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PubMed2000

Frequency dependence of the functional MRI response after electrical median nerve stimulation.

Localizing sensorimotor areas with high resolution functional MRI is of considerable interest for a wide range of medical applications from the preoperative planning of neurosurgical interventions to determining the course of neuroplastic reorganisation after brain lesions. We examined the effect of the stimulation frequency on the blood oxygen level dependent (BOLD) fMRI response and on perfusion weighted fMRI using electrical median nerve stimulation at 5, 15, 40, and 100 Hz. BOLD fMRI was performed using a single shot gradient echo EPI sequence to acquire 15 contiguous slices. For the qualitative flow sensitive studies, a single slice inversion recovery prepared spin echo echoplanar sequence (IR-SE EPI) was used. In the primary sensorimotor cortex, a linear increase of the fMRI-BOLD response, affecting both the number of activated pixels and the amplitude of the signal changes, was seen with increasing stimulation frequencies. The qualitative in-flow sensitive studies, using the IR-SE EPI sequence, indicate that the tissue perfusion also increases over the same range of frequencies. This implicates that larger fMRI responses can be obtained if electrical median nerve stimulation is performed at higher frequencies. The results are compared with electrophysiological data, which show a decrease of the early somatosensory evoked potentials at higher frequencies.

Adult↗

PubMed2000

Imaging pulmonary blood flow and perfusion using phase-sensitive selective inversion recovery.

A technique is described for imaging pulmonary blood flow using a phase-sensitive selective inversion recovery (PS-SIR) sequence. PS-SIR image reconstruction provides excellent contrast, differentiating fully relaxed inflowing blood from inverted blood and lung tissue. The magnetization of the inverted tissues remains negative at any inversion delay less than that at which the magnetization of the lung tissue is nulled, whereas that of the fully relaxed inflowing blood is always positive. Pulmonary blood flow can be observed by tracking the propagation of the pixels with positive values. Five healthy volunteers were imaged. The normal pattern of blood flow advancing from the central arteries toward the peripheries and into the lung parenchyma with return toward the center via draining veins was depicted. The method offers promise for evaluating pulmonary blood flow without the need for image subtraction or contrast administration. Magn Reson Med 43:793-795, 2000.

Adult↗

PubMed2001

Two-dimensional time correlation relaxometry of skeletal muscle in vivo at 3 Tesla.

A hybrid two-dimensional relaxometry (2DR) sequence was used to simultaneously measure both the spin-spin (R2) and spin-lattice relaxation rates (R1) of skeletal muscle in vivo. The 2DR sequence involved a 180 degrees inversion pulse followed by a variable delay time (30 values from 40 to 7000 ms); a projection presaturation (PP) scheme to localize a 16-ml cylindrical voxel; and a CPMG sequence (950 even echoes, effective echo spacing = 1.2 ms, equilibrium time = 12 s). The 2DR data were collected at 3.0 Tesla from the flexor digitorum profundus of eight healthy males, 26 +/- 2 years old. Analysis was performed with a 2D version of the non-negative least-squares algorithm and a one-way ANOVA. All subjects exhibited at least three spin-groups (R2 < 200 s(-1)), designated B, C, and D, with R2 values of 42.7, 26.5, and 8.1 s(-1), and fractional volumes of 52, 35, and 7%, respectively. The R1 values of B and C were similar, congruent with0.7 s(-1), but different from that of D (P < 0.001), which had an R1 of 1.0 s(-1). The results suggest that exchange between B and C ranges from 0.7-16.2 s(-1), while exchange between either of these spin-groups with D is slower. If the data are interpreted with a compartment model, in which spin-groups with short and long R2 values are attributed to extra- and intracellular fluid, respectively, the exchange of water across the cell membrane in living skeletal muscle is slow or intermediate relative to both R1 and R2.

Adult↗

PubMed2004

DNA sequence alterations affect nucleosome array formation of the chicken ovalbumin gene.

