTRODUCTION: The progressive growth of malignancies is accompanied by a decline in the immune response through mechanisms which are poorly understood. Apoptosis and induction of inflammation by tumor released cytokines as tumor escape mechanisms have been proposed to play an important role in colorectal carcinogenesis. METHODS: Expression of Tumor necrosis factor-alpha (TNF-α) was analyzed in colorectal cancer specimen and the cancer cell line HT-29 by immunohistochemistry and RT-PCR. TNF-α expression on protein and mRNA level were correlated with clinical characteristics and impact on survival. TNFR-1 was co-labelled with TNF-α and CD8+ cytotoxic T cells in immunofluorescence double staining experiments. RESULTS: 94% (n = 98/104) of the patients with CRC expressed TNF-α. High TNF-α expression was significantly associated with positive lymph node stage and recurrence of the tumor. Multivariate analysis revealed high TNF-α expression as an independent prognostic factor. Immunohistochemistry was correlated with RT-PCR results (т = 0.794). Immunofluorescence double staining experiments revealed increased TNFR-1 expression by CD8+ cells. CONCLUSIONS: TNF-α expression by tumor cells may be an efficient immunological escape mechanism by inflammation-enhanced metastases and probably by induction of apoptosis in tumor-infiltrating CD8+ immune cells resulting in a down regulation of the tumoral immune response. Our data support the role of tumor-derived TNF-α expression as an important promoter of tumoral immune escape mechanisms and malignant progression, and suggest that analysis on either protein (immunohistochemistry) or RNA level (RT-PCR) can be used effectively in this respect. Targeting TNF-α may be a promising option, especially in cases with high TNF-α expression and positive lymph node metastases.


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Lymph Nodes/pathology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Aged , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/pathology , Colorectal Neoplasms/genetics , Disease Progression , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunohistochemistry , Lymph Nodes/drug effects , Lymph Nodes/metabolism , Lymphatic Metastasis/pathology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/pathology , Male , Middle Aged , Observer Variation , Prognosis , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recurrence , Survival Analysis , Tumor Necrosis Factor-alpha/genetics
8.
Anal Cell Pathol (Amst) ; 33(3): 151-63, 2010.
Article in English | MEDLINE | ID: mdl-20978325

ABSTRACT

INTRODUCTION: The progressive growth of malignancies is accompanied by a decline in the immune response through mechanisms which are poorly understood. Apoptosis and induction of inflammation by tumor released cytokines as tumor escape mechanisms have been proposed to play an important role in colorectal carcinogenesis. METHODS: Expression of Tumor necrosis factor-alpha (TNF-α) was analyzed in colorectal cancer specimen and the cancer cell line HT-29 by immunohistochemistry and RT-PCR. TNF-α expression on protein and mRNA level were correlated with clinical characteristics and impact on survival. TNFR-1 was co-labelled with TNF-α and CD8+ cytotoxic T cells in immunofluorescence double staining experiments. RESULTS: 94% (n=98/104) of the patients with CRC expressed TNF-α. High TNF-α expression was significantly associated with positive lymph node stage and recurrence of the tumor. Multivariate analysis revealed high TNF-α expression as an independent prognostic factor. Immunohistochemistry was correlated with RT-PCR results (τ=0.794). Immunofluorescence double staining experiments revealed increased TNFR-1 expression by CD8+ cells. CONCLUSIONS: TNF-α expression by tumor cells may be an efficient immunological escape mechanism by inflammation-enhanced metastases and probably by induction of apoptosis in tumor-infiltrating CD8+ immune cells resulting in a down regulation of the tumoral immune response. Our data support the role of tumor-derived TNF-α expression as an important promoter of tumoral immune escape mechanisms and malignant progression, and suggest that analysis on either protein (immunohistochemistry) or RNA level (RT-PCR) can be used effectively in this respect. Targeting TNF-α may be a promising option, especially in cases with high TNF-α expression and positive lymph node metastases.


