Intraoperative MR systems. Low-field approaches.

Numerous different concepts in low- and mid-field intervention have been described and applied for percutaneous intervention and intraoperative MR imaging, and each has demonstrated usefulness in specific applications. As the use of interventional methods under MR imaging expands, the imaging systems used for intervention will continue to evolve. More than likely, different system designs will be advocated for different interventional applications. The ultimate choice of interventional system will depend on each institution's optimal balance among the types of procedures to be performed, the required image quality, and the financial constraints.

Optimizing brain tumor resection. Low-field interventional MR imaging.

In this article, the authors describe their strategy for intraoperative MR image guidance of tumor resection at low-field (0.2 T) strength. It is their opinion that intraoperative imaging is most useful in assessing the resection of intrinsic, infiltrating brain tumors; boundaries of the tumor cannot be clearly distinguished with the surgeon's eyes, and therefore, this discussion predominantly applies to the surgical resection of gliomas.

Multiple genetic aberrations including evidence of chromosome 11q13 rearrangement detected in pituitary adenomas by comparative genomic hybridization.

OBJECT: This study was conducted to determine whether comparative genomic hybridization (CGH) is a more sensitive method for detecting genetic aberrations than other tests currently in use. METHODS: The authors used CGH to examine 40 primary and 13 recurrent adenomas obtained from 52 patients for loss and gain of genetic material. Copy number aberrations (CNAs) were detected in 25 (48%) of the 52 patients studied. The chromosomes affected were, in order of decreasing frequency, 11, 7, X, 1, 8, 13, 5, 14, 2, 6, 9, 10, 12, 3, 18, 21, 4, 16, 15, 19, 22, and Y. Endocrinologically active adenomas were more likely to contain (p = 0.009) and had a greater number (p = 0.003) of CNAs. Of 26 adenomas with CNAs, 18 showed multiple aberrations involving entire chromosomes or chromosome arms. The most frequent CNA involving a chromosome subregion, which was present in four (8%) of 53 adenomas, was the loss of all chromosome 11 material except for a preserved common segment containing 11q13. Immunoperoxidase staining did not detect cyclin D1 expression in those four cases, making cyclin D1 an unlikely target of this rearrangement. CONCLUSIONS: These findings indicate that genetic abnormalities are present in pituitary adenomas at a higher rate than previously reported, are associated with endocrinological activity, and often involve several chromosomes. Rearrangement at 11q13 may inactivate a tumor suppressor gene or activate an oncogene that is important in the initiation or progression of sporadic pituitary adenomas.

A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations.

p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay allows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.

Regulation of transcription of genes required for fatty acid transport and unsaturated fatty acid biosynthesis in Escherichia coli by FadR.

Fatty acid biosynthesis and fatty acid degradation in Escherichia coli are co-ordinately regulated at the level of transcription by the product of the fadR gene, FadR. In the present work we investigate FadR interaction with the fabA and fadL promoters. The FadR-responsive operator within fabA, OA, was localized to a region -47 to -31 base pairs relative to the start of transcription using DNase I protection studies. The promoter and untranslated leader within fadL had two binding sites for FadR, OL1 at -25 to -9 and OL2 at -1 to +16 relative to the start of transcription. The binding affinity of FadR for OA and OL1 or OL2 was lower than that for the single site within fadB (OB) as measured using protein-DNA gel retention assays. Overall, these experiments demonstrated that the affinity of FadR binding for DNA containing the fadB, fadL and fabA promoters was OB > OL1, OL2 > OA. We could not distinguish separate binding affinities for OL1 or OL2. We demonstrated repression of fadL transcription and activation of fabA transcription in vitro using run-off transcription assays containing purified FadR and RNA polymerase.

Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase.

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of beta-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be M(r) = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.

Characterization of FadR, a global transcriptional regulator of fatty acid metabolism in Escherichia coli. Interaction with the fadB promoter is prevented by long chain fatty acyl coenzyme A.

