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Breakpoints in alpha, beta, and satellite III DNA sequences of chromosome 9 result in a variety of pericentric inversions.

Human chromosome 9 with a pericentric inversion involving the qh region is considered normal. It has probably evolved through breakage and reunion and is retained through mendelian inheritance without any apparent phenotypic consequences. Fluorescent in situ hybridisation (FISH) technique using alpha, beta, and satellite III DNA probes showed that the breakpoints are variable and can be localised in the alpha or in the satellite III and beta DNA regions or both. Three types of inversions are proposed which appear similar by CBG banding: pericentric inversions with two alphoid, one beta, and one satellite III hybridisation signals were classified as type A. Type B were those with two beta, one alpha, and one satellite III hybridisation signals, while type C was complex, and most likely involved two inversions, since two separate hybridisation signals were detected in each of the alphoid, beta satellite, and satellite III DNA regions. Based on eight cases, type A is likely to be the most frequent, but the frequencies, which at present appear non-random for these different types of inversions in the population, can only be estimated by studying a larger sample size. Inversion heteromorphisms may promote reshuffling of tandem arrays of DNA repeat sequences, thereby giving rise to new heteromorphic domains. Alternatively, the repetitive nature of the sequences lends to the structural variations observed within the inv(9) chromosomes (or any other abnormal chromosome that is the result of recombination between, or breakage within, repetitive DNA).

Chromosome Inversion↗

Transcription modulation in vitro of the fibroin gene exerted by a 200-base-pair region upstream from the "TATA" box.

We have previously reported that the 5'-flanking sequence upstream from the "TATA" box modulates the faithful transcription initiation of the fibroin gene in a homologous whole cell extract prepared from the silk glands, whereas such a modulating effect is not observed in a HeLa cell extract. Subsequently we have determined that major signals responsible for the modulating effect are located within a 200-base-pair region upstream from the TATA box, mainly in a distal region between nucleotide positions -238 and -116 and in a proximal one between -73 and -53. Inversion of the sequence element -234 to -66 did not alter its modulating effect. A similar modulating effect by the upstream region of the sericin gene was also observed in the silk gland extract but not in the HeLa cell extract. In contrast, a modulating effect by the upstream region of the adenovirus 2 major late gene was observed in the HeLa cell extract but not in the silk gland extract. Thus, the modulating effect by the upstream regions of these genes is exerted only in their own homologous extracts.

Animals↗

Patellar articular cartilage lesions: in vitro MR imaging evaluation after placement in gadopentetate dimeglumine solution.

PURPOSE: To evaluate T1-weighted magnetic resonance (MR) imaging after diffusion of gadopentetate dimeglumine for visualization of articular cartilage lesions. MATERIALS AND METHODS: MR imaging was performed in eight human cadaveric patella specimens immediately and 4 hours after placement into a vessel filled with gadopentetate dimeglumine solution (2.5 mmol/L). T1-weighted spin-echo and inversion-recovery turbo spin-echo MR sequences with nulled cartilage signal (inversion time of 300 msec) were used. In a total of 128 articular cartilage areas, MR imaging findings were compared with macroscopic and histopathologic findings. Pathologic evaluation was performed by one musculoskeletal pathologist. With knowledge of pathologic observations, MR images were analyzed by one musculoskeletal radiologist with regard to intrinsic signal intensity characteristics and surface abnormalities of articular cartilage. RESULTS: Histopathologic findings demonstrated 67 areas of normal articular cartilage and 66 cartilage lesions (grade 1, n = 19; grade 2, n = 15; grade 3, n = 26; grade 4, n = 6). All grade 3 and 4 lesions could be identified on MR images obtained immediately after submersion and after 4 hours. Ninety-four percent of grade 1 and 2 lesions were identified as areas of predominantly decreased contrast enhancement on delayed MR images obtained with both sequences. MR images obtained immediately after submersion demonstrated abnormal signal intensity in only 9% and 12% of grade 1 and 2 lesions, respectively. CONCLUSION: T1-weighted MR images obtained in vitro after gadopentetate dimeglumine diffusion allow demonstration of articular cartilage surface lesions and early stages of cartilage degradation.

Aged↗

Cloning, sequencing, and characterization of the cgmB gene of Sinorhizobium meliloti involved in cyclic beta-glucan biosynthesis.

Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346-6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

Base Sequence↗

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).

Base Sequence↗

Evaluation of ischemic heart disease on a 1.5 Tesla scanner: combined first-pass perfusion and viability study.

PURPOSE: We implemented a fast gradient echo (GRE) sequence with an echo-planar imaging (EPI) read-out (FGRE-ET) to conduct myocardial perfusion studies on a conventional scanner. The accuracy of combined perfusion and viability studies is evaluated in comparison with coronary angiography (CAG). MATERIALS AND METHODS: We enrolled 33 patients suspected of having coronary artery disease in this study. Short-axis perfusion images of the left ventricles were acquired following intravenous bolus injection of gadolinium-DTPA (0.05 mml/kg), both after myocardial loading by dipyridamole (0.56 mg/kg) and at rest. Viability studies were obtained using an inversion-recovery FGRE sequence. Radiologists performed blinded film readings. The findings with perfusion and the viability studies were compared with CAG on a segment-to-segment basis corresponding to the coronary arteries' territories. Stenosis equal to or greater than 75% in diameter was considered significant on CAG. The results were also compared with single photon emission computed tomography (SPECT) in 23 patients. RESULTS: The combination of perfusion and viability studies showed 85.7% sensitivity, 88.9% specificity, and 87.2% accuracy in comparison with CAG. SPECT revealed respective rates of 71.7%, 78.3%, and 73.9% in 23 patients of this group. CONCLUSION: Myocardial perfusion studies using FGRE-ET were feasible and accurate, even on a conventional scanner.

Adult↗

Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12.

The trxA gene of Escherichia coli K-12 has been cloned into multicopy plasmids on DNA fragments of varying sizes. The smallest of these was a 1-kb fragment resulting from partial digestion with Sau3A (pBHK10). The complete nucleotide sequence of the trxA gene and its promoter was determined. Comparison of the DNA sequence with the published amino acid sequence revealed the inversion of two amino acid pairs and the possibility of a leader peptide 18 amino acids in length. Three-factor P1 transductional crosses and physical mapping experiments have determined a map order of ilv-trxA-uvrD-corA-metE.

Bacterial Proteins↗

Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition.

We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.

Bacteriophage mu↗

Inversion of the IDS gene resulting from recombination with IDS-related sequences is a common cause of the Hunter syndrome.

We have recently described the identification of a second IDS locus (IDS-2) located within 90 kb telomeric of the IDS gene (Bondeson et al. submitted). Here, we show that this region is involved in a recombination event with the IDS gene in about 13% of patients with the Hunter syndrome. Analysis of the resulting rearrangement at the molecular level showed that these patients have suffered a recombination event that results in a disruption of the IDS gene in intron 7 with an inversion of the intervening DNA. Interestingly, all of the six cases with a similar type of rearrangement showed recombination between intron 7 of the IDS gene and sequences close to exon 3 at the IDS-2 locus implying that these regions are hot spots for recombination. Analysis by nucleotide sequencing showed that the inversion is caused by recombination between homologous sequences present in the IDS gene and the IDS-2 locus. No detectable deletions or insertions were observed as a result of the recombination event. The results in this study have practical implications for diagnosis of the Hunter syndrome.

Base Sequence↗

Perfusion-modulated MR imaging-guided radiofrequency ablation of the kidney in a porcine model.

OBJECTIVE: This study was performed to test the hypothesis that temporary renal ischemia will result in increased thermal lesion size during radiofrequency thermal ablation in the kidney. MATERIALS AND METHODS: Twelve kidneys were treated in six pigs that were placed under general anesthesia in the MR suite, using a 0.2-T open C-shaped MR imaging system. A 4-cm-long, 14-mm-diameter balloon catheter was placed into the aorta using a transfemoral approach, and the balloon was positioned proximal to the renal arteries via guidance with MR imaging. A 2-cm exposed-tip MR-compatible 17-gauge radiofrequency electrode was placed into one kidney under MR fluoroscopy using fast imaging with steady-state free precession (FISP) sequences. Thermal ablation was performed with the electrode tip temperature maintained at 90 +/- 2 degrees C for 10 min. This procedure was repeated in the contralateral kidney. The balloon was inflated during one ablation. Postablation images were obtained, the pigs were sacrificed, and both kidneys of each animal were harvested for pathologic correlation. RESULTS: Technical success was achieved in all animals. The lesion measured 14.2 +/- 2.2 mm (mean +/- standard deviation) for the ischemic kidney versus 8.0 +/- 2.6 mm in the normally perfused kidney (p = 0.00002). No significant complications were noted. In all images, thermal lesions displayed low signal intensity with a sharp rim of high signal intensity best visualized using short tau inversion recovery (STIR) sequences with a mean accuracy of 1.3 +/- 1.2 mm when compared with pathologic findings and a mean contrast-to-noise ratio of 4.9 +/- 2.5. CONCLUSION: We accept the hypothesis that temporary renal ischemia leads to a significantly increased radiofrequency ablation lesion size. We conclude that catheter-based balloon perfusion reduction is feasible, that the procedure does not lead to major complications, and that it can be performed using MR imaging as the sole imaging modality.