Subject(s)
Colorectal Neoplasms/metabolism , Lymphatic Metastasis/physiopathology , Neoplasm Recurrence, Local/physiopathology , Tumor Necrosis Factor-alpha/metabolism , Colorectal Neoplasms/genetics , HT29 Cells , Humans , Immunohistochemistry , Neoplasm Recurrence, Local/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/genetics
10.
Cancer Biomark ; 7(6): 285-94, 2010.
Article in English | MEDLINE | ID: mdl-21694467

ABSTRACT

BACKGROUND: Esophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barrett's esophagus (BE) regarded as precancerous lesion. Glucocorticoid-induced TNFR family-related Receptor (GITR)-mediated inflammation of tumor infiltrating leucocytes (TILs) in the tumor microenvironment might play a role during the multistep carcinogenetic process as either tumor promoting factor according to an inflammatory microenvironment or as a feature of anti-tumor activity. METHODS: Immunohistochemical analysis of GITR expression was analyzed in esophageal cancer (n=70: 41 EAC with BE, 19 EAC without BE, and n=10 esophageal squamous-cell carcinomas, ESCC), the adenocarcinoma cell line OE-33, and peripheral blood leucocytes (PBLs) of EAC patients, furthermore in biopsies of BE without intraepithelial neoplasia (IN) (n=18). Results were correlated with clinicopathological parameters and five-year survival rates. Immunohistochemical GITR expression results were confirmed on mRNA level (RT-PCR). RESULTS: Quantification showed a significant increase of 25% GITR positive TILs in EAC with BE (p< 0.05) compared to 13% in adjacent BE, 24% in EAC without BE, 14% in ESCC, and 1% in BE without IN. High GITR levels were not significantly associated with clinicopathologic features which may predict worse clinical outcome and had no impact on survival (p= 0.7878). Increased GITR expression of peripheral blood leucocytes (PBLs) in EAC patients was shown on protein level (32%) and confirmed by RT-PCR (3.7-fold difference compared to normal tissue). CONCLUSIONS: This study provides for the first time evidence that GITR expression of TILs is associated in the pathogenesis of Barrett's esophagus. Our findings suggest that GITR-expression of TILs is associated with cancer progression. Its role as either tumor promoting factor %according to an in the inflammatory microenvironment or as a feature of anti-tumor activity and promising target for molecular therapies needs to be substantiated in further investigations.


Subject(s)
Adenocarcinoma/metabolism , Barrett Esophagus/metabolism , Esophageal Neoplasms/metabolism , Glucocorticoid-Induced TNFR-Related Protein/biosynthesis , Leukocytes/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Aged , Barrett Esophagus/genetics , Barrett Esophagus/pathology , Cell Line, Tumor , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Glucocorticoid-Induced TNFR-Related Protein/genetics , Humans , Immunohistochemistry , Leukocytes/pathology , Male , Mucous Membrane/metabolism , Mucous Membrane/pathology , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis
11.
Protoplasma ; 226(3-4): 137-46, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16333572

ABSTRACT

The determination of protein-protein interactions is becoming more and more important in the molecular analysis of signal transduction chains. To this purpose the application of a manageable and simple assay in an appropriate biological system is of major concern. Bimolecular fluorescence complementation (BiFC) is a novel method to analyze protein-protein interactions in vivo. The assay is based on the observation that N- and C-terminal subfragments of the yellow-fluorescent protein (YFP) can only reconstitute a functional fluorophore when they are brought into tight contact. Thus, proteins can be fused to the YFP subfragments and the interaction of the fusion proteins can be monitored by epifluorescence microscopy. Pairs of interacting proteins were tested after transient cotransfection in etiolated mustard seedlings, which is a well characterized plant model system for light signal transduction. BiFC could be demonstrated with the F-box protein EID1 (empfindlicher im dunkelroten Licht 1) and the Arabidopsis S-phase kinase-related protein 1 (ASK1). The interaction of both proteins was specific and strictly dependent on the presence of an intact F-box domain. Our studies also demonstrate that etiolated mustard seedlings provide a versatile transient assay system to study light-induced subcellular localization events.


