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PubMed1998

Cloning of the Xenopus laevis aldolase C gene and analysis of its promoter function in developing Xenopus embryos and A6 cells.

A Xenopus aldolase C gene (XAClambda3-1), much longer (9.6 kb) than human and rat genes (3.7-3.6 kb), was isolated and characterized, and expression studies were performed using Xenopus embryos and A6 cells, a kidney cell line constitutively expressing aldolase C gene. The Xenopus gene contained nine exons, and in its proximal 5'-upstream region a GC box and a 16 bp long aldolase C-specific element (ACSE), and in addition, a CCAAT box and a TATA-like element, both missing in mammalian genes. The lacZ gene connected to the 5'-upstream region (1.6 kb) of the aldolase gene containing many potentially regulative sequence elements was expressed in embryos temporally and spatially like the endogenous aldolase C gene. Deletion experiments using embryos and A6 cells suggested that this 5'-upstream DNA contained in its distal part a region which negatively affected on its expression in embryos, but not in A6 cells. The proximal-most region contained a basal promoter (68 bp) essential for expression in both embryos and A6 cells. Deletion experiments using A6 cells failed to detect such regulative regions within the first intron (spanning ca. 4 kb). Analyses with mutated promoters in A6 cells revealed that the GC box was the crucial element in the basal promoter, although the TATA-like element appeared to have a slightly stimulative effect on the GC box functioning. Gel retardation and foot-printing assays revealed the occurrence in A6 cells of a nuclear factor(s) that binds specifically to the GC box. Since Xenopus aldolase C gene has several unique structural features, we expect that it will provide an interesting material for studying the evolution and developmental control of the aldolase C gene.

Amino Acid Sequence↗

PubMed2002

Multiple spatially distributed stimulators and timing programs for entrainment of activation during ventricular fibrillation.

Activation sequences during ventricular fibrillation (VF) display complex pattern and fast rate. Recent evidence suggests that even during VF an excitable gap may exist. Existence of excitable gap lead us to hypothesize that it should be possible to entrain activation patterns during VF by using spatially distributed and temporally phased pacing strength stimuli. We describe here the electronics hardware and software that were developed to test our hypothesis. Eight biphasic stimulators were designed and fabricated, each addressable via a TTL input and thus independently triggered. To minimize electrical interference from stimulus pulses the stimulators were optically isolated. A program written in C was used to deliver TTL inputs to time the sequence of stimulation. The parameters that could be varied were, pulse intensity, polarity, and duration, inter pulse interval and activation pattern cycle length. Restitution's of action potential duration, conduction velocity, and complex activation patterns make the timing of stimulators complex. To aid in optimal timing of these stimulators, we used a Luo-Rudy ionic model of cellular activation to simulate VF in a matrix of 400 x 400 cells. Entrainment of activation was verified using animated displays of activation sequences. Using results of simulation we verified the function of our stimulators experimentally using electrically induced VF in canines and by using electrograms recorded from 121-electrode patch. Our results show that the developed hardware and software can be used to deliver distributed stimuli in a flexible and effective pattern, which may aid in development of approaches for treatment of VF.

Animals↗

PubMed2006

Diversity of microcystin-producing cyanobacteria in spatially isolated regions of Lake Erie.

The diversity of microcystin-producing cyanobacteria in the western basin of Lake Erie was studied using sequence analysis of mcyA gene fragments. Distinct populations of potentially toxic Microcystis and Planktothrix were found in spatially isolated locations. This study highlights previously undocumented diversity of potentially toxic cyanobacteria.

Amino Acid Sequence↗

PubMed1996

Primary sequence and developmental expression pattern of mRNAs and protein for an alpha1 subunit of the sodium pump cloned from the neural plate of Xenopus laevis.