Animals↗

Physical maps for genome analysis of serotype A and D strains of the fungal pathogen Cryptococcus neoformans.

The basidiomycete fungus Cryptococcus neoformans is an important opportunistic pathogen of humans that poses a significant threat to immunocompromised individuals. Isolates of C. neoformans are classified into serotypes (A, B, C, D, and AD) based on antigenic differences in the polysaccharide capsule that surrounds the fungal cells. Genomic and EST sequencing projects are underway for the serotype D strain JEC21 and the serotype A strain H99. As part of a genomics program for C. neoformans, we have constructed fingerprinted bacterial artificial chromosome (BAC) clone physical maps for strains H99 and JEC21 to support the genomic sequencing efforts and to provide an initial comparison of the two genomes. The BAC clones represented an estimated 10-fold redundant coverage of the genomes of each serotype and allowed the assembly of 20 contigs each for H99 and JEC21. We found that the genomes of the two strains are sufficiently distinct to prevent coassembly of the two maps when combined fingerprint data are used to construct contigs. Hybridization experiments placed 82 markers on the JEC21 map and 102 markers on the H99 map, enabling contigs to be linked with specific chromosomes identified by electrophoretic karyotyping. These markers revealed both extensive similarity in gene order (conservation of synteny) between JEC21 and H99 as well as examples of chromosomal rearrangements including inversions and translocations. Sequencing reads were generated from the ends of the BAC clones to allow correlation of genomic shotgun sequence data with physical map contigs. The BAC maps therefore represent a valuable resource for the generation, assembly, and finishing of the genomic sequence of both JEC21 and H99. The physical maps also serve as a link between map-based and sequence-based data, providing a powerful resource for continued genomic studies

Chromosomes, Artificial, Bacterial↗

Nucleotide sequence and organization of the adeno-associated virus 2 genome.

The complete nucleotide sequence of the adeno-associated virus 2 genome was determined. The single-stranded genome is 4,675 nucleotides in length and contains inverted terminal repeats of 145 nucleotides, the first 125 nucleotides of which form a palindromic sequence. Within the inverted terminal repetitions, there are two distinct sequences representing an inversion of 43 nucleotides that can exist on either terminus. The 5' and 3' termini of three major mRNA transcripts, which are present in both spliced and unspliced forms, were also mapped on the viral genome. Potential initiation and termination codons for efficient protein synthesis were identified, and genome segments were assigned that code for three major viral capsid proteins and, possibly, some as-yet-unidentified, nonstructural viral proteins.

Amino Acid Sequence↗

Transcriptional regulation of the rat bradykinin B2 receptor gene: identification of a silencer element.

Kinins are involved in a variety of physiological and pathophysiological processes related to cardiovascular homeostasis, inflammation, blood flow, and nociception. Under physiological conditions, the bradykinin B2 (BKB2) receptor is constitutively expressed and mediates most of kinins' actions. However, the mechanisms regulating BKB2 receptor gene expression are still poorly understood. In this study, 4.6 kilobases of the 5'-flanking region from the rat BKB2 receptor gene were sequenced, and computer analysis revealed several sites for transcriptional factors. Nine promoter mutants were cloned in luciferase reporter gene vectors and transfected in NG108-15 cells and rat aorta vascular smooth muscle cells (VSMCs), showing several positive and negative regulatory elements. A classical silencer with 56 base pairs (bp) caused a decrease in reporter gene activity in NG108-15 cells and VSMCs and was able to inhibit the thymidine kinase promoter. Using electrophoretic mobility shift assay and surface plasmon resonance assay, protein-DNA interactions in the silencer region were determined and specific sets of protein-silencer complexes were detected in both cell types. More intense complexes were observed in the central 21 bp of the silencer and mutation in a putative SRE-1 site strongly impaired the protein-DNA binding. Down-regulation of the BKB2 receptor population in NG108-15 cells promoted by N(6), 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate was paralleled by an increase in the amount of nuclear proteins bound to the silencer sequence showing an inverse relationship between protein-silencer complexes and the transcription of the BKB2 receptor gene. In summary, these data highlight the cell-specific regulation of the BKB2 receptor and the importance of a silencer element present in the regulatory region of the gene.

