Immunological and genetic characterization of 2-deoxygalactose-resistant, galactokinase-deficient mutants of Chinese hamster cells: evidence for structural mutations at the galK locus.

Ten independent mutants resistant to 2-deoxygalactose and without any detectable galactokinase activity (null-galactokinase mutations) were isolated from mutagenized Chinese hamster somatic cells. They were analyzed for the presence of serologically cross-reacting material (CRM) with antiserum generated against highly purified Chinese hamster galactokinase. All 10 mutants contain cross-reacting material (i.e., were CRM+), indicating that all the mutations affect the correct expression of a product of the galactokinase structural gene. Complementation analysis among them shows that the 10 mutations fall in one functional genetic unit.

Isolation of variants resistant to methylglyoxal bis(guanylhydrazone) from adenovirus-transformed rat cells.

It is presumed that proteins encoded by viral oncogenes interact with proteins encoded by cellular genes to bring about the transformed phenotype. To demonstrate the existence of such cellular genes we attempted to isolate mutants with a nontransformed phenotype from an adenovirus-transformed rat cell line (F4) which contains multiple copies of the transforming E1 region. F4 cells were mutagenized with ethyl methanesulfonate and variants resistant to the anticancer drug methylglyoxal bis(guanylhydrazone) were selected. The proportion of such variants was about one in 10(6) and increased 5-fold after mutagenesis. Two variant clones (G1 and G2) were isolated and characterized: they were 5-fold more resistant to methylglyoxal bis(guanylhydrazone); they had a stable phenotype; they showed decreased drug uptake; they had a reduced ability to grow in soft agar, low serum, and nude mice; there was no detectable change in the restriction pattern of integrated viral genes or in the expression of the E1a and E1b proteins. These properties suggest that selection for methylglyoxal bis(guanylhydrazone) resistance may result in the isolation of variants with phenotypic characteristics of nontransformed cells. It was likely that these variants were altered in a cellular function required for the maintenance of the transformed phenotype.

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition.

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.

Alcohol dehydrogenase mutants of Chinese hamster somatic cells resistant to allyl alcohol.

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) mutants of Chinese hamster somatic cells were isolated as resistant to allyl alcohol (ALLR). The ALLR phenotypes of the mutant clones were reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Several mutants, Adh-1, Adh-2, Adh-9 and Adh-13, resistant to allyl alcohol were characterized. They have between 15 and 40% of the alcohol dehydrogenase activity of the wild-type cell lines. This phenotype is therefore a useful marker to analyze gene segregation of somatic cell mutations and to study the expression of the genes involved in the metabolism of ethanol in mammalian cells.

On some genetic aspects of phage lambda resistance in E. coli K12.

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.

Galactokinase mutants of Chinese hamster somatic cells resistant to 2-deoxygalactose.

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.

Mutations of Chinese hamster somatic cells from 2-deoxygalactose sensitivity to resistance.

In Chinese hamster somatic cells, the spontaneous change of phenotype from 2-deoxygalactose sensitivity to resistance was studied using fluctuation test experiments à la Luria and Delbrück (1943) for four Chinese hamster cell strains derived from V79. The results are consistent with true mutational events. The mutation rates are in the range of 1 to 3.5 X 10(-5) per cell per generation. The relationship between the 2-deoxyglactose resistance and the galactokinase markers is discussed.

Monoclonal antibodies against metallothioneins and metalloproteins.

Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

Identification and characterization of four novel large deletions in the human neurofibromatosis type 1 (NF1) gene.

We studied 20 unrelated NF1 patients by Southern blots with seven cDNA probes and loss of heterozygosity (LOH) analysis with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Four novel large deletions (178, 184, 236, and 237) have been identified and characterized. The breakpoint of deletion 178 was located in between exons 23-2 and 27b and the sequences downstream of the breakpoint were deleted. For deletion 184, the breakpoint was in between exons 27b and 29, and the region upstream of the breakpoint was deleted. With deletion 236, the breakpoint was in between exons 14 and 18 and the region downstream of the breakpoint was deleted. The breakpoint of deletion 237 was in between exons 38 and 45 and the sequences upstream of the breakpoint were deleted. These deletions were distributed randomly across the NF1 gene and no deletion hot spot was found. Our study suggests that the combination of analyses of loss of heterozygosity, southern blotting and southern blot densitometry can be used as a powerful method to detect large deletions, especially when family record is not available or the patient is a sporadic case.