The role of the large amount (more than half of the genome) of noncoding DNA in higher organisms is not well understood. DNA evolved to function in the context of chromatin, and the possibility exists that some of the noncoding DNA serves to influence chromatin structure and function. In this age of genomics and bioinformatics, genomic DNA sequences are being searched for informational content beyond the known genetic code. The discovery that period-10 non-T, A/T, G (VWG) triplets are among the most abundant motifs in human genomic DNA suggests that they may serve some function in higher organisms. In this paper, we provide direct evidence that the regular oscillation of period-10 VWG that occurs in the chicken ovalbumin gene sequence with a dinucleosome-like period facilitates nucleosome array formation. Using a linker histone-dependent in vitro chromatin assembly system that spontaneously aligns nucleosomes into a physiological array, we show that nucleosomes tend to avoid DNA regions with low period-10 VWG counts. This avoidance leads to the formation of an array with a nucleosome repeat equal to half the period value of the oscillation in period-10 VWG, as determined by Fourier analysis. Two different half-period deletions in the wild-type DNA sequence altered the nucleosome array, as predicted computationally. In contrast, a full-period deletion had an insignificant effect on the nucleosome array formed, also consistent with the prediction. An inversion mutation, with no DNA sequences deleted, again altered the nucleosome array formed, as predicted computationally. Hence, a VWG dinucleosome signal is plausible.

Animals↗

PubMed1991

A complex regulatory DNA element associated with a major histocompatibility complex class I gene consists of both a silencer and an enhancer.

A novel regulatory element which contributes to the regulation of quantitative, tissue-specific differences in gene expression has been found between -771 and -676 bp upstream of the major histocompatibility complex (MHC) class I gene, PD1. Molecular dissection of this element reveals the presence of two overlapping functional activities: an enhancer and a silencer. Distinct nuclear factors bind to the overlapping enhancer and silencer DNA sequence elements within the regulatory domain. The levels of factors binding the silencer DNA sequence in different cell types are inversely related to levels of class I expression; in contrast, factors binding the enhancer DNA sequence can be detected in all cells. In cultured cell lines, inhibition of protein synthesis leads to the rapid loss of silencer complexes, with a concomitant increase in both enhancer complexes and MHC class I RNA. From these data, we conclude that a labile silencer factor competes with a constitutively expressed, stable enhancer factor for overlapping DNA-binding sites; the relative abundance of the silencer factor contributes to establishing steady-state levels of MHC class I gene expression.

Animals↗

PubMed1993

Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.

Base Sequence↗

PubMed1999

MR imaging of experimentally induced intracranial hemorrhage in rabbits during the first 6 hours.

PURPOSE: To evaluate the MR appearance of intracranial, especially intraparenchymal, hemorrhage during the first 6 hours after bleeding with various pulse sequences in an animal model. MATERIAL AND METHODS: Intracerebral hematomas and subarachnoid hemorrhage were created by injecting autologous blood in 9 rabbits. MR studies were performed using a 1.5 T scanner with pixel size and slice thickness comparable to those used in clinical practice before blood injection, immediately after injection, and at regular intervals during 6 hours. The images were compared with the hematoma sizes on formalin-fixed brain slices. RESULTS: In every animal, susceptibility-weighted gradient-echo (GRE) pulse sequences depicted the intraparenchymal hematomas and blood escape in the ventricles or subarachnoid space best as areas of sharply defined, strong hypointensity. The findings remained essentially unchanged during follow-up. The sizes corresponded well to the post-mortem findings. Gradient- and spin-echo (GRASE) imaging revealed some hypointensities, but these were smaller and less well defined. Spin-echo (SE) sequences (proton density-, T1- and T2-weighted) as well as a fluid-attenuated inversion recovery turbo spin-echo sequence (fast FLAIR) depicted the hemorrhage sites as mostly isointense to brain. CONCLUSION: Susceptibility-weighted GRE imaging at 1.5 T is highly sensitive to both hyperacute hemorrhage in the brain parenchyma and to subarachnoid and intraventricular hemorrhage.