Subject(s)
F-Box Proteins/metabolism , Light , Plants/metabolism , Base Sequence , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Luminescent Proteins , Molecular Sequence Data , Mustard Plant/genetics , Petroselinum/genetics , Phytochrome A/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified , Plasmids , Protoplasts/metabolism , Recombinant Fusion Proteins/metabolism , Seedlings/genetics , Signal Transduction , Spectrometry, Fluorescence/methods , Transfection , Two-Hybrid System Techniques
12.
J Inherit Metab Dis ; 28(5): 743-57, 2005.
Article in English | MEDLINE | ID: mdl-16151906

ABSTRACT

Glycosaminoglycans are accumulated in both mucopolysaccharidoses (MPS) and mucolipidoses (ML). MPS I, II, III and VII and ML II and ML III patients cannot properly degrade heparan sulphate (HS). In spite of the importance of HS storage in the metabolic pathway in these diseases, blood and urine HS levels have not been determined systematically using a simple and economical method. Using a new ELISA method using anti-HS antibodies, HS concentrations in blood and urine were determined in MPS and ML II and ML III patients. HS concentrations were determined in 156 plasma samples from MPS I (n = 23), MPS II (n = 26), MPS III (n = 24), MPS IV (n = 62), MPS VI (n = 5), MPS VII (n = 5), ML II (n = 8) and ML III (n = 3), and 205 urine samples from MPS I (n = 33), MPS II (n = 33), MPS III (n = 30), MPS IV (n = 82), MPS VI (n = 7), MPS VII (n = 9), ML II (n = 8) and ML III (n = 3). The ELISA method used monoclonal antibodies against HS. MPS I, II, III and VII and ML II and III patients had significant elevation in plasma HS, compared to the age-matched controls (p < 0.0001). Eighty-three out of 89 (93.3%) of individual values in the above MPS types and ML were above the mean +2SD of the controls. In urine samples, 75% of individual values in patients with those types were above the mean +2SD of the controls. In contrast to the previous understanding of the HS metabolic pathway, plasma HS levels in all five MPS VI and 15% of MPS IV patients were elevated above the mean +2SD of the controls. These findings suggest that HS concentration determined by ELISA, especially in plasma, could be a helpful marker for detection of the most severe MPS I, II, III, VI and VII and ML II, distinguishing them from normal populations.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Heparitin Sulfate/chemistry , Mucolipidoses/diagnosis , Mucopolysaccharidoses/diagnosis , Adolescent , Biomarkers/metabolism , Chemistry, Clinical/methods , Child , Child, Preschool , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Glycosaminoglycans/chemistry , Heparin/chemistry , Heparitin Sulfate/blood , Heparitin Sulfate/urine , Humans , Infant , Infant, Newborn , Mucolipidoses/blood , Mucolipidoses/urine , Mucopolysaccharidoses/blood , Mucopolysaccharidoses/urine
13.
J Inherit Metab Dis ; 28(2): 187-202, 2005.
Article in English | MEDLINE | ID: mdl-15877208

ABSTRACT

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.


Subject(s)
Keratan Sulfate/blood , Keratan Sulfate/urine , Mucolipidoses/metabolism , Mucopolysaccharidoses/metabolism , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Antibody Specificity , Biomarkers , Child , Child, Preschool , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Infant , Infant, Newborn , Keratan Sulfate/immunology , Middle Aged , Mucolipidoses/diagnosis , Mucopolysaccharidoses/diagnosis , Sensitivity and Specificity
15.
Mol Genet Genomics ; 265(6): 964-76, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11523788

ABSTRACT

The common plant regulatory factors (CPRFs) from parsley are transcription factors with a basic-leucine-zipper motif that bind to cis-regulatory elements frequently found in promoters of light-regulated genes. Proposed to function in concert with members of other transcription factor families, CPRFs regulate the transcriptional activity of many target genes. Here, we report that, in contrast to CPRF2, which operates as a transcriptional activator, CPRF1 functions as repressor in vivo. Two-hybrid screens using CPRF1 and CPRF2 as "baits" resulted in the isolation of four novel parsley proteins which interact with either CPRF1 or CPRF2 in vivo. Three of these factors represent new parsley bZIP factors, designated CPRF5-CPRF7, whereas the fourth, named CPRF1-interacting protein (CIP), shows no homology to any other known protein. CPRF5 and CIP specifically interact with CPRF1, whilst CPRF6 and CPRF7 exclusively form heterodimers with CPRF2. CPRF5, CPRF6 and CPRF7 are transcription factors that exhibit sequence-specific DNA-binding as well as transactivation abilities, whereas the function of CIP remains elusive. The newly isolated CPRFs and CIP are constitutively localized in the nucleus in parsley protoplasts. Furthermore, mRNA accumulation studies revealed that the expression of these novel bZIP genes and CIP is not altered by exposure to light. We discuss the possible roles of the newly identified proteins in CPRF1- and CPRF2-dependent target gene expression.


