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1.
Ann ICRP ; 44(1): 9-127, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26116562

ABSTRACT

The objective of this publication is to provide guidance on radiological protection in the new technology of cone beam computed tomography (CBCT). Publications 87 and 102 dealt with patient dose management in computed tomography (CT) and multi-detector CT. The new applications of CBCT and the associated radiological protection issues are substantially different from those of conventional CT. The perception that CBCT involves lower doses was only true in initial applications. CBCT is now used widely by specialists who have little or no training in radiological protection. This publication provides recommendations on radiation dose management directed at different stakeholders, and covers principles of radiological protection, training, and quality assurance aspects. Advice on appropriate use of CBCT needs to be made widely available. Advice on optimisation of protection when using CBCT equipment needs to be strengthened, particularly with respect to the use of newer features of the equipment. Manufacturers should standardise radiation dose displays on CBCT equipment to assist users in optimisation of protection and comparisons of performance. Additional challenges to radiological protection are introduced when CBCT-capable equipment is used for both fluoroscopy and tomography during the same procedure. Standardised methods need to be established for tracking and reporting of patient radiation doses from these procedures. The recommendations provided in this publication may evolve in the future as CBCT equipment and applications evolve. As with previous ICRP publications, the Commission hopes that imaging professionals, medical physicists, and manufacturers will use the guidelines and recommendations provided in this publication for implementation of the Commission's principle of optimisation of protection of patients and medical workers, with the objective of keeping exposures as low as reasonably achievable, taking into account economic and societal factors, and consistent with achieving the necessary medical outcomes.


Subject(s)
Cone-Beam Computed Tomography/standards , Environmental Exposure , Radiation Dosage , Radiation Monitoring , Radiation Protection/standards , Humans , Occupational Exposure , Radiation Monitoring/standards
2.
Lab Anim ; 48(2): 105-13, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24468712

ABSTRACT

Cardiac perfusion studies using computed tomography are a common tool in clinical practice. Recent technical advances and the availability of dedicated small animal scanners allow the transfer of these techniques to the preclinical sector in general and to mouse models of cardiac diseases in particular. This necessitates new requirements for contrast injection techniques as a rapid transport of contrast media from the intravenous access to the animal heart. Clinical contrast agents containing high iodine concentrations are used within small animal studies although they exhibit a high viscosity which might limit their transport within the vasculature. The authors provide a comparison of the transport of contrast media following an injection into the lateral tail vein and an injection into the retrobulbar sinus and discuss the anatomy involved. The temporal evolution of a contrast bolus and its in vivo distribution is visualized. It is demonstrated that injecting contrast agents into the lateral tail vein of mice results in a retrograde blood flow to the liver veins and therefore does not deliver a detectable contrast bolus to the heart, and thus it cannot be used for cardiac perfusion studies. By contrast, boli injected into the retrobulbar sinus are rapidly transported to the heart and provide ventricular contrast enabling perfusion studies similar to those in human patients. The results demonstrate that an injection into the retrobulbar sinus is superior to an injection into the lateral tail vein for the delivery of contrast boli to the animal heart, while all drawbacks of an injection into the lateral tail vein are overcome.


Subject(s)
Contrast Media/metabolism , Injections, Intravenous/methods , Iohexol/analogs & derivatives , Myocardial Perfusion Imaging/methods , X-Ray Microtomography/methods , Animals , Blood Circulation , Contrast Media/administration & dosage , Iohexol/administration & dosage , Iohexol/metabolism , Mice , Mice, Inbred C57BL
4.
Phys Med Biol ; 58(10): 3283-300, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23615179

ABSTRACT

Image-guided interventions are an increasingly important part of clinical minimally invasive procedures. However, up to now they cannot be performed under 4D (3D + time) guidance due to the exceedingly high x-ray dose. In this work we investigate the applicability of compressed sensing reconstructions for highly undersampled CT datasets combined with the incorporation of prior images in order to yield low dose 4D intervention guidance. We present a new reconstruction scheme prior image dynamic interventional CT (PrIDICT) that accounts for specific image features in intervention guidance and compare it to PICCS and ASD-POCS. The optimal parameters for the dose per projection and the numbers of projections per reconstruction are determined in phantom simulations and measurements. In vivo experiments in six pigs are performed in a cone-beam CT; measured doses are compared to current gold-standard intervention guidance represented by a clinical fluoroscopy system. Phantom studies show maximum image quality for identical overall doses in the range of 14 to 21 projections per reconstruction. In vivo studies reveal that interventional materials can be followed in 4D visualization and that PrIDICT, compared to PICCS and ASD-POCS, shows superior reconstruction results and fewer artifacts in the periphery with dose in the order of biplane fluoroscopy. These results suggest that 4D intervention guidance can be realized with today's flat detector and gantry systems using the herein presented reconstruction scheme.