Animals↗

PubMed1997

Complex genomic rearrangement within the 12q15 multiple aberration region induced by integrated human papillomavirus 18 in a cervical carcinoma cell line.

Human papillomavirus (HPV) DNA is integrated into the host genome in cervical cancer. The cervical carcinoma cell line SW756 has integrated HPV-18 DNA in chromosome region 12q15, in the papillomavirus-associated locus-2 (PAL2). By polymerase chain reaction and hybridization of an arrayed cosmid library with oligonucleotides from the rearranged allele, we determined the pre-integration germline structure of the region. PAL2 was located approximately 10 kb from sequence-tagged site marker U27131, which was the marker most proximal to the 3' flank of the integrated viral DNA. HPV-18 DNA integration induced a complex genomic rearrangement resulting in inversion and deletion of cellular sequences. PAL2 is within the multiple aberration region, which has been shown to be affected in several types of benign tumors of mesenchymal origin. The integrated viral DNA was located 50 kb from a CpG island and 150 kb upstream of the high-mobility group I-C (HMGI-C) gene. The HMGI-C gene and the integrated HPV-18 DNA had opposite transcriptional orientations. No overexpression or altered message of the HMGI-C gene was detected in three cervical carcinoma cell lines. The integrated viral DNA did not affect any other known gene in the region and may be a marker for an unknown gene associated with malignant tumor phenotypes.

Base Sequence↗

PubMed2004

Overexpression and affinity chromatography purification of the Type III restriction endonuclease EcoP15I for use in transcriptome analysis.

The Type III restriction endonuclease EcoP15I is a multifunctional hetero-oligomeric enzyme that recognizes the non-symmetric DNA sequence 5'-CAGCAG. For efficient cleavage, EcoP15I needs the interaction with two copies of the recognition sequence that have to be inversely oriented in the DNA double strand. The enzyme cuts the upper DNA strand 25-26 bp and the lower DNA strand 27-28 bp, respectively, downstream of the recognition sequence-a distinct feature that makes the enzyme particularly valuable for gene expression profiling methods relying on the SAGE procedure (Matsumura et al., PNAS 100, 15718, 2003). Because the broader use of this transcriptome analysis method requires the availability of larger amounts of restriction endonuclease EcoP15I and the enzyme is not commercially available, we have cloned the genes coding for the EcoP15I restriction endonuclease into pQE-16 plasmid vector that provides the enzyme with a C-terminal 6xHis-tag. After Ni-NTA affinity chromatography and ion exchange chromatography on heparin sepharose, we obtained 5mg homogeneous EcoP15I per gram cell pellet within 1-2 day(s). Moreover, the C-terminally 6xHis-tagged EcoP15I restriction endonuclease shows comparable enzymatic activity as the untagged enzyme.

Amino Acid Sequence↗

PubMed2004

A novel method for multiple alignment of sequences with repeated and shuffled elements.

We describe ABA (A-Bruijn alignment), a new method for multiple alignment of biological sequences. The major difference between ABA and existing multiple alignment methods is that ABA represents an alignment as a directed graph, possibly containing cycles. This representation provides more flexibility than does a traditional alignment matrix or the recently introduced partial order alignment (POA) graph by allowing a larger class of evolutionary relationships between the aligned sequences. Our graph representation is particularly well-suited to the alignment of protein sequences with shuffled and/or repeated domain structure, and allows one to construct multiple alignments of proteins containing (1) domains that are not present in all proteins, (2) domains that are present in different orders in different proteins, and (3) domains that are present in multiple copies in some proteins. In addition, ABA is useful in the alignment of genomic sequences that contain duplications and inversions. We provide several examples illustrating the applications of ABA.