Cloning of DNA complementary to bovine prolactin mRNA.

We have cloned DNA complementary to mRNA coding for bovine prolactin (bPrl). Double-stranded cDNA prepared from bovine pituitary mRNA was inserted into the Pst I site of plasmid bPR322 by the dC x dG tailing technique and amplified in E. coli chi 1776. A recombinant plasmid containing bPrl cDNa was identified by hybridization to cloned rat Prl cDNA. It contains cDNA corresponding to the region of the mRNA coding for the carboxy terminal 101 amino acids of bPrl, as well as 42 nucleotides in the 3' untranslated region of the mRNA. Nucleotide sequencing confirmed the amino acid sequencing of this region of bPrl, and permitted the assignment of asparagine or glutamic acid at seven previously equivocal loci. Codon use in bPrl mRNA is comparable to that found in rat and human Prl mRNA's and differs from that in bovine, rat, and human growth hormone mRNA's.

Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility.

Two new mouse cell mutants, resistant to either 80 or 100 mM CdCl2, were isolated to study the regulation of transcription by the glucocorticoid hormones. Their metallothionein mt-1% and mt-2+ genes were amplified coordinately to a maximum of 30 copies per cell. By Southern blot analysis, no gross rearrangement was detectable near the mt+ loci. Contrary to other mutants previously isolated, the metallothionein-specific mRNAs of these mutants are inducible by dexamethasone.

Myocardial tracking, a new method to calculate ejection fraction with gated SPECT: validation with (201)Tl versus planar angiography.

UNLABELLED: Left ventricular ejection fraction (LVEF) and viability are essential variables for the prognosis of myocardial infarction and can be measured simultaneously by (201)Tl gated SPECT; however, most algorithms tend to underestimate LVEF. This study aimed to evaluate a new myocardial tracking algorithm, MyoTrack (MTK), for automatic LVEF calculation. METHODS: A rest/redistribution (20 min/4 h) (201)Tl gated SPECT protocol followed immediately by a (99m)Tc equilibrium radionuclide angiography (ERNA) was performed in 75 patients with history of myocardial infarction. Quality of myocardial uptake was evaluated from count statistics and automatic quantification of defect sizes and severities (CardioMatch). LVEFs were calculated both with Germano's quantitative gated SPECT (QGS) algorithm and with MTK. Briefly, the originality of this algorithm resides in the unique end-diastole segmentation, matching to a template and motion field tracking throughout the cardiac cycle. RESULTS: ERNA LVEF averaged 33% +/- 14%. QGS significantly underestimated this value at 20 min (30% +/- 13%, P < 0.001) and at 4 h (30% +/- 13%, P < 0.0001). By contrast, MTK did not miscalculate LVEF at 20 min (34% +/- 14%, probability value was not significant) though a similar underestimation occurred at 4 h (31% +/- 13%, P < 0.02). Individual differences between early and late gated SPECT values and differences between gated SPECT and ERNA values did not correlate with the extension of perfusion defects, count statistics, or heart rate. CONCLUSION: MTK algorithm accurately calculates LVEF on early/high-count images compared with ERNA [corrected], even in patients with severe perfusion defects, but tends to underestimate LVEF on delayed/low-contrast images, as other algorithms do.

Genotype analysis of the NF1 gene in the French Canadians from the Québec population.

We genotyped 19 NF1 families from the French Canadians of the Québec population with six intragenic polymorphic markers including 2 RFLPs (EcoRI and RsaI) and 4 microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Genotype analysis indicated families 7610 and 7473 bear deletions. In Family 7610 the deletion removed the entire NF1 gene except exons 1 to 4b. The breakpoint of the deletion is located between exons 4a and 4b. The deletion 7473 was derived from the maternal chromosome and exons 1 to 5 were deleted. The breakpoint of the deletion is located between exons 7 and 13. Their phenotypes are reported. The allele frequencies of microsatellites IVS27AC28.4 and IVS38GT53.0 are compared to previously reported data from Caucasians, including Spanish and Italians. The difference is statistically significant (P < 0.0036) for marker IVS27AC28.4 between the Québec French Canadian and the Italian population.