Subject(s)
Four-Dimensional Computed Tomography/methods , Radiology, Interventional/methods , Algorithms , Animals , Phantoms, Imaging , Radiation Dosage , Swine
5.
Eur Respir J ; 38(5): 1060-70, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21478215

ABSTRACT

The onset and spontaneous development of cystic fibrosis (CF) lung disease remain poorly understood. In the present study, we used volumetric computed tomography (VCT) as a new method for longitudinal in vivo monitoring of early lesions and disease progression in CF-like lung disease in ß-epithelial Na(+) channel (ENaC)-transgenic (TG) mice. Using a VCT scanner prototype (80 kV, 50 mA·s, scan time 19 s and spatial resolution 200 µm), ßENaC-TG mice and wild-type (WT) littermates were examined longitudinally at 10 time-points from neonatal to adult ages, and VCT images were assessed by qualitative and quantitative morphological parameters. We demonstrate that VCT detected early-onset airway mucus obstruction, diffuse infiltrates, atelectasis and air trapping as characteristic abnormalities in ßENaC-TG mice. Furthermore, we show that early tracheal mucus obstruction predicted mortality in ßENaC-TG mice and that the density of lung parenchyma was significantly reduced at all time-points in ßENaC-TG compared with WT mice (median ± sem -558 ± 8 HU in WT versus -686 ± 16 HU in ßENaC-TG at 6 weeks of age; p < 0.005). Our study demonstrates that VCT is a sensitive, noninvasive technique for early detection and longitudinal monitoring of morphological abnormalities of CF-like lung disease in mice, and may thus provide a useful tool for pre-clinical in vivo evaluation of novel treatment strategies for CF.


Subject(s)
Cone-Beam Computed Tomography , Cystic Fibrosis/diagnostic imaging , Lung/diagnostic imaging , Airway Obstruction/diagnostic imaging , Animals , Bronchography , Cystic Fibrosis/complications , Cystic Fibrosis/pathology , Cystic Fibrosis/physiopathology , Disease Progression , Lung/pathology , Mice , Mice, Transgenic , Mucus , Pulmonary Emphysema/diagnostic imaging , Pulmonary Emphysema/etiology , Pulmonary Emphysema/physiopathology , Trachea/diagnostic imaging
6.
Phys Med Biol ; 55(7): 2069-85, 2010 Apr 07.
Article in English | MEDLINE | ID: mdl-20299735

ABSTRACT

A fully automated, intrinsic gating algorithm for small animal cone-beam CT is described and evaluated. A parameter representing the organ motion, derived from the raw projection images, is used for both cardiac and respiratory gating. The proposed algorithm makes it possible to reconstruct motion-corrected still images as well as to generate four-dimensional (4D) datasets representing the cardiac and pulmonary anatomy of free-breathing animals without the use of electrocardiogram (ECG) or respiratory sensors. Variation analysis of projections from several rotations is used to place a region of interest (ROI) on the diaphragm. The ROI is cranially extended to include the heart. The centre of mass (COM) variation within this ROI, the filtered frequency response and the local maxima are used to derive a binary motion-gating parameter for phase-sensitive gated reconstruction. This algorithm was implemented on a flat-panel-based cone-beam CT scanner and evaluated using a moving phantom and animal scans (seven rats and eight mice). Volumes were determined using a semiautomatic segmentation. In all cases robust gating signals could be obtained. The maximum volume error in phantom studies was less than 6%. By utilizing extrinsic gating via externally placed cardiac and respiratory sensors, the functional parameters (e.g. cardiac ejection fraction) and image quality were equivalent to this current gold standard. This algorithm obviates the necessity of both gating hardware and user interaction. The simplicity of the proposed algorithm enables adoption in a wide range of small animal cone-beam CT scanners.