5' Flanking Region↗

Short TI short TR inversion recovery imaging using reduced flip angles.

In this paper the effects of reducing the flip angle of the 90-degree observation pulse in inversion recovery imaging are described and analyzed. When incorporated in an IR sequence with a short inversion time (STIR), reduction to the 90-degree pulse allows a significant shortening of the repetition time without loss in contrast, although at the expense of some signal/noise. The generalized STIR sequence thus combines the previously reported advantages of a conventional STIR sequence--suppression of ghost artifacts from abdominal wall movement, suppression of chemical shift and boundary artifacts, additive effects of N(H), T1 and T2 on image contrast--with reduced power deposition and the advantages resulting from shorter repetition times, viz, single heart-beat triggering, increased number of signal averages for suppression of motion artifacts, acquisition of interleaved contiguous slices without cross-talk or considerable time savings when the number of required slices is limited. The proposed method is demonstrated and experimentally verified by imaging experiments on phantoms and human subjects. In principle the method is applicable to all cases where STIR imaging has been proven to be successful.

Brain↗

Analyses of the ribosomal DNA region in Nosema bombycis NIS 001.

Ribosomal DNA (rDNA) containing small subunit (SSU) rDNA and both flanking regions in the entomopathogenic microsporidian Nosema bombycis NIS 001 was amplified from genomic DNA with a primer set based on the sequence of an inverse polymerase chain reaction (PCR)-derived fragment. In this fragment, SSU rDNA was divided by a 618-bp insert at nt 599, and 5S rDNA was located downstream of the SSU rDNA, fragmented by 284-bp intergenic spacer. In addition, the 48-bp 3'-end of large subunit (LSU) rDNA was located 118 bp upstream of the fragmented SSU rDNA. In the amplicon, the region upstream of the LSU rDNA was a homologue of the C-terminal CHARLIE8 transposon-like element of human GTF2IRD2. In this organism, another fragmented SSU rDNA, which was divided by a 231-bp insert at nt 50, was also detected. Both the intact (insertless) and fragmented SSU rDNAs clustered with LSU rDNA and 5S rDNA and the intergenic sequences between SSU rDNA and 5S rDNA were divergent in an organism. Reverse transcription (RT)-PCR assay indicated that not only the intact SSU rDNA but also the fragmened SSU rDNA were transcribed in N. bombycis.

Animals↗

Disruption of a rhodaneselike gene results in cysteine auxotrophy in Saccharopolyspora erythraea.

A 3,373-base-pair DNA segment from a clone fortuitously isolated from Saccharopolyspora erythraea by hybridization to an oligodeoxynucleotide probe was sequenced. Computer-assisted analysis of the nucleotide sequence reveals three closely linked Streptomyces open reading frames plus a fourth converging on the others. The deduced product of one of them, ORF2, shows considerable similarity to bovine liver rhodanese. orf2, and the closely linked orf3 located just downstream of it, were disrupted by insertion of an apramycin resistance cassette into the orf2 coding sequence along with inversion of the fragment carrying most of orf2 and orf3 via two successive recombinational events in the wild-type strain. The mutant strain thus created contains wild-type levels of rhodanese activity but cannot grow on minimal medium. It is a cysteine auxotroph, capable of utilizing efficiently only thiosulfate among the inorganic sulfur sources tested. orf2 has been designated cysA. The possible role of the rhodaneselike cysA gene product in thiosulfate formation is discussed.

Base Sequence↗

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. II. Isolation of the gene and characterization of the 5' flanking region.

Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and microsomal HMG-CoA reductase are sequential enzymes in the cholesterol biosynthetic pathway; both are negatively regulated by cholesterol. In this paper, we report the isolation of overlapping bacteriophage lambda clones that encompass the gene for hamster cytoplasmic HMG-CoA synthase. The gene spans 20 kilobases and contains 11 exons and 10 introns. Under conditions of high-level expression in cultured hamster cells and hamster liver, approximately 50% of the mRNAs contain two introns in the region of the gene corresponding to the 5' untranslated region. The remaining 50% contain only one intron as a result of the direct splicing of exon 1 to exon 3. The optional exon (exon 2) in the 5' untranslated region contains a 26-nucleotide sequence that is homologous to the 5' untranslated region of the mRNA for HMG-CoA reductase. Approximately 350 base pairs upstream of the transcription initiation site, the HMG-CoA synthase gene contains a sequence that is strongly homologous to the 72-base pair enhancer region of the SV40 virus. Approximately 125 nucleotides downstream of this region, the sequence CCGCCC, or its inverse complement GGGCGG, is repeated four times. Multiple copies of this sequence are present in the SV40 promoter and in the 5' flanking region of HMG-CoA reductase and several other cellular housekeeping genes. The availability of cloned genes for two consecutive negatively regulated enzymes of the cholesterol synthetic pathway should allow elucidation of the mechanism for this coordinate expression.