Expression of a catalytic alpha subunit of the sodium pump was followed in early Xenopus embryos for correlation with physiological experiments showing that the sodium pump controls cavity expansion and the differentiation of neurones from the neural plate. Two cDNAs (one full length, one partial) for alpha1 subunit isoforms were cloned from a neural plate stage Xenopus library and sequenced. Other isoforms were not detected. Temporal and spatial expression patterns for alpha1 subunit transcripts and protein revealed extensive developmental regulation. At all stages, cells involved in cavity generation (outer ectoderm and cells lining the archenteron) expressed alpha1, transcripts with protein confined to the lateral and basal membranes. Before gastrulation, transcript levels were low and predominantly in animal cells. During gastrulation, alpha1 mRNAs rose significantly. Transcripts and protein were down-regulated in future outer neural plate cells as the mesoderm invaginated. Protein appeared at the blastopore on apical surfaces of lip cells and apposing surfaces of invaginating cells, suggesting that the Na pump opposes entry of fluid. In early neurulae, alpha1 mRNAs rose sharply. Transcript expression remained low in outer neural plate cells and increased in the endoderm, and protein appeared in the notochord. In midneurulae, transcripts returned in outer neural plate cells. Protein expression appeared on basal surfaces of deep neural plate cells and the floor plate, matching physiological observations. After neural tube closure, transcripts were detected in all dorsal structures. Protein was retained in the notochord and floor plate, was eliminated from the outer layer of the neural tube, and appeared on ependymal cells. The results are discussed in relation to previous physiological observations.

Amino Acid Sequence↗

PubMed2007

Fast 3D 1H spectroscopic imaging at 3 Tesla using spectroscopic missing-pulse SSFP with 3D spatial preselection.

Three-dimensional (3D) (1)H MR spectroscopic imaging (SI) allows metabolic changes in human tissue to be identified. In clinical practice, fast acquisition techniques are required to achieve an adequate spatial resolution within acceptable total measurement times. In this study a novel fast pulse sequence for 3D (1)H SI based on the condition of steady-state free precession (SSFP), termed "spectroscopic missing-pulse SSFP" (spMP-SSFP), is proposed. It combines 3D spatial preselection with the acquisition of full spin echoes (SEs), and thus makes subsequent phase correction of spectra redundant. The sequence was applied to a phantom and healthy human brains in vivo at 3 Tesla. Metabolic images are acquired with a spatial resolution of 1.8 cm(3) within a total measurement time of about 6 min. With a lower signal-to-noise ratio (SNR) per unit measurement time compared to previous spectroscopic SSFP implementations, 3D spatial preselection can now be realized with spMP-SSFP. Since the method does not require separate techniques for water and lipid suppression, and employs a simple data-processing approach, spMP-SSFP is a robust, fast SI method that requires only minimal user interaction.

Brain↗

PubMed2006

Reverse replay of behavioural sequences in hippocampal place cells during the awake state.

The hippocampus has long been known to be involved in spatial navigational learning in rodents, and in memory for events in rodents, primates and humans. A unifying property of both navigation and event memory is a requirement for dealing with temporally sequenced information. Reactivation of temporally sequenced memories for previous behavioural experiences has been reported in sleep in rats. Here we report that sequential replay occurs in the rat hippocampus during awake periods immediately after spatial experience. This replay has a unique form, in which recent episodes of spatial experience are replayed in a temporally reversed order. This replay is suggestive of a role in the evaluation of event sequences in the manner of reinforcement learning models. We propose that such replay might constitute a general mechanism of learning and memory.

Action Potentials↗

PubMed2000

Sinorhizobium meliloti nfe (nodulation formation efficiency) genes exhibit temporal and spatial expression patterns similar to those of genes involved in symbiotic nitrogen fixation.