Subject(s)
Algorithms , Cardiac-Gated Imaging Techniques/veterinary , Cone-Beam Computed Tomography/veterinary , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Pattern Recognition, Automated/methods , Respiratory-Gated Imaging Techniques/veterinary , Animals , Image Enhancement/methods , Reproducibility of Results , Sensitivity and Specificity
7.
AJNR Am J Neuroradiol ; 30(7): 1419-24, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19369606

ABSTRACT

BACKGROUND AND PURPOSE: A recent development in radiology is the use of flat panel detectors in CT to obtain higher-resolution images. This technique is known as flat panel volume CT (fpVCT). We sought to compare the image quality and diagnostic value of 2 different flat panel detector-equipped scanners: one is a prototype fpVCT scanner, and the other is a so-called flat panel digital volume tomography (fpDVT) scanner, which is routinely used in clinical setup with current state-of-the-art multisection CT (MSCT) scanners. MATERIALS AND METHODS: Five explanted temporal bones and 2 whole-head cadaveric specimens were scanned with fpVCT, fpDVT, and MSCT scanners. The image series were blindly evaluated by 3 trained observers who rated 38 anatomic structures with regard to their delineation/appearance. RESULTS: Although the image quality obtained with fpVCT and fpDVT was rated significantly better compared with MSCT on isolated temporal bones, the differences were not significant when whole cadaveric heads were scanned. CONCLUSIONS: Theoretic and practical advantages exist for flat panel detector-equipped scanners, including improved image quality. However, when imaging whole cadaveric heads, no significant difference could be demonstrated between them and standard-of-care MSCT.


Subject(s)
Imaging, Three-Dimensional/instrumentation , Radiographic Image Enhancement/instrumentation , Temporal Bone/diagnostic imaging , Tomography, X-Ray Computed/instrumentation , X-Ray Intensifying Screens , Equipment Design , Equipment Failure Analysis , Humans , Reproducibility of Results , Sensitivity and Specificity
8.
Laryngorhinootologie ; 87(1): 18-22, 2008 Jan.
Article in German | MEDLINE | ID: mdl-17713878

ABSTRACT

BACKGROUND: The accuracy of navigation systems can be improved significantly by using high-resolution flat panel-based Volume Computed Tomography (fpVCT) so that new surgical therapeutic concepts become feasible. A navigation-guided minimally-invasive cochleostomy places highest requirements on the accuracy of intraoperative navigation. METHODS: A flat-panel Volume Computed Tomograph (fpVCT) was used to scan four human temporal bones. The isometric voxel size was 200 microm. The preoperative planning was used to define an optimized drilling channel from the mastoid surface to the round window niche and the scala tympani providing a safety margin to critical anatomical structures such as facial nerve, chorda tympani, sigmoid sinus and posterior wall of auditory canal. The canal was drilled hand-operated with a navigated drill following the previously planned trajectory. Afterwards the drilled canal was imaged by fpVCT. Conventional dissection including mastoidectomy and posterior tympanotomy assured correct localization of the cochleostomy. RESULTS: Path planning took an average of 54 minutes (range 35-85 minutes). Installation took an average of 16 minutes (range 14-19 minutes). The drilling procedure itself took an average of 7.75 min (range 5-12 minutes.) The RMSE-values varied between 0.1 and 0.2 mm (Table 1). All four specimens showed a cochleostomy located at the scala tympani anterior inferior to the round window. The chorda tympani was damaged in one specimen--this was preoperatively planned as a narrow facial recess was encountered. The time needed for planning and system-installation could be reduced continuously. CONCLUSIONS: This feasibility study demonstrates that using current image-guided surgery technology in combination with fpVCT allows drilling of a minimally invasive channel to the cochlea with loco typico cochleostomy. The necessary accuracy of intraoperative navigation can be achieved by use of fpVCT (technical accuracy between 0.1 and 0.2 mm). Our results demonstrate the feasibility of a navigation-guided minimally-invasive cochleostomy loco typico. While we are enthused by this preliminary work, we recognize the barriers which exist in translation to clinical application. These include surgical issues (e.g. control of unexpected bleeding) and electrode issues (e.g. development of insertion tools).


Subject(s)
Cochlear Implantation/instrumentation , Cone-Beam Computed Tomography/instrumentation , Minimally Invasive Surgical Procedures/instrumentation , Petrous Bone/surgery , Surgery, Computer-Assisted/instrumentation , Calibration , Electrodes, Implanted , Equipment Design , Humans , Software
9.
Rofo ; 179(12): 1236-42, 2007 Dec.
Article in German | MEDLINE | ID: mdl-18004691

ABSTRACT

Modern imaging modalities such as computed tomography (CT) and magnetic resonance imaging (MRI) allow high-resolution imaging of the abdomen. Modern scanners made high temporal as well as high spatial resolution available. Therapeutic approaches to the treatment of renal cell carcinoma have been improved over the recent years. Besides conventional and open laparoscopic tumor nephrectomy and nephron sparing, surgical approaches such as local tumor cryotherapy and radiofrequency ablation (RF) are ablative modalities and are used increasingly. Improved anesthesiological methods and new surgical approaches also allow curative treatment in extended tumors. Prerequisites for preoperative imaging modalities include visualization of the kidney tumor as well as its staging. Tumor-related infiltration of the renal pelvis or invasion of the perinephric fat and the renal hilus has to be excluded prior to nephron sparing surgery. In cases with extended tumors with infiltration of the inferior vena cava, it is necessary to visualize the exact extension of the tumor growth towards the right atrium in the vena cava. The radiologist should be informed about the diagnostic possibilities and limitations of the imaging modalities of CT and MRI in order to support the urologist in the planning and performance of surgical therapeutical approaches.


Subject(s)
Carcinoma, Renal Cell/diagnosis , Carcinoma, Renal Cell/surgery , Kidney Neoplasms/diagnosis , Kidney Neoplasms/surgery , Magnetic Resonance Imaging , Tomography, X-Ray Computed , Carcinoma, Renal Cell/diagnostic imaging , Carcinoma, Renal Cell/pathology , Catheter Ablation , Cryotherapy , Humans , Kidney Neoplasms/diagnostic imaging , Kidney Neoplasms/pathology , Laparoscopy , Magnetic Resonance Imaging/methods , Neoplasm Invasiveness , Neoplasm Staging/methods , Nephrectomy/methods , Nephrons , Tomography, X-Ray Computed/methods
10.
Dentomaxillofac Radiol ; 36(6): 317-27, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17699701

ABSTRACT

OBJECTIVES: To assess the feasibility of flat-panel detector based volumetric CT (fpVCT) scanning of the whole human skull base and maxillofacial region, which has thus far only been demonstrated on small, excised specimens. Flat-panel detectors offer more favourable imaging properties than image intensifiers. It is therefore likely that they will replace them in cone-beam CT scanners that are currently used to scan parts of the skull base and maxillofacial region. Furthermore, the resolution of current CT imaging limits diagnosis, surgical planning and intraoperative navigation within these regions. fpVCT might overcome these limitations because it offers higher resolution of high contrast structures than current CT. METHODS: Three embalmed cadaver heads were scanned in two scanners: an experimental fpVCT that offers a scan field large enough for a whole human head, and in a current multislice CT (MSCT). 28 structures were compared. RESULTS: Both scanners produced bone images of diagnostic quality. Small high contrast structures such as parts of the ossicular chain and thin bony laminas were better delineated in fpVCT than in MSCT. fpVCT of maxillofacial region and skull base was rated superior to MSCT (P=0.002) as found in this limited, experimental study. CONCLUSIONS: High spatial resolution fpVCT scanning of both regions in a whole human head is feasible and might be slightly superior to MSCT. fpVCT could improve diagnostic accuracy in selected cases, as well as surgical planning and intraoperative navigation accuracy.


Subject(s)
Facial Bones/diagnostic imaging , Skull Base/diagnostic imaging , Tomography Scanners, X-Ray Computed , Tomography, X-Ray Computed/methods , Aged , Cadaver , Ear, Inner/diagnostic imaging , Equipment Design , Feasibility Studies , Humans , Image Processing, Computer-Assisted/instrumentation , Image Processing, Computer-Assisted/methods , Mandibular Nerve/diagnostic imaging , Maxillary Nerve/diagnostic imaging , Middle Aged , Radiographic Image Enhancement/instrumentation , Radiographic Image Enhancement/methods , Temporal Bone/diagnostic imaging , Tomography, X-Ray Computed/instrumentation , Tooth/diagnostic imaging
11.
Laryngorhinootologie ; 85(10): 755-9, 2006 Oct.
Article in German | MEDLINE | ID: mdl-17031763

ABSTRACT

A 53-year-old patient with myotonic dystrophy presented to our clinic with progressive bilateral hearing loss. The ENT status and particularly the otological examination were without pathological signs. Pure tone audiograms showed a bilateral moderate to severe sensorineural hearing loss. Routinely performed computed tomography of the temporal bones revealed the rare picture of exostosis of the internal auditory canals and the medial surface of the petrous bones. To our knowledge, this is the first report describing exostosis of the internal auditory canal in a patient with myotonic dystrophy, although at present it remains unclear in how far there is a causal connection between these two pathologies.