Changes in chromatin conformation regulate the 5-lipoxygenase gene expression during differentiation of HL60 cells.

The HL60 cell line does not express the 5-lipoxygenase gene prior to differentiation with various agents. In this paper we have shown by DNase I sensitivity that the chromatin conformation of the 5-lipoxygenase gene changes following differentiation of HL60 cells with dimethyl sulfoxide (DMSO). Moreover, run-on analysis suggests that transcription of the 5-lipoxygenase gene is enhanced after differentiation. We proposed that the chromatin conformation represses the expression of the 5-lipoxygenase gene in HL60 cell line and that differentiation of these cells with DMSO changes the chromatin conformation, which allows the expression of the 5-lipoxygenase gene. The HeLa cells which, like the HL60 cells, do not express the 5-lipoxygenase, were insensitive to DNase I treatment. These results suggest that chromatin structure might represent a form of regulation for the 5-lipoxygenase gene.

Direct extraction of boundaries from computed tomography scans.

Presents a method, based on the filtered backprojection technique (FBP), to extract directly the boundaries of X-ray images, without previous image reconstruction. The author preprocess the raw data in order to compute directly the reconstructed values of the gradient or of the Laplacian at any location in the plane (defined with real coordinates). The reconstructed value of the gradient and of the Laplacian correspond to the exact mathematical definition of the differentials of the image. For noisy data, the author proposes also to use an extension of existing FBP techniques, adapted to the computation of the gradient and of the Laplacian. Finally, the author shows how to use the corresponding operators to perform the segmentation of a slice, without image reconstruction. Images of the reconstructed gradient, Laplacian, and segmented objects are presented.

Segmentation of tomographic data without image reconstruction.

Geometric tomography (GT), a technique for processing tomographic projections in order to reconstruct the external and internal boundaries of objects, is presented. GT does not necessitate the reconstruction of an image of the slice of the object. It is shown that the segmentation can be performed directly with the raw data, the sinogram produced with the scanner, and that those segmented shapes can be geometrically transformed into reconstructed shapes in the usual space. If one is interested in only the boundaries of the objects, they do not need to reconstruct an image, and therefore the method needs much less computation than those using traditional computed tomography techniques. Experimental results are presented for both synthesized and real data, leading to subpixel positioning of the reconstructed boundaries. GT gives its best results for sparse, highly contrasted objects such as bones or blood vessels in angiograms, it allows ;on the fly' processing of the data, and real time tracking of the object boundaries.

Deformation analysis to detect and quantify active lesions in three-dimensional medical image sequences.

Evaluating precisely the temporal variations of lesion volumes is very important for at least three types of practical applications: pharmaceutical trials, decision making for drug treatment or surgery, and patient follow-up. In this paper we present a volumetric analysis technique, combining precise rigid registration of three-dimensional (3-D) (volumetric) medical images, nonrigid deformation computation, and flow-field analysis. Our analysis technique has two outcomes: the detection of evolving lesions and the quantitative measurement of volume variations. The originality of our approach is that no precise segmentation of the lesion is needed but the approximative designation of a region of interest (ROI) which can be automated. We distinguish between tissue transformation (image intensity changes without deformation) and expansion or contraction effects reflecting a change of mass within the tissue. A real lesion is generally the combination of both effects. The method is tested with synthesized volumetric image sequences and applied, in a first attempt to quantify in vivo a mass effect, to the analysis of a real patient case with multiple sclerosis (MS).

Automatic 3-D segmentation of internal structures of the head in MR images using a combination of similarity and free-form transformations: Part I, Methodology and validation on normal subjects.

The study presented in this paper tests the hypothesis that the combination of a global similarity transformation and local free-form deformations can be used for the accurate segmentation of internal structures in MR images of the brain. To quantitatively evaluate our approach, the entire brain, the cerebellum, and the head of the caudate have been segmented manually by two raters on one of the volumes (the reference volume) and mapped back onto all the other volumes, using the computed transformations. The contours so obtained have been compared to contours drawn manually around the structures of interest in each individual brain. Manual delineation was performed twice by the same two raters to test inter- and intrarater variability. For the brain and the cerebellum, results indicate that for each rater, contours obtained manually and contours obtained automatically by deforming his own atlas are virtually indistinguishable. Furthermore, contours obtained manually by one rater and contours obtained automatically by deforming this rater's own atlas are more similar than contours obtained manually by two raters. For the caudate, manual intra- and interrater similarity indexes remain slightly better than manual versus automatic indexes, mainly because of the spatial resolution of the images used in this study. Qualitative results also suggest that this method can be used for the segmentation of more complex structures, such as the hippocampus.

Automatic analysis of cerebral atrophy.

3D MR data obtained for 10 healthy control subjects have been used to build a brain atlas. The atlas is built in four stages. First, a set of features that are unambiguously definable and anatomically relevant need to be computed for each item in the database. The chosen features are crest lines along which the maximal principal curvature of the surface of the brain is maximal in its associated principal direction. Second, a nonrigid registration algorithm is used to determine the common crest lines among the subjects in the database. These crest lines form the structure of the atlas. Third, a set of crest lines is taken as a reference set and a modal analysis is performed to determine the fundamental deformations that are necessary to bring the individual data in line with the reference set. The deformations are averaged and the set of mean crest lines becomes the atlas. Finally, the standard deviation of the deformations between the atlas and the items in the database defines the normal variation in the relative positions of the crest lines in a healthy population. In a fully automatic procedure, the crest lines on the surface of the brain adjacent to the cerebral ventricles in a patient with primary progressive aphasia were compared to the atlas; confirmation that the brain of this patient demonstrates atrophy was provided by stereological analysis that showed that the volume of the left cerebral hemisphere is 48.8 ml (CE 2.8%) less than the volume of the right cerebral hemisphere in the region of the temporal and frontal lobes. When the amplitude of the deformations necessary to register the crest lines obtained for the patient with the atlas were greater than three standard deviations beyond the variability inherent in the atlas, the deformation was considered significant. Four of the main deformation modes of the longest crest line of the surface of the brain adjacent to the cerebral ventricles were significantly different in the patient with primary progressive aphasia compared to the atlas. The ventricles are preferentially enlarged in the left cerebral hemisphere. Furthermore, they are closer together posteriorly and further apart anteriorly than in the atlas. These observations may be indicative of the atrophy of the temporal and frontal lobes of the left cerebral hemisphere noted in the patient. Ultimately, the approach may provide a useful screening technique for identifying brain diseases involving cerebral atrophy. Serial studies of individual patients may provide insights into the processes controlling or affected by particular disease.

Automatic detection of hippocampal atrophy on magnetic resonance images.

An automatic method for identifying hippocampal atrophy on magnetic resonance (MR) images obtained from patients with clinical evidence of temporal lobe epilepsy (TLE) is described. The method is based on the analysis of image intensity differences between patients and controls within a volume of interest (VOI) centred on the hippocampus. The core of the method is a fully automatic signal intensity-based inter-subject image registration technique. In particular, a global affine registration to a reference image is performed, followed by a local affine registration within the VOI. A mask produced by manual segmentation of the mean hippocampus for 30 control subjects enabled investigations to be restricted to a specified region of the VOI approximately corresponding to the hippocampus. Normal variations of hippocampal signal intensity were computed from images obtained for the 30 control subjects. The manual method of hippocampal volumetry, currently an important component of the pre-surgical evaluation of patients with clinical evidence of medically intractable TLE, is used to determine the lower 1st percentile limits of normal hippocampal volume. Hippocampi with volumes below this limit are defined as atrophic. We investigated whether the automatic method can correctly distinguish between 15 patients with significant hippocampal atrophy according to absolute volumes and a further 14 controls. ROC curves enabled evaluation of sensitivity and specificity in respect of an intensity threshold. 100% specificity is required when determining suitability of patients for neurosurgery, resulting in levels of 50% and 70% sensitivity in detecting atrophy in the right and left hippocampus, respectively. We propose that the method can be developed as an automatic screening procedure.