The nfe genes (nfeA, nfeB, and nfeD) are involved in the nodulation efficiency and competitiveness of the Sinorhizobium meliloti strain GR4 on alfalfa roots. The nfeA and nfeB genes are preceded by functional nif consensus sequences and NifA binding motifs. Here, we determined the temporal and spatial expression patterns of the nfe genes in symbiosis with alfalfa. Translational fusions of the nfe promoters with the gusA gene and reverse transcription-polymerase chain reaction analyses indicate that they are expressed and translated within mature nitrogen-fixing nodules and not during early steps of nodule development. Within the nodules the three nfe genes exhibit a spatial expression pattern similar to that of genes involved in symbiotic nitrogen fixation. We show that nfeB and nfeD genes are expressed not only from their own promoters but also from the upstream nfe promoter sequences. Furthermore, with the use of specific antibodies the NfeB and NfeD proteins were detected within the root nodule bacteroid fraction. Finally, NfeB was inmunolocalized in the bacteroid cell membrane whereas NfeD was detected in the bacteroid cytoplasm.

Bacterial Proteins↗

PubMed1998

Excitation of complicated shapes in three dimensions.

We experimentally verified a recently proposed technique for the excitation of a complicated three-dimensional profile (CARVE, completely arbitrary regional volume excitation). CARVE is based on a generalized DANTE RF pulse sequence and a synchronous string of gradient steps. Provided there is no limitation in the number of pulses, CARVE can generate an excitation profile of any shape with any resolution. However, hardware limitations and sample properties restrict the number of RF pulses and gradient steps and, thus, limit attainable resolution of the excitation profile. We theoretically and experimentally showed that spatial resolution can be increased by distributing a long sequence among several CARVE experiments and summing up their signals. This is particularly important for three-dimensional excitation profiles where an n-fold increase in resolution requires an n3-fold increase of the number of events in the sequence. The potential use of three-dimensional CARVE might be in spectroscopic imaging where the excitation profile can be tailored to match the shape of a selected organ or body part.

Magnetic Resonance Spectroscopy↗

PubMed2000

Responses of rat subicular neurons to convergent stimulation of lateral entorhinal cortex and CA1 in vivo.

There has been little electrophysiological examination of the afferent projection from lateral entorhinal cortex to dorsal subiculum. Here we provide evidence that synaptic inputs from lateral entorhinal cortex and CA1 converge onto single dorsal subicular neurons in vivo. Subicular responses to CA1 stimulation consisted of excitation and/or long-duration inhibition. Neurons excited by CA1 activation usually showed inhibition to entorhinal stimulation. The latter inhibition was usually of short duration, however, long duration inhibition was seen in a significant proportion of responses. Entorhinal stimulation produced excitatory responses in four bursting cells and it was these cells that also tended to show the longest inhibition. Only bursting cells could be driven antidromically by entorhinal stimulation. Biocytin-filled multipolar and pyramidal cells displayed excitation-inhibition sequences to CA1 and inhibition to entorhinal stimulation. These data strongly suggest that subicular inhibitory neurons receive excitatory input from CA1 and display mutual inhibition. The source of entorhinal-evoked inhibition is less clear. The relative sparseness of observed entorhinal-evoked responses suggests that the input to dorsal subiculum from any one part of lateral entorhinal cortex is spatially restricted. These data show that excitation-inhibition sequences can be seen in subicular pyramidal and multipolar cells and that single subicular neurons receive convergent inputs from CA1 and entorhinal cortex. We show for the first time that bursting cells can be driven both orthodromically and antidromically by direct entorhinal stimulation. These data support the existence of a reciprocal excitatory connection between lateral entorhinal cortex and dorsal subiculum and suggest further that this connection may involve only bursting subicular neurons.

Action Potentials↗

PubMed1999

Expression of Mmp-9 and related matrix metalloproteinase genes during axolotl limb regeneration.