Animals↗

[Laparoscopic and general surgery guided by open interventional magnetic resonance].

Interventional magnetic resonance (IMR) machines have produced unique opportunity for image-guided surgery. The open configuration design and fast pulse sequence allow virtual real time intraoperative scanning to monitor the progress of a procedure, with new images produced every 1.5 sec. This may give greater appreciation of anatomy, especially deep to the 2-dimensional laparoscopic image, and hence increase safety, reduce procedure magnitude and increase confidence in tumour resection surgery. The aim of this paper was to investigate the feasibility of performing IMR-image-guided general surgery, especially in neoplastic and laparoscopic field, reporting a single center -- St. Mary's Hospital (London, UK) -- experience. Procedures were carried out in a Signa 0.5 T General Elettric SP10 Interventional MR (General Electric Medical Systems, Milwaukee, WI, USA) with magnet-compatible instruments (titanium alloy instruments, plastic retractors and ultrasonic driven scalpel) and under general anesthesia. There were performed 10 excision biopsies of palpable benign breast tumors (on female patients), 3 excisions of skin sarcoma (dermatofibrosarcoma protuberans), 1 right hemicolectomy and 2 laparoscopic cholecystectomies. The breast lesions were localized with pre- and postcontrast (intravenous gadolinium DPTA) sagittal and axial fast multiplanar spoiled gradient recalled conventional Signa sequences; preoperative real time fast gradient recalled sequences were also obtained using the flashpoint tracking device. During right hemicolectomy intraoperative single shot fast spin echo (SSFSE) and fast spoiled gradient recalled (FSPGR) imaging of right colon were performed after installation of 150 cc of water or 1% gadolinium solution, respectively, through a Foley catheter; imaging was also obtained in an attempt to identify mesenteric lymph nodes intraoperatively. Concerning laparoscopic procedures, magnetic devices (insufflator, light source) were positioned outside scan room, the tubing and light head being passed through penetration panels. Intraoperative MR-cholangiography was performed using fast spin echo (SSFSE) techniques with minimal intensity projection 3-dimensional reconstruction. About skin sarcomas, 2 of them were skin recurrences of previously surgically treated sarcomas (all of them received preoperative biopsy) and the extent of the lesion was then determined using short tau inversion recovery (STIR) sequence. The skin was closed in each case without need for any plastic reconstruction. The breast lesions were visualized with both Signa and real-time imaging and all enhanced with contrast: 2 (20%) were visualized only after contrast enhancement; intraoperative real time imaging clearly demonstrated a resection margin in all cases. Maximum dimensions of breast specimens (range 8-50 mm, median 24.5 mm) were not significantly different from those measured by Signa (p>0.17, Student's paired t-test) or real time images (p>0.4): also there was no significant difference in lesion size between Signa and real time images (p>0.25). All postprocedure scans clearly demonstrated complete excision. The extent of the tumor at MR imaging was greater in each case than suggested by clinical examination. Adequate resection margins were planned using STIR sequences. Histological examination confirmed clear surgical margins of at least 1 cm in each case. During right hemicolectomy, both intraoperative SSFSE and FSPGR contrast imaging revealed the lesion and details of the colonic surface; imaging of the lymph node draining right colon was only partially successful, due to movement artifact. Concerning laparoscopic procedures, both FSE and SSFSE techniques produced reasonable images of the gallbladder and intrahepatic ducts, but the FSE imaging was of poor quality due to respiration artifact; however, SSFSE allowed visualization of the gallbladder and part of the common bile duct. About skin sarcomas, the extent of the tumor at MR imaging was greater in each case than suggested by clinical examination and in each case the complete tumor excision was confirmed. Histological examination confirmed clear surgical margins of at least 1 cm in each case. Intraoperative MR scanning reliably identifies palpable breast tumours and skin sarcomas and is sufficiently accurate to guide their surgical excision. Further work may be done to develop laparoscopic and open abdominal surgery as well.

Anesthesia, General↗