Algorithms↗

PubMed2001

Biased distribution of chromosomal breakpoints involving the MLL gene in infants versus children and adults with t(4;11) ALL.

Derivative chromosomes of 40 patients diagnosed with t(4;11) acute lymphoblastic leukemia (ALL) were analysed on the genomic DNA level. Chromosomal breakpoints were identified in most cases within the known breakpoint cluster regions of the involved MLL and AF4 genes. Due to our current knowledge of the primary DNA sequences of both breakpoint cluster regions, specific features were identified at the chromosomal fusion sites, including deletions, inversions and duplications of parental DNA sequences. After separation of all t(4;11) leukemia patients into two age classes (below and above 1 year of age), the analysis of chromosomal fusion sites revealed significant differences in the distribution of chromosomal breakpoints and led to the definition of two hotspot areas within the MLL breakpoint cluster region. This may point to the possibility of different age-linked mechanisms that were leading to t(4;11) chromosomal translocations.

Adult↗

PubMed1979

Structure of simian virus 40 recombinants that contain both host and viral DNA sequences. I. The structure of variant CVPS/1/P2 (EcoRI res).

The entire nucleotide sequence (1210-base-pair repeating units) of a defective variant of simian virus 40 is presented. Within this variant there are deletions of large portions of the wild type genome and an inversion within the remaining wild type viral sequences. In addition, the defective variant contains DNA sequences derived from the permissive monkey cells in which the virus was propagated. The monkey sequences include a portion that is homologous to sequences within highly repeated monkey DNA (alpha component) as well as portions derived from sequences that are infrequently repeated in the monkey genome. One out of every three to four of the tandem 1210-base-pair repeat units contains in addition, a duplication of a part of the monkey sequences. The sequence information defines the structures of a number of recombinational joints which result from deletions, inversions, duplications, and insertions of host sequences into the viral genome. The data demonstrate that the various recombinational events that resulted in the formation of this defective variant did not depend on extensive homology between recombining segments.

Animals↗

PubMed1994

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction.

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.

Base Sequence↗

PubMed1991

Effect of altered activation sequence on epicardial QRST area and refractory period in dogs.

BACKGROUND: We investigated the effects of activation sequence on cardiac surface QRST areas and refractory periods in experiments on dogs. METHODS AND RESULTS: Right and left ventricular pacings were performed, and the pacing site was altered every 6 minutes. After 4 minutes of a given pacing, 54 unipolar electrograms distributed over the entire cardiac surface were recorded. Next, refractory periods at electrode sites near pacing electrodes were measured. Paired right ventricular/left ventricular (RV/LV) pacing data were obtained six or seven times in each sample. Although the QRST isoarea maps during the two activation orders were qualitatively similar, it was recognized consistently from the right ventricle-left ventricle difference map that leads around the RV free wall had positive values and that leads around the LV free wall and apex had negative values. Compared with the same leads at RV and LV pacing, QRST areas were larger when pacing sites were near the leads. The local QRST areas of individual leads at which we measured local refractory period were consistently larger during drive from proximal pacing sites than during drive from distant pacing sites. Refractory periods were consistently longer during proximal pacing than during distal pacing, and there was a positive correlation between change in local QRST area and change in refractory period (r = 0.64) during altered activation sequence, whereas there was an inverse correlation between change in QRST area and change in refractory period (r = -0.91) during localized myocardial warming. CONCLUSIONS: Both local QRST areas and local refractory periods were dependent on the activation sequence, and there was a positive correlation between QRST areas and refractory periods during various activation sequences compared with localized myocardial warming.

Animals↗

PubMed1985

Rearrangements between the operators in the bacteriophage lambda.

The left operator mutant lambda v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of lambda v2s, designated lambda iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators OL and OR. Formation of the inversion is facilitated by a translocation of right operator OcR mutant sequence to the left operator in lambda v2s. The inversion in lambda iri positions wild-type OR sequence at OL returning control of the left operon to repression by the lambda cro repressor.

Bacteriophage lambda↗

PubMedDate not supplied

MR imaging of anisotropically restricted diffusion in the brain of neonates and infants.

Brain MR examinations were performed on one normal and 30 abnormal neonates and infants with a variety of clinical problems using pulsed gradient spin echo (PGSE) sequences sensitized in different directions to detect changes due to anisotropically restricted diffusion of water within the brain. Anisotropically restricted diffusion was demonstrated within white matter, which appeared to be myelinated with T1-weighted inversion recovery or spin echo sequences and within white matter where the presence of myelin was not demonstrated with these sequences. Cysts and fluid collections were recognised by their low signal intensity using PGSE sequences in the subcortical regions, lentiform nuclei, and periventricular regions. An intracerebral hematoma and a subdural hematoma showed a high signal intensity with PGSE sequences consistent with isotropically restricted diffusion. Chronic infarction displayed a low signal intensity consistent with relatively free isotropic diffusion. Patients with leukodystrophy associated with congenital muscular dystrophy showed an anisotropic pattern with different components of the abnormality present on images with sensitization in different directions. The corticospinal tracts failed to show highlighting in three cases where conventional imaging was normal. In several patients with a history of birth asphyxia, asymmetry and a relative reduction in signal intensity were seen in the corticospinal tracts.

Asphyxia Neonatorum↗

PubMed1999

The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR.

Functional chimeric TCR chains, encoded by V gamma J gamma C beta or V gamma J beta C beta hybrid gene TCR, are expressed at the surface of a small fraction of alpha beta T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR gamma and beta loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between D beta and J gamma genic segments generates C gamma V beta and C beta V gamma hybrid loci. Within the C gamma V beta locus, secondary rearrangements between V gamma and J gamma or V gamma and J beta elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the C beta V gamma locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same D beta-3' recombination signal sequence can be used in successive rearrangements with J gamma and J beta genic segments, thus showing that a signal joint has been involved in a secondary recombination event.

Ataxia Telangiectasia↗

PubMed1993

De novo and inverse folding predictions of protein structure and dynamics.

In the last two years, the use of simplified models has facilitated major progress in the globular protein folding problem, viz., the prediction of the three-dimensional (3D) structure of a globular protein from its amino acid sequence. A number of groups have addressed the inverse folding problem where one examines the compatibility of a given sequence with a given (and already determined) structure. A comparison of extant inverse protein-folding algorithms is presented, and methodologies for identifying sequences likely to adopt identical folding topologies, even when they lack sequence homology, are described. Extension to produce structural templates or fingerprints from idealized structures is discussed, and for eight-membered beta-barrel proteins, it is shown that idealized fingerprints constructed from simple topology diagrams can correctly identify sequences having the appropriate topology. Furthermore, this inverse folding algorithm is generalized to predict elements of supersecondary structure including beta-hairpins, helical hairpins and alpha/beta/alpha fragments. Then, we describe a very high coordination number lattice model that can predict the 3D structure of a number of globular proteins de novo; i.e. using just the amino acid sequence. Applications to sequences designed by DeGrado and co-workers [Biophys. J., 61 (1992) A265] predict folding intermediates, native states and relative stabilities in accord with experiment. The methodology has also been applied to the four-helix bundle designed by Richardson and co-workers [Science, 249 (1990) 884] and a redesigned monomeric version of a naturally occurring four-helix dimer, rop. Based on comparison to the rop dimer, the simulations predict conformations with rms values of 3-4 A from native. Furthermore, the de novo algorithms can assess the stability of the folds predicted from the inverse algorithm, while the inverse folding algorithms can assess the quality of the de novo models. Thus, the synergism of the de novo and inverse folding algorithm approaches provides a set of complementary tools that will facilitate further progress on the protein-folding problem.

Algorithms↗