One of the earliest events in limb regeneration is the extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) are a family of matrix degrading enzymes that have been identified in both normal and disease states. Using RT-PCR and cDNA library screening, we have isolated sequences homologous to four different Mmp genes. The spatial and temporal expression of one of these, Mmp-9, has been analyzed during axolotl limb regeneration. Northern blot analysis identifies a 3.8 kb transcript that is abundantly expressed during regeneration, and whole-mount in situ hybridization has uncovered an unusual bi-phasic expression pattern. The first phase begins at 2 hours after amputation, and expression is confined to the healed wound epithelium. This phase continues for 2 days, showing peak expression at 14 hours after amputation. This early phase may be needed to retard reformation of the basal lamina of the epidermis, and thereby facilitate the epidermal-mesenchymal interactions required for successful regeneration. The second phase begins a few days later when a small blastema has formed. During this phase, expression is in the mesenchyme, localized to cells around the tips of the cut skeletal elements. This expression is maintained through several stages until redifferentiation begins. The timing and position of the second phase of expression is consistent with a role for Mmp-9 in the removal of damaged cartilage matrix. We have also discovered that the time of onset of Mmp-9 expression is sensitive to denervation, which causes a delay of several hours. Finally, retinoids, known for their dramatic effects on the pattern of regenerating limbs, can cause a down regulation of Mmp-9 expression. Dev Dyn 1999;216:2-9.

Ambystoma↗

PubMed1997

Extraordinary conservation of cysteines among homologous Chironomus silk proteins sp185 and sp220.

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20-28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20-28 residues, 61% of which can be described by the motif X5-8-Cys-X5-(Trp/Phe/Tyr)-X4-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5'- and 3'-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures.

Amino Acid Sequence↗

PubMed1997

Autowave tunneling through a non-excitable area of active media.

The mechanisms of autowaves propagation through local non-homogeneities in active media relevant to diverse class of physiological systems were studied by means of a computer simulation. The model proposed by Zel'dovich and Frank-Kamenetsky and that of FitzHugh-Nagumo were used for studying autowave tunneling, which in a broad sense implies underbarrier passing. It was shown that for every fixed parameter value corresponding to the degree of non-excitability of local area a critical value for non-excitable zone latitude exists. An autowave overcomes the barrier and continues to propagate when the value of zone latitude is less than critical. Critical conditions for origination of a source of periodical sequence of impulses in excitable medium were found. The source properties, as shown, can be modified by regulation of size of a non-excitable zone and a zone of higher excitability. In particular, the conditions when spatial irregularity behaves as a source of unidirectional and/or asynchronous sequence of impulses were explored.

Animals↗

PubMed2002

Mismatch responses to randomized gradient switching noise as reflected by fMRI and whole-head magnetoencephalography.

The central auditory system of the human brain uses a variety of mechanisms to analyze auditory scenes, among others, preattentive detection of sudden changes in the sound environment. Electroencephalography (EEG) and magnetoencephalography (MEG) provide a measure to monitor neuronal cortical currents. The mismatch negativity (MMN) or field (MMNm) reflect preattentive activation in response to deviants within a sequence of homogenous auditory stimuli. Functional magnetic resonance imaging (fMRI) allows for a higher spatial resolution as compared to the extracranial electrophysiological techniques. The image encoding gradients of echo planar imaging (EPI) sequences, however, elicit an interfering background noise. To circumvent this shortcoming, the present study applied multi-echo EPI mimicking an auditory oddball design. The gradient trains (SOA = 800 msec, 94.5 dB SPL, stimulus duration = 152 msec) comprised amplitude (-9 dB) and duration (76 msec) deviants in a randomized sequence. Moreover, the scanner noise was recorded and applied in a whole-head MEG device to validate the properties of this specific material. Robust fMRI activation patterns emerged in response to the deviant gradient switching. Changes in amplitude activated the entire auditory cortex, whereas the duration deviants elicited right-lateralized signal increase in secondary areas. The recorded scanner noise evoked reliably right-lateralized mismatch MEG responses. Source localization was in accordance with activation of secondary auditory cortex. The presented paradigm provides a robust and feasible tool to study the functional anatomy of early cognitive auditory processing in clinical populations such as schizophrenia.

Acoustic Stimulation↗

PubMed1993

Molecular homology among members of the R gene family in maize.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.

Amino Acid Sequence↗

PubMed1997

Laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS) to detect and characterise HPV DNA sequences by FISH in HeLa cells.