Subject(s)
Ear Canal , Exostoses , Hearing Loss, Sensorineural/etiology , Myotonic Dystrophy/complications , Petrous Bone , Audiometry, Pure-Tone , Exostoses/complications , Exostoses/diagnostic imaging , Hearing Loss, Sensorineural/diagnosis , Humans , Male , Middle Aged , Petrous Bone/diagnostic imaging , Temporal Bone/diagnostic imaging , Tomography, X-Ray Computed
12.
Dentomaxillofac Radiol ; 35(4): 227-31, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16798916

ABSTRACT

OBJECTIVES: Three-dimensional computed tomography (3D-CT) of facial fractures has been reported as beneficial using surface (SR) and volume rendering (VR). There are controversial statements concerning the preferable algorithm. The purpose of this study was to evaluate and compare SR and VR for clinical 3D-CT in facial fractures on an experimental basis. METHODS: Multislice CT was obtained in 22 patients with facial fractures using two data acquisition protocols. Five SR and VR post-processing protocols were applied. Five assessors independently evaluated the quality of visualization of the fracture gap and dislocated fragments as well as the overall image quality using a five-point rating scale. The potential benefit of the 3D-images for radiological diagnosis and presentation was evaluated. The influence of the data acquisition protocol was analysed. RESULTS: SR in general achieved better evaluation scores than VR at corresponding thresholds. Variation of evaluation scores for all criteria was found for SR and VR depending on the segmentation threshold. Apart from the overall image quality no significant influence of the data acquisition technique was found for the evaluated criteria. CONCLUSIONS: SR provided sufficient and time efficient means for 3D-visualization of facial fractures in this study. No diagnostic benefit of VR over SR was found.


Subject(s)
Facial Bones/injuries , Imaging, Three-Dimensional/methods , Radiographic Image Enhancement/methods , Skull Fractures/diagnostic imaging , Tomography, Spiral Computed/methods , Adolescent , Adult , Algorithms , Analysis of Variance , Child , Child, Preschool , Facial Bones/diagnostic imaging , Female , Frontal Bone/diagnostic imaging , Frontal Bone/injuries , Humans , Infant , Male , Mandibular Fractures/diagnostic imaging , Maxillary Fractures/diagnostic imaging , Middle Aged , Nasal Bone/diagnostic imaging , Nasal Bone/injuries , Radiation Dosage , Retrospective Studies , Zygomatic Fractures/diagnostic imaging
14.
Laryngorhinootologie ; 83(7): 438-44, 2004 Jul.
Article in German | MEDLINE | ID: mdl-15257492

ABSTRACT

Post-processing of CT-data allows non-invasive 3D-Visualisation of the middle ear for diagnosis and surgical planning. In this study different post-processing techniques and the clinical application of a 3D-postprocessing algorithm in a large number of patients are presented. 20 normal patients, 6 dissected temporal bones and 213 patients with suspected middle ear pathology were examined using a low-dosage Multi-Slice CT protocol. Virtual endoscopic views of the middle ear and 3D-images of the ossicles were generated using a standardised algorithm. Evaluation of the image quality was performed. The virtual views of the dissected temporal bones were compared to real views. In 32 patients high-quality 3D-models of the individual anatomical structures were generated and displayed using different visualisation techniques. The standardised and evaluated method enabled visualisation of the normal middle ear anatomy. Assessment of different pathologies, especially malformation, trauma, implants and postoperative alterations, was facilitated. The high-quality 3D-models allowed precise imaging of the anatomical structures. 3D-Visualisation of the middle ear using CT-data is beneficial for radiological diagnosis and surgical planning in cases of complex middle ear pathology as a complementary examination technique.


Subject(s)
Ear Diseases/diagnostic imaging , Ear Ossicles/diagnostic imaging , Ear, Middle/diagnostic imaging , Image Enhancement , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Otoscopy , Tomography, Spiral Computed , User-Computer Interface , Adolescent , Adult , Aged , Ear Diseases/surgery , Ear Ossicles/abnormalities , Ear Ossicles/injuries , Ear Ossicles/surgery , Ear, Middle/abnormalities , Ear, Middle/injuries , Ear, Middle/surgery , Female , Humans , Male , Middle Aged , Petrous Bone/injuries , Sensitivity and Specificity , Skull Fractures/diagnostic imaging , Skull Fractures/surgery , Technology Assessment, Biomedical
15.
Neuroradiology ; 44(9): 783-90, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12221454

ABSTRACT

The 3D imaging of the middle ear facilitates better understanding of the patient's anatomy. Cross-sectional slices, however, often allow a more accurate evaluation of anatomical structures, as some detail may be lost through post-processing. In order to demonstrate the advantages of combining both approaches, we performed computed tomography (CT) imaging in two normal and 15 different pathological cases, and the 3D models were correlated to the cross-sectional CT slices. Reconstructed CT datasets were acquired by multi-slice CT. Post-processing was performed using the in-house software "3D Slicer", applying thresholding and manual segmentation. 3D models of the individual anatomical structures were generated and displayed in different colours. The display of relevant anatomical and pathological structures was evaluated in the greyscale 2D slices, 3D images, and the 2D slices showing the segmented 2D anatomy in different colours for each structure. Correlating 2D slices to the 3D models and virtual endoscopy helps to combine the advantages of each method. As generating 3D models can be extremely time-consuming, this approach can be a clinically applicable way of gaining a 3D understanding of the patient's anatomy by using models as a reference. Furthermore, it can help radiologists and otolaryngologists evaluating the 2D slices by adding the correct 3D information that would otherwise have to be mentally integrated. The method can be applied to radiological diagnosis, surgical planning, and especially, to teaching.


Subject(s)
Ear, Middle/diagnostic imaging , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Tomography, X-Ray Computed , Adolescent , Adult , Aged , Child , Child, Preschool , Cochlear Implants , Female , Humans , Male , Middle Aged
16.
Mayo Clin Proc ; 72(8): 749-56, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9276603

ABSTRACT

Asthma is a common, chronic inflammatory disorder of the airways associated with pronounced health and economic consequences. Consistent and effective education that promotes an active partnership with patients remains the cornerstone for managing asthma. Identification and control of asthma triggers, regular monitoring of lung function, and adequate pharmacologic therapy are three other critical components. In this article, we describe several practical considerations for developing a collaborative, multidisciplinary approach to asthma care that emphasizes patient education and strengthens the partnership between patients and health-care professionals.


Subject(s)
Asthma/therapy , Activities of Daily Living , Administration, Inhalation , Adrenal Cortex Hormones/administration & dosage , Adult , Asthma/drug therapy , Health Knowledge, Attitudes, Practice , Humans , Life Style , Middle Aged , Patient Care Planning , Patient Education as Topic , Physician's Role , Primary Health Care , Self Care
17.
Curr Genet ; 29(5): 474-81, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8625428

ABSTRACT

Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.


Subject(s)
Aspergillus/metabolism , Erwinia/enzymology , Gene Expression , Polysaccharide-Lyases/metabolism , Amino Acid Sequence , Aspergillus/genetics , Base Sequence , Erwinia/genetics , Molecular Sequence Data , Polysaccharide-Lyases/genetics , Protein Processing, Post-Translational , Protein Sorting Signals/genetics , Recombinant Fusion Proteins/metabolism , Species Specificity , Transformation, Genetic
18.
Microbiology (Reading) ; 141 ( Pt 4): 873-81, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7773390

ABSTRACT

Phytopathogenic Erwinia bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from Erwinia carotovora subsp. atroseptica established the organization of a 7.5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the E. chrysanthemi KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the pel genes in the Escherichia coli-pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture)-1 with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31% esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.


Subject(s)
Erwinia/enzymology , Isoenzymes/metabolism , Polysaccharide-Lyases/metabolism , Amino Acid Sequence , Base Sequence , DNA, Bacterial/genetics , Erwinia/genetics , Erwinia/pathogenicity , Genes, Bacterial , Isoenzymes/chemistry , Isoenzymes/genetics , Kinetics , Molecular Sequence Data , Molecular Structure , Pectins , Plant Diseases/microbiology , Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
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