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Results for “Deep sequencing”

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At least 505 records · Page 28Linked to original sources
PubMed1996

Intrinsic connections in the anterior dorsal ventricular ridge of the lizard Psammodromus algirus.

We have studied the intrinsic connections of the anterior dorsal ventricular ridge (ADVR) in the lacertid lizard Psammodromus algirus by means of retrograde transport of horseradish peroxidase (HRP) and fluorescent labeling with the lipophilic carbocyanine dye DiI. We injected HRP into different regions in the ADVR arrayed in a medial-to-lateral sequence, with each consisting of three distinct superficial-to-deep zones. When HRP was injected into a given region, many labeled neurons (always located ipsilateral to the injection site) were found at all mediolateral regions of ADVR in locations rostrally distant from the injection site. DiI crystals were applied on different superficial-to-deep zones within each region. Two patterns could be recognized: DiI crystals applied on the periventricular (most superficial) zone resulted in a labeling of cells widely distributed throughout the ADVR independently of the mediolateral region of the application site, whereas DiI crystals applied on deeper zones resulted in a staining of cells mostly restricted to a narrow radial area. Results from both types of labeling confirm that the ADVR has a prominent radial component in its intrinsic organization, but they also demonstrate that some areas of the ADVR receive projections from distant, rostrally located neurons in every ipsilateral region of the ridge itself, which establishes a clear non-radial component. This organization may have important functional properties with regard to a putative integration of different sensory modalities conveyed by thalamic afferent fibers to the ADVR. Last, we analyzed some evolutionary implications of our results.

Animals↗
PubMed1999

Impaired respiratory mechanics in pulmonary emphysema: evaluation with dynamic breathing MRI.

To evaluate impaired respiratory mechanics in pulmonary emphysema, dynamic breathing magnetic resonance imaging (BMRI) was acquired with fast-gradient echo pulse sequences at fixed thoracic planes over two to three slow, deep respiratory cycles in 6 controls and 28 patients with pulmonary emphysema including 9 patients undergoing lung volume reduction surgery (LVRS). Respiratory motions of the diaphragm and chest wall (D/CW) were assessed by a cine-loop view, a fusion display of maximal inspiratory and expiratory images, and the time-distance curves. By contrast with normal subjects with regular synchronous D/CW motions, the patients frequently showed reduced, irregular, or asynchronous motions, with significant decreases in the maximal amplitude of D/CW motions (MAD and MACW), and the length of apposition of the diaphragm (LAD) (P < 0.0001, P < 0.001, P < 0. 01, respectively). After LVRS, nine patients showed improvements in D/CW configuration and mobility, with significantly increased MAD, MACW, and LAD (P < 0.01, P < 0.0001, and P < 0.05, respectively). In 40 studies of 28 patients including the post-LVRS examinations, the normalized MAD and MACW significantly correlated with %FEV(1) (r = 0. 881 and r = 0.906; P < 0.0001, respectively). BMRI seems useful for noninvasively and directly assessing the impaired respiratory mechanics associated with abnormal ventilation in pulmonary emphysema, and also for monitoring the effects of LVRS. J. Magn. Reson. Imaging 1999;10:510-520.

Aged↗
PubMed2006

Mutations and polymorphisms in the human ornithine transcarbamylase (OTC) gene.

Ornithine transcarbamylase (OTC) deficiency is the most common inherited disorder of the urea cycle and is transmitted as an X-linked trait. Defects in the OTC gene cause a block in ureagenesis resulting in hyperammonemia, which can lead to brain damage and death. Three previous mutation updates for the OTC gene have been published, in 1993, 1995, and 2002. The most recent comprehensive update, in 2002, contained 244 mutations including 13 nondisease-causing mutations and polymorphisms. This current update reports 341 mutations, of which 93 have not been previously reported, and an additional 29 nondisease-causing mutations and polymorphisms. Out of the 341 mutations, 149 were associated with neonatal onset of hyperammonemia (within the first week of life), 70 were seen in male patients with later onset of hyperammonemia, and 121 were found in heterozygous females (one unknown). Along with the reported mutations, residual enzyme activities and other pertinent clinical information are included whenever available. Most mutations in the OTC gene are specific to a particular family ("private" mutations). They are distributed throughout the gene, with a significant paucity of mutations in the 32 first codons encoding the "leader" peptide (exon 1 and the beginning of exon 2). Almost all mutations in consensus splice sites confer a neonatal onset phenotype. Using the current molecular screening methods, mutations are found in about 80% of the patients. The remaining patients may have mutations in regulatory domains or mutations deep in the introns, which constitute 98.5% of the genomic sequence. In addition, a phenocopy of OTC deficiency caused by mutations in another unknown gene cannot be excluded.

Amino Acid Sequence↗
PubMed2004

Active deep brain stimulation during MRI: a feasibility study.

The goal of this study was to evaluate the feasibility of active deep brain stimulation (DBS) during the application of standard clinical sequences for functional MRI (fMRI) in phantom measurements. During active DBS, we investigated induced voltage, temperature at the electrode tips and lead, forces on the electrode and lead, consequences of defective leads and loose connections, proper operation of the neurostimulator, and image quality. Sequences for diffusion- and perfusion-weighted imaging, fMRI, and morphologic MRI were used. The DBS electrode and lead were placed in a NaCl solution-filled phantom. The results indicate that there are severe potential hazards for patients. Strong heating, high induced voltage, and even sparking at defects in the connecting cable could be observed. However, it was demonstrated that under certain conditions, safe MR examinations during active DBS are feasible. Certain safety precautions are recommended in this report.

Body Temperature↗
PubMed2005

Arthrobacter ardleyensis sp. nov., isolated from Antarctic lake sediment and deep-sea sediment.

Three psychrotrophic Arthrobacter strains, isolated from Antarctic lake sediment (An24, An25T) and deep-sea sediment (ZX6) were studied. Their 16S rRNA gene sequences showed highest similarities (97.0-97.9%) with those of A. nicotianae and A. protophormiae. All three strains underwent rod-coccus morphological change, had high mol% G+C content, were aerobic to slightly anaerobic, and grew between 0 degrees C and 30 degrees C, with optimal growth temperature around 25 degrees C. The cell wall peptidoglycan was A4alpha variant. DNA-DNA hybridization, physiological and chemotaxonomic studies indicated that these three strains constituted a new homogeneous genomic species within the genus Arthrobacter, for which the name Arthrobacter ardleyensis, with the type strain An25T (CGMCC 1.3685, JCM 12921) was proposed.

Aerobiosis↗
PubMed2007

Prediction of recovery from a post-traumatic coma state by diffusion-weighted imaging (DWI) in patients with diffuse axonal injury.

INTRODUCTION: To determine whether diffusion-weighted magnetic resonance (MR) imaging findings combined with initial clinical factors indicate the depth of shearing lesions in the brain structure and therefore relate to coma duration in diffuse axonal injury (DAI). METHODS: A total of 74 adult patients (48 male and 26 female) with DAI were examined with conventional MR imaging and diffusion-weighted MR imaging between 2 hours and 20 days after injury. Apparent diffusion coefficient (ADC) maps were obtained and the mean ADC values of each region of interest (ROI) were measured using MRI console software. The involvement of the brainstem, deep gray matter, and corpus callosum was determined for each sequence separately as well as for the combination of all sequences. The correlations between MR imaging findings indicating the presence of apparent brain injury combined with initial clinical factors were determined. RESULTS: Clinical characteristics, such as initial score on the Glasgow coma scale (GCS), age and number of all lesions, and ADC scores were predictive of the duration of coma. CONCLUSION: It was possible to predict post-traumatic coma duration in DAI from cerebral MR imaging findings combined with clinical prognostic factors in the acute to subacute stage after head injury. Age, ADC scores, GCS score and number of lesions were highly significant in predicting coma duration. The technique presented here might provide a tool for in vivo detection of DAI to allow the prediction of the coma duration during the early stages in patients with traumatic brain injury.

Accidents, Traffic↗
PubMed1977

Single unit recording in the midbrain of rats during shock-elicited fighting behavior.

Single unit activity was recorded extracellularly from the midbrain of rats during fighting behavior and during non-fighting control manipulations. Fighting behavior was elicited by footshock or startle stimuli or occurred spontaneously as a result of prior footshock presentations. Seven cells were found in the midbrain reticular formation and central gray which displayed maximum firing rates during fighting behavior. These cells also fired to a limited extent to some of the control manipulations, particularly contralateral vibrissae stimulation. These cells fired phasically during fighting behavior and their firing was correlated with either the approach or paw-strike of the opponent animal or to the response of the recording animal to a tactic of the opponent animal. However, no specific movement or sensory event reliably predicted the firing of these cells during fight sequences. Cells located in other midbrain areas, such as the deep tectum or the area of the red nucleus, also responded during fighting behavior. However, the discharge of these cells was correlated with specific body movements or sensory events. The activity during fighting was similar in rate and pattern to activity during control manipulations whenever similar movements or sensory stimulation were produced. Cells were also found which did not discharge during fighting behavior although they fired under a variety of other conditions.

Action Potentials↗
PubMed2004

Differences in malate dehydrogenases from the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 and the psychrophilic bacterium Moritella sp. strain 5710.

The gene encoding malate dehydrogenase (MDH) of the obligately piezophilic deep-sea bacterium Moritella sp. strain 2D2 was cloned and sequenced. There were two positions [close to the active site (Ala-180) and in the subunit interaction site (His-229)] with 2D2-specific substitutions. The MDH genes of strain 2D2 and a psychrophilic bacterium Moritella sp. strain 5710 exhibiting the highest sequence similarity were overexpressed in Escherichia coli. The 2D2 MDH was more heat-stable than the 5710 MDH. The apparent Km value at 62.1 MPa for NADH of the 2D2 MDH was higher than that of the 5710 MDH. The 2D2 MDH in which a His-Gln substitution was introduced at position 229 decreased the thermal stability and Km value at 62.1 MPa. The 5710 MDH that was substituted Gln-229 with His increased the thermal stability and Km value at 62.1 MPa. These results indicate that the His residue at position 229 of the 2D2 MDH may play a role in the thermal stability and the MDH function at high pressure.

Amino Acid Sequence↗
PubMed2006

Haplotype of thrombomodulin gene associated with plasma thrombomodulin level and deep vein thrombosis in the Japanese population.

INTRODUCTION: Thrombomodulin (TM) is an essential cofactor in protein C activation by thrombin. Here, we evaluated the contribution of genetic variations in the TM gene to soluble TM (sTM) level and deep vein thrombosis (DVT) in Japanese. PATIENTS AND METHODS: We sequenced the TM putative promoter, exon, and 3'-untranslated region in DVT patients (n=118). Among 17 genetic variations we identified, two missense mutations (R385K, D468Y) and three common single nucleotide polymorphisms (-202G>A, 2487A>T, 2729A>C) were genotyped in a general population of 2247 subjects (1032 men and 1215 women) whose sTM levels were measured. We then compared the frequency of these mutations in DVT patients with that in the age, body mass index-adjusted population-based controls. RESULTS: We identified one neutral mutation (H381) and three missense mutations (R385K; n=2, A455V; n=53 heterozygous, n=14 homozygous, D468Y; n=2) of TM in the DVT patients. Age-adjusted mean values of sTM were lower in C-allele carriers of 2729A>C than in noncarriers in the Japanese general population (women: 16.7+/-0.3 U/ml vs. 17.9+/-0.2 U/ml, p<0.01, men: 19.4+/-0.3 U/ml vs. 20.4+/-0.3 U/ml, p=0.03). Additionally, the CC genotype of this mutation was more common in the male DVT patients than in the male individuals of the general population (odds ratio=2.76, 95% confidence interval=1.14-6.67; p=0.02). This mutation was in linkage disequilibrium (r-square>0.9) with A455V mutation. CONCLUSIONS: TM mutations, especially those with a haplotype consisting of 2729A>C and A455V missense mutation, affect sTM levels, and may be associated with DVT in Japanese.

3' Untranslated Regions↗
PubMed2005

Definitive fossil evidence for the extant avian radiation in the Cretaceous.

Long-standing controversy surrounds the question of whether living bird lineages emerged after non-avian dinosaur extinction at the Cretaceous/Tertiary (K/T) boundary or whether these lineages coexisted with other dinosaurs and passed through this mass extinction event. Inferences from biogeography and molecular sequence data (but see ref. 10) project major avian lineages deep into the Cretaceous period, implying their 'mass survival' at the K/T boundary. By contrast, it has been argued that the fossil record refutes this hypothesis, placing a 'big bang' of avian radiation only after the end of the Cretaceous. However, other fossil data--fragmentary bones referred to extant bird lineages--have been considered inconclusive. These data have never been subjected to phylogenetic analysis. Here we identify a rare, partial skeleton from the Maastrichtian of Antarctica as the first Cretaceous fossil definitively placed within the extant bird radiation. Several phylogenetic analyses supported by independent histological data indicate that a new species, Vegavis iaai, is a part of Anseriformes (waterfowl) and is most closely related to Anatidae, which includes true ducks. A minimum of five divergences within Aves before the K/T boundary are inferred from the placement of Vegavis; at least duck, chicken and ratite bird relatives were coextant with non-avian dinosaurs.

Animals↗
PubMed2001

gmhX, a novel gene required for the incorporation of L-glycero-D-manno-heptose into lipooligosaccharide in Neisseria meningitidis.

Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of gmhX mutants by homologous recombination with the wild-type gmhX restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes; however, in N. meningitidis, gmhX was found in a location distinct from that of gmhA, rfaD, rfaE, aut and rfaC. GmhX is a novel enzyme required for the incorporation of L-glycero-D-manno-heptose into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-heptose phosphatase of the heptose biosynthesis pathway.

Bacterial Proteins↗
PubMed2006

Ochrobactrum oryzae sp. nov., an endophytic bacterial species isolated from deep-water rice in India.

A non-pigmented, motile, Gram-negative bacterium designated MTCC 4195(T) was isolated from surface-sterilized seeds and plant tissue from deep-water rice (Oryza sativa) cultivated in Suraha Tal Lake in northern India. This isolate was shown to reinfect and colonize deep-water rice endophytically. The highest level of 16S rRNA sequence similarity (96.8 %) to strain MTCC 4195(T) was shown by Ochrobactrum gallinifaecis DSM 15295(T). Strain MTCC 4195(T) utilized gamma-hydroxybutyric acid, adonitol, d-glucosaminic acid and arabinose as carbon sources, but failed to use gentiobiose or citrate. The cell-wall fatty acids of strain MTCC 4195(T) were characterized by the presence of a relatively large proportion of C(18 : 1)omega7c and a relative small proportion of C(16 : 0) in comparison with Ochrobactrum species. DNA-DNA relatedness studies showed less than 52 % binding with the DNAs of type strains of other species of the genus Ochrobactrum. On the basis of phenotypic and genotypic characteristics and the results of 16S rRNA gene sequence analysis, the novel species Ochrobactrum oryzae sp. nov. is proposed, with MTCC 4195(T) (=DSM 17471(T)) as the type strain.

Carbohydrate Metabolism↗
PubMed2025

Dosa: A method to covalently barcode proteins for high throughput biochemistry.

Deep mutational scanning couples a protein's activity to DNA sequencing for high throughput assessment of the effects of all single amino acid substitutions, but it largely uses indirect assays, like growth, as proxy for protein activity. Here, we covalently link variant proteins in vivo to an RNA barcode by fusing them to E. coli tRNA (m5U54) methyltransferase TrmA (E358Q), which forms a covalent bond with a tRNA stem-loop. Following cell lysis, variant proteins are separated in vitro according to their biochemical properties and identified by their barcodes. We use this method, Dosa, to analyze a large pool of FLAG epitope variants for binding to an anti-FLAG antibody, to profile the cleavage preferences of variants of enteropeptidase and human rhinovirus 3C protease, and to measure the solubility of several hundred A&#x3b2;(1-42) variants. This method should be amenable to numerous biochemical assays with proteins produced in E. coli or mammalian cells.

Protein display↗
PubMed1992

A maize protein associated with the G-box binding complex has homology to brain regulatory proteins.

The G-box element is a moderately conserved component of the promoter of many inducible genes, including the alcohol dehydrogenase genes of Arabidopsis and maize. We used monoclonal antibodies generated against partially purified G-box binding factor (GBF) activity to characterize maize proteins that are part of the DNA binding complex. Antibodies interacted with partially purified maize GBF complexes to produce a slower migrating complex in the gel retardation assay. Immunoprecipitation experiments suggested that the protein recognized by the antibody is not a DNA binding protein in and of itself, but rather is associated with the DNA binding complex. These monoclonal antibodies were used to isolate cDNA clones encoding a protein that we have designated GF14. Maize GF14 contains a region resembling a leucine zipper and acidic carboxy and amino termini, of which the latter can form an amphipathic alpha-helix similar to known transcriptional activators such as VP16 and GAL4. Protein gel blot analysis of cell culture extract showed that a single, major protein of approximately 30 kD is recognized by anti-GF14; the protein is also present predominantly in the kernel and root. The deduced amino acid sequence of maize GF14 is more than 80% identical to Arabidopsis GF14 and Oenothera PHP-O, and is more than 60% identical to a class of mammalian brain proteins described as both protein kinase C inhibitors and activators of tyrosine and tryptophan hydroxylases. GF14 is found in a variety of monocotyledons and dicotyledons, gymnosperms, and yeast. This suggests a deep evolutionary conservation of a potential regulatory protein associated with a core sequence found in the promoter region of many genes.

14-3-3 Proteins↗
PubMed2001

Impact of cultivation on characterisation of species composition of soil bacterial communities.

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Journal Article↗
PubMed2000

Isolation and piezoresponse of the rpoA gene encoding the RNA polymerase alpha subunit from the deep-sea piezophilic bacterium Shewanella violacea.

The rpoA gene encoding the alpha subunit of RNA polymerase from the deep-sea piezophilic bacterium Shewanella violacea DSS12 was cloned and sequenced. The rpoA gene was found to encode a polypeptide consisting of 329 amino acids with a molecular mass of 36238 Da. S. violacea alpha protein was expressed in a ts Escherichia coli mutant, to confirm whether the rpoA gene is functional. It complemented this mutation, indicating a chimeric RNA polymerase is assembled at the non-permissive temperature. Recombinant alpha protein was overexpressed using an expression plasmid harboring the rpoA gene and purified to near homogeneity. Primer extension analysis revealed that two transcriptional initiation sites are recognized by sigma(70) RNA polymerase. It also indicated that pressure response (piezoresponse) in the alpha operon occurred at the transcriptional level, suggesting some positive regulators may interact with the transcriptional apparatus and regulate the expression of the operon at different pressure conditions.

Amino Acid Sequence↗
PubMed1992

Atomic structure of the DNA repair [4Fe-4S] enzyme endonuclease III.

The crystal structure of the DNA repair enzyme endonuclease III, which recognizes and cleaves DNA at damaged bases, has been solved to 2.0 angstrom resolution with an R factor of 0.185. This iron-sulfur [4Fe-4S] enzyme is elongated and bilobal with a deep cleft separating two similarly sized domains: a novel, sequence-continuous, six-helix domain (residues 22 to 132) and a Greek-key, four-helix domain formed by the amino-terminal and three carboxyl-terminal helices (residues 1 to 21 and 133 to 211) together with the [4Fe-4S] cluster. The cluster is bound entirely within the carboxyl-terminal loop with a ligation pattern (Cys-X6-Cys-X2-Cys-X5-Cys) distinct from all other known [4Fe-4S] proteins. Sequence conservation and the positive electrostatic potential of conserved regions identify a surface suitable for binding duplex B-DNA across the long axis of the enzyme, matching a 46 angstrom length of protected DNA. The primary role of the [4Fe-4S] cluster appears to involve positioning conserved basic residues for interaction with the DNA phosphate backbone. The crystallographically identified inhibitor binding region, which recognizes the damaged base thymine glycol, is a seven-residue beta-hairpin (residues 113 to 119). Location and side chain orientation at the base of the inhibitor binding site implicate Glu112 in the N-glycosylase mechanism and Lys120 in the beta-elimination mechanism. Overall, the structure reveals an unusual fold and a new biological function for [4Fe-4S] clusters and provides a structural basis for studying recognition of damaged DNA and the N-glycosylase and apurinic/apyrimidinic-lyase mechanisms.

Bacterial Proteins↗
PubMed2003

Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.

A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAb's were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAb's before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAb's was associated with enhanced levels of IL-4, IL-6, and IFN-gamma in the lungs of infected mice. The mAb's increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAb's bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B-like protein. This study establishes that mAb's to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.

Amino Acid Sequence↗