Visualisation and localisation of specific DNA sequences were performed by fluorescence in situ hybridisation (FISH), confocal laser scanning microscopy (CLSM), and factor analysis of biomedical image sequences (FAMIS). HeLa cells containing 10-50 copies per cell of human papillomavirus (HPV) DNA type 18 integrated in cellular DNA were used as a model. HPV-DNA was identified by DNA probes and DNA-DNA hybrids were revealed by alkaline phosphatase and Fast Red (FR) TR salt/naphtol-MX phosphate. Cell nuclei were counterstained with thiazole orange (TO). FAMIS summarises image sequences into a reduced number of images called factor images and curves called factors. Factor images correspond to spatial distributions of the different factors. Factors estimate different individual physical behaviours in the sequence (extinction velocity, spectral emission, depth emission profiles). We verified that HPV-DNA hybridisation signals are specific to the spectrum of FR, and distinguished between FR and TO. The latter result was found by taking into account differences in their extinction velocities. The focus of CLSM was improved on 3D image sequences, and the location of fluorescent signals inside the preparations was determined. Factor images showed that FR stained targets were located on different focal planes at the periphery of the nuclei.

DNA, Viral↗

PubMed2000

Pulse sequences for interventional magnetic resonance imaging.

Interventional magnetic resonance imaging (iMRI) is different from diagnostic magnetic resonance imaging (MRI) in its spatial, temporal, and contrast resolution requirements due to its specific clinical applications. As a result, the pulse sequences used in iMRI often are significantly different than those used in the more conventional diagnostic arena. The focus of this article is to summarize how iMRI is different from diagnostic MRI, to describe a variety of MRI pulse sequences and sequence strategies that have evolved because of these differences, and to describe some MRI sequence strategies that are in development and may be seen in future iMRI applications.

Body Temperature↗

PubMed2003

Spatio-temporal image correlation (STIC): new technology for evaluation of the fetal heart.

Spatio-temporal image correlation (STIC) is a new approach for clinical assessment of the fetal heart. It offers an easy to use technique to acquire data from the fetal heart and to aid in visualization with both two-dimensional and three-dimensional (3D) cine sequences. The acquisition is performed in two steps: first, images are acquired by a single, automatic volume sweep. Second, the system analyzes the image data according to their spatial and temporal domain and processes an online dynamic 3D image sequence that is displayed in a multiplanar reformatted cross-sectional display and/or a surface rendered display. The examiner can navigate within the heart, re-slice, and produce all of the standard image planes necessary for a comprehensive diagnosis. The advantages of STIC for use in evaluation of the fetal heart are as follows: the technique delivers a temporal resolution which corresponds to a B-mode frame rate of approximately 80 frames/s; it provides the examiner with an unlimited number of images for review; it allows for correlation between image planes that are perpendicular to the main image acquisition plane; it may shorten the evaluation time when complex heart defects are suspected; it enables the reconstruction of a 3D rendered image that contains depth and volume which may provide additional information that is not available from the thin multiplanar image slices (e.g. for pulmonary veins, septal thickness); it lends itself to storage and review of volume data by the examiner or by experts at a remote site; it provides the examiner with the ability to review all images in a looped cine sequence.

Artifacts↗

PubMed2006

Image-type dependent eigen-regions-of-interest define conspicuity operators for predicting human scanpath fixation.

Top-down informativeness elaboration and bottom-up conspicuity processing are intimately interconnected in visual perception. An internal cognitive model of the external world must necessarily control not only our recognition but also the scanpath sequence of eye movement/shift of attention jumps. A self-organizing process based on principal component analysis and scanpath experimental data are used in this paper to define spatial visual conspicuity from the eigen-features of the scanpath sequence image loci. Eight different classes of images are used as both training and testing set. We first demonstrate that cognitive-driven scanpath loci can be discriminated in terms of these bottom-up eigen conspicuity features. We can finally define a conspicuity processing algorithm and measure its ability to predict human scanpaths as evidenced by the positional similarity measure Sp. Some computer vision applications will also be discussed.

Algorithms↗