Subject(s)
Apiaceae/genetics , Apiaceae/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Apiaceae/radiation effects , Basic-Leucine Zipper Transcription Factors , Cells, Cultured , Cloning, Molecular , Conserved Sequence , DNA-Binding Proteins/chemistry , G-Box Binding Factors , Leucine Zippers , Light , Molecular Sequence Data , Plant Proteins/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transcription Factors/metabolism , Ultraviolet Rays
16.
J Cell Sci ; 114(Pt 3): 475-80, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11171317

ABSTRACT

Plants monitor changes in the ambient light environment by highly specialised photoreceptors, which include the red/far-red photoreversible phytochromes, the blue-light-absorbing cryptochromes and phototropin and the so-far-unidentified UVB photoreceptor(s). Light easily penetrates plant organs/tissues and reaches even the subcellular compartments of various cell types. Therefore, it is not surprising that the determination of the intracellular localisation of photoreceptors has been, for many years, a major, and often controversial, subject of plant photobiology and cell biology research. Phototropin, one of the blue-light photoreceptors of higher plants, controls phototropism by monitoring the direction of light, and it is localised in or at the plasmalemma. In contrast, the subcellular localisation of phytochromes changes dynamically and exhibits a very complex pattern. These photoreceptors are localised in the cytosol in dark- grown tissues. Irradiation, however, induces import of phytochromes into the nucleus. The import occurs in a light-quality- and light-quantity-dependent fashion and, as such, seems to be unique to higher plants. Light-induced accumulation of phytochromes in the nuclei correlates well with various physiological responses mediated by these photoreceptors. These observations indicate that light-dependent intracellular redistribution of phytochrome photoreceptors is one of the major regulatory steps in photomorphogenesis.


Subject(s)
Light , Photosynthetic Reaction Center Complex Proteins/metabolism , Signal Transduction/radiation effects , Darkness , Plants/metabolism , Plants/ultrastructure , Protein Transport
17.
Plant J ; 22(2): 125-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792828

ABSTRACT

Phytochromes (phy) are a family of photoreceptors that control various aspects of light-dependent plant development. Phytochrome A (phyA) is responsible for the very low fluence response (VLFR) under inductive light conditions and for the high irradiance response (HIR) under continuous far-red light. We have recently shown that nuclear import of rice phyA:GFP is regulated by VLFR in transgenic tobacco. The import is preceded by very fast, light-induced formation of sequestered areas of phyA:GFP in the cytosol. Here we report that expression of the Arabidopsis phyA:GFP fusion protein in phyA-deficient Arabidopsis plants complements the mutant phenotype. In these transgenic Arabidopsis lines, both light-dependent cytosolic formation of sequestered areas of the phyA:GFP as well as VLFR or HIR-mediated nuclear import of the fusion protein was observed. By contrast, light-dependent nuclear import of the same fusion protein was induced only by continuous far-red light (HIR) but not by pulses of far-red light (VLFR) in transgenic tobacco. These results demonstrate that photoregulation of intracellular partitioning of the Arabidopsis phyA:GFP differs significantly in different genetic backgrounds.


Subject(s)
Arabidopsis/physiology , Cell Nucleus/metabolism , Light , Nicotiana/physiology , Phytochrome/metabolism , Plants, Toxic , Recombinant Fusion Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins , Cell Compartmentation/physiology , Cell Nucleus/physiology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Microscopy, Fluorescence , Phytochrome/genetics , Phytochrome/physiology , Phytochrome A , Plants, Genetically Modified , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Nicotiana/metabolism
18.
Plant J ; 22(2): 135-45, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792829

ABSTRACT

Photomorphogenesis of higher plants is regulated by photoreceptors including the red/far-red light-absorbing phytochromes, blue-UV/A sensing cryptochromes and as yet uncharacterized UV/B receptors. Specific phototransduction pathways that are controlled by either individual or interacting photoreceptors mediate regulation. Phytochrome B (phyB) is the major red light-sensing photoreceptor. Phototransduction mediated by this light sensor has been shown to include light-dependent nuclear import and interaction of phyB with transcription factor-like proteins in the nucleus. Here we report that nuclear import of phyB and physiological responses regulated by this photoreceptor exhibit very similar wavelength- and fluence rate-dependence. Nuclear import of phyB is insensitive to single red, blue and far-red light pulses. It is induced by continuous red light and to a lesser extent by continuous blue light, whereas far-red light is completely ineffective. The data presented indicate that light-dependent partitioning of phyB exhibits features characteristic of blue light responsiveness amplification, a phenomenon that is thought to be mediated by interaction of phyB with CRY1.


Subject(s)
Cell Nucleus/metabolism , Light , Nicotiana/physiology , Photoreceptor Cells , Phytochrome/metabolism , Plants, Toxic , Recombinant Fusion Proteins/metabolism , Transcription Factors , Cell Compartmentation , Cell Nucleus/physiology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Microscopy, Fluorescence , Phytochrome/genetics , Phytochrome/physiology , Phytochrome B , Plants , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/physiology , Subcellular Fractions/metabolism , Nicotiana/metabolism
19.
Exp Neurol ; 161(1): 174-82, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10683283

ABSTRACT

Ribosomes are integral constitutens of the protein synthesis machinery. Polymerase I (POL I) is located in the nucleolus and transcribes the large ribosomal genes. POL I activity is decreased in ischemia but nothing is known so far on POL I in perinatal asphyxia. We investigated the involvement of POL I in a well-documented model of graded systemic asphyxia at the level of activity, mRNA, protein, and morphology. Caeserean section was performed at the 21st day of gestation. Rat pups still in the uterus horns were immerged in a water bath for asphyctic periods from 5-20 min. Brain was taken for measurement of pH, nuclear POL I activity, and mRNA steady state, and protein levels of RPA40, an essential subunit of POL I and III. Silver staining and transmission electron microscopy with morphometry when appropriate were used to examine the nucleolus. Brain pH and nuclear POL I activity decreased with the length of the asphyctic period while POL-I mRNA and protein levels were unchanged. Accompanying the decrease in brain pH we found significant changes of nucleolar structure in the course of perinatal asphyxia at the light and electron microscopic level. As early as ten min following the asphyctic insult, morphological disintegration of the nucleolus was observed. The changes became more dramatic with longer duration of perinatal asphyxia. We conclude that severe acidosis may be responsible for decreased POL activity and for disintegration of nucleoli in neurons. This condition may lower the ribosome content in neonatal neurons and impair protein synthesis.


Subject(s)
Asphyxia Neonatorum/metabolism , Cell Nucleolus/enzymology , Frontal Lobe/enzymology , RNA Polymerase I/metabolism , Animals , Animals, Newborn , Blotting, Northern , Cell Nucleolus/ultrastructure , Disease Models, Animal , Female , Gene Expression Regulation, Enzymologic , Humans , Hydrogen-Ion Concentration , Infant, Newborn , Microscopy, Electron , Pregnancy , RNA Polymerase I/analysis , RNA Polymerase I/genetics , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Silver Staining , Transcription, Genetic/physiology
20.
Semin Cell Dev Biol ; 11(6): 505-10, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11145880

ABSTRACT

Phytochromes in harmony with blue light photoreceptors play a major role in controlling plant growth and development from germination to seed maturation. Light absorption by phytochromes triggers a signaling cascade, phototransduction, which culminates in regulated gene expression. A major regulatory step at the cellular level, which affects specificities of light-induced physiological responses, seems to be the light-quality and light-quantity dependent nuclear import of the phytochromes themselves. The correlations found between the nuclear import of phytochromes (phyA and phyB) and various physiological responses regulated by these photoreceptors provides strong support for this hypothesis.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Photoreceptor Cells , Phytochrome/metabolism , Plants/metabolism , Transcription Factors , Active Transport, Cell Nucleus , Darkness , Light , Phytochrome A , Phytochrome B , Plant Cells , Plants/enzymology , Protein Kinases/metabolism , Signal Transduction
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