The Escherichia coli fadR gene product, FadR, is a multifunctional regulator of fatty acid metabolism. In this work we have purified FadR by a two-step procedure employing two ion-exchange columns. The amino-terminal sequence of the purified protein confirms the sequence of the protein derived from analysis of the DNA sequence (DiRusso, C. C. (1988) Nucleic Acids Res. 16, 7995-8009) and indicates that the initiating methionine is cleaved from the mature protein. Purified FadR binds to a 326-base pair HaeIII fragment of fadB DNA which carries the fadB promoter. DNase I footprinting localizes the operator to a sequence, 5' ATCTGGTACGACCAGAT 3', at +1 to +17 nucleotides relative to the start of transcription. Using protein-DNA gel retention assays, we estimate the Keq of FadR binding to the fadB operator to be approximately 3 x 10(-10) M. Binding of FadR is specifically inhibited by long chain fatty acyl-CoA compounds. The apparent Ki values for oleoyl-CoA, palmitoyl-CoA, and palmitoleoyl-CoA are each 5 nM while that of myristoyl-CoA is 250 nM. Decanoyl-CoA, crotonoyl-CoA, and free fatty acids inhibited binding only at concentrations above 1 microM.

Involvement of multiple chromosome 17p loci in medulloblastoma tumorigenesis.

Loss of heterozygosity for sequences located on chromosome 17p in several tumor types is often associated with mutations in the tumor suppressor gene p53. We previously showed consistent deletion of chromosome 17p12-13.1 in medulloblastoma, a common childhood brain tumor. Using denaturing gradient gel electrophoresis and direct sequencing, we have detected p53 mutations in only two of 20 medulloblastoma specimens. Moreover, additional RFLP studies of these 20 specimens showed loss of heterozygosity at a more distal and distinct site, 17p13.3. Deletion of 17p almost invariably signified a negative prognosis. Our results suggest that p53 mutations may contribute to the pathogenesis of medulloblastoma in relatively few cases. The consistent deletion of other discrete loci on 17p suggests that additional or alternative tumor suppressor genes may contribute to the tumor's phenotype.

Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma.

We detected a germ-line mutation of the p53 gene in a patient with a malignant ependymoma of the posterior fossa. This mutation, which was found at codon 242, resulted in an amino acid substitution in a highly conserved site of exon 7 of the p53 gene; the same mutation was found in both the germ-line and the tumor tissue. This is the most common region of previously described somatic p53 mutations in tumor specimens and of the germ-line p53 mutations in patients with the Li-Fraumeni cancer syndrome. Evaluation of the patient's family revealed several direct maternal and paternal relatives who had died at a young age from different types of cancer. The association of a germ-line p53 mutation with an intracranial malignancy and a strong family history of cancer suggests that p53 gene mutations predispose a person to malignancy and, like retinoblastoma mutations, may be inherited.

Loss of heterozygosity for chromosome 22 DNA sequences in human meningioma.

Monosomy of chromosome 22 in meningioma was the first consistent cytogenetic anomaly reported for a solid tumor. Although most meningiomas are isolated sporadic lesions, multiple and familial occurrences have been reported, usually in cases of documented neurofibromatosis 2 (NF2). Previous reports have placed the NF2 locus on chromosome 22, flanked by the markers D22S1 and D22S28. We report a restriction fragment-length polymorphism study of 16 meningiomas conducted using chromosome 22 probes. None of the patients had clinical findings or a family history of NF2, although two of them eventually developed multiple intracranial meningiomas. Detectable loss of chromosome 22 sequences was observed in 50% of informative patients. Deletion mapping of tumors with preserved sequences showed that the loss of chromosome 22 DNA overlapped the region previously linked to NF2, but also included a sequence distal to the NF2 locus. These results suggest that the oncogenesis of human meningioma involves inactivation of a chromosome 22 locus that may be in close proximity to the gene for NF2.

Deletion mapping of the medulloblastoma locus on chromosome 17p.

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-p13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the p53 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance.