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Results for “second generation sequencing”

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At least 991 records · Page 55Linked to original sources

PubMed2000

The chi-Chi earthquake sequence: active, out-of-sequence thrust faulting in taiwan

We combined precise focal depths and fault plane solutions of more than 40 events from the 20 September 1999 Chi-Chi earthquake sequence with a synthesis of subsurface geology to show that the dominant structure for generating earthquakes in central Taiwan is a moderately dipping (20 degrees to 30 degrees ) thrust fault away from the deformation front. A second, subparallel seismic zone lies about 15 kilometers below the main thrust. These seismic zones differ from previous models, indicating that both the basal decollement and relic normal faults are aseismic.

Journal Article↗

PubMed2007

Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells.

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer.

Animals↗

PubMed2003

Modulation of Toll-like Receptor 9 Responses through Synthetic Immunostimulatory Motifs of DNA.

Bacterial, plasmid, and synthetic DNA containing unmethylated CpG dinucleotides in specific sequence contexts activate the vertebrate innate immune system. A pattern recognition receptor (PRR), toll-like receptor 9 (TLR9), recognizes CpG DNA and activates signaling cascade leading to the secretion of a number of cytokines and chemokines. Our extensive structure-immunostimulatory activity relationship studies showed that a number of synthetic pyrimidine (Y) and purine (R) nucleotides are accepted by the receptor as substitutes for natural deoxycytidine and deoxyguanosine in a CpG dinucleotide. These studies permitted development of synthetic immunostimulatory motifs YpG, CpR, and YpR and established the nucleotide motif recognition pattern of the receptor. A number of site-specific chemical modifications in the flanking sequences to the CpG dinucleotide permitted modulation of immunostimulatory affects in a predictable manner. Our studies also showed that TLR9 recognizes and reads the CpG DNA sequence from the 5'-end. Design of oligonucleotides with two 5'-ends, immunomers, resulted in potent immunomodulatory agents with distinct cytokine profiles. Immunomers containing synthetic immunostimulatory motifs produced different cytokine induction profiles compared with natural CpG motifs. Importantly, some of these synthetic motifs showed optimal activity in both mouse and human systems without requiring to change sequences, suggesting overriding the species-dependent specificity of the receptor by the use of synthetic motifs. In this article, we review current understanding of structural recognition and functional modulation of TLR9 receptor by second-generation immunomodulatory oligonucleotides and their potential application as wide spectrum therapeutic agents.

3' Flanking Region↗

PubMed2002

Transfection of pancreatic-derived beta-cells with a minigene encoding for human glucagon-like peptide-1 regulates glucose-dependent insulin synthesis and secretion.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the proglucagon gene, capable of regulating the transcription of the three major genes that determine the pancreatic beta-cell-specific phenotype: insulin, GLUT-2, and glucokinase. The aim of this study was to investigate the potential role of GLP-1 for the gene therapy of glucose-insensitive pancreatic beta-cells. We transfected mouse insulinoma cells with a DNA fragment of the human proglucagon gene containing the nucleotide sequence encoding for human GLP-1 but lacking the coding region for glucagon. Two constructs were generated: In one, the expression of GLP-1 was under the control of the cytomegalovirus (CMV) promoter (CMV/GLP-1), and the second was regulated by the rat insulin II promoter (RIP)/GLP-1). Northern blot, HPLC, and RIA analyses confirmed that the minigene was transcribed and the protein appropriately translated, processed, and secreted in the extracellular environment. Gene expression studies revealed that although CMV/GLP-1 cells did not gain a greater glucose sensitivity as a result of the transfection with GLP-1, compared with cells transfected with the plasmid alone, RIP/GLP-1 was capable of regulating the gene expression of insulin and GLP-1 based on the concentration of glucose in the culture medium. Detection of the counterpart proteins (insulin and GLP-1) in the culture medium paralleled the observation derived from the Northern blot analysis. GLP-1 action was mediated by an IDX-1 (islet/duodenum homeobox-1) dependent transactivation of the endogenous insulin promoter, as demonstrated by gel shift analysis. This was further suggested by a significant increase of the glucose-dependent binding of IDX-1 to the insulin promoter in RIP/GLP-1 cells but not in CMV/GLP-1 cells or control cells. Finally, we observed that although the GLP-1-dependent secretion of insulin was mediated by an increase in cAMP levels, the transcription of the insulin gene, in response to GLP-1, was in large part cAMP independent. The present study lays the research foundation to investigate the potential use of GLP-1 for the gene or cell therapy of diabetes.

Animals↗

PubMed2000

Method for cloning restriction fragments containing the termini of BAC inserts.

We have developed a method to isolate the termini of BAC clones. The method is based on the two unique NotI sites located approximately 300 bp on either side of the EcoRI cloning site of the BAC vector pECS-BAC4. Our strategy includes the following steps: (i) generation of Southern blots with BAC clones digested with NotI and a second restriction enzyme; (ii) identification of the termini attached to the NotI/EcoRI fragment of the BAC vector via hybridization with a probe derived from sequences located between one NotI site (left or right arm) and the cloning site; (iii) ligation of the doubly digested BAC clone (NotI and the selected second restriction enzyme) with an equally doubly digested cloning plasmid vector; and (iv) confirmation of the clone as a terminus. This strategy has allowed us to begin the construction of a contig near a common bean gene that controls resistance to a group of potyviruses.

Blotting, Southern↗

PubMed1984

Two introns define functional domains of a neuropeptide precursor in Aplysia.

Biologically active peptides are synthesized as parts of precursor proteins which are proteolytically processed to generate active molecules. The structure of the gene encoding peptides expressed in Aplysia neurons R3-14 suggests that two intervening sequences split the transcript into functional domains. The first exon encodes the 5' untranslated region, the second exon the signal sequence and the bulk of the negative charge of the precursor protein while the third exon encodes the remainder of the precursor and the 3' untranslated region.

Animals↗

PubMed1988

Preferential amplification of rearranged sequences near amplified adenylate deaminase genes.

In a previous study of three independent families of mutants selected for overproduction of adenylate deaminase (AMPD), we were not able to isolate a cDNA probe for the gene and so could not demonstrate its amplification directly. In addition to overproduction of AMPD, four proteins of unknown function, designated W, X, Y1, and Y2, accumulated, and by using the corresponding cDNA probes, we demonstrated amplification of all four genes. In independent mutant clones, sometimes all and sometimes only a subset of these genes were amplified. Assuming that all five genes are linked, the pattern of their coamplification suggested a genetic map in which AMPD lies between W and Y1. We show here that a two-step chromosome walk joins the W and Y1 genes, that the AMPD gene is the only expressed sequence between them, and that its amplification is indeed responsible for overproduction of the AMPD protein. In the course of this work, we cloned and studied two novel joints which mark rearrangements on either side of the AMPD gene. Each joint was generated independently in a single first-step mutant at single or low copy number. Remarkably, each joint was amplified preferentially in every second- and third-step mutant derived from the first-step line in which it was originally present, suggesting that the two independent rearrangements each generated amplification-prone structures.

AMP Deaminase↗

PubMedDate not supplied

Molecular characterisation of antidepressant effects in the mouse brain using gene expression profiling.

Antidepressants are widely used for the treatment of psychiatric disorders, including depression and anxiety. Although they are efficient drugs, there are several unsolved questions regarding their clinical pharmacology. Furthermore, the molecular mechanisms of action of antidepressants are still poorly understood and the molecular targets and pathways remain to be identified. To address these issues, we performed a gene expression analysis in mice treated with two commonly used antidepressants with differing pharmacology (paroxetine or mirtazapine) for 1, 7 or 28 days. We quantified the effects of these treatments on gene expression in the mouse brain with cDNA-microarrays containing 3624 expressed sequence tags (ESTs) representing murine genes expressed in the brain. We found that both drugs led to downregulation of four common genes. In addition, although it was possible to identify common targets for the two drugs, the expression profiles of the drugs differed in a fundamental manner, and the longer the treatment duration, the greater the difference in the profiles. These findings suggest that antidepressants with different pharmacologies can share molecular targets even though the primary pathways at which they act are different.

Animals↗

PubMed2006

A decision model to compare health care costs of olanzapine and risperidone treatment for schizophrenia in Germany.

Second-generation atypical antipsychotics such as clozapine, olanzapine, risperidone, quetiapine, ziprasidone, amisulpride and ariprazole offer the potential to reduce the significant health care resource demands in the treatment of schizophrenia through improved levels of initial clinical response and reduced levels of long-term acute relapse. However, the optimal sequencing of these drugs remains unclear. To consider this issue from a health economic viewpoint a decision model approach was used comparing healthcare costs and clinical outcomes when treating patients with alternative sequences of atypical antipsychotic treatment. Treated patients were assumed to be in a current acute episode with at least a 10-year history of disease and to be naive to previous atypical treatments. Treatment strategies were based on either first-line olanzapine or risperidone with switching to the alternative drug as second-line treatment following an inadequate clinical response to first-line drug therapy. Clinical response data were derived from a pivotal published comparative study of both olanzapine and risperidone. Published data on the long-term use of antipsychotic drugs where used wherever possible to populate the model for relapse rates during the maintenance phase. Health care resource data were defined for Germany based on expert clinical opinion. A treatment strategy of first-line olanzapine was shown to be cost saving over a 1-year period, with additional clinical benefits in the form of avoided relapses. The model suggests that over the first year of treatment a strategy of first-line olanzapine is associated with lower risk of additional relapse (0.33 fewer acute relapses per 100 patients per year) and with cost savings (euro 35,306 per 100 patients per year). There is a need for longer term direct in-trial comparisons of atypical antipsychotics to confirm these indicative results.

Antipsychotic Agents↗

PubMed2006

Temporal grouping affects the automatic processing of deviant sounds.

The present study investigates the influence of auditory temporal grouping on automatic sound processing. We compared the mismatch negativity (MMN) event-related potential elicited by successive deviant tones in a condition in which sounds were organized in tone pairs with respective MMN obtained in a non-paired sequence. When successive deviants belonged to different tone pairs the second deviant elicited a higher MMN amplitude than the corresponding deviant in the non-paired condition. Moreover, this temporal grouping had immediate effects on the extraction of rules as revealed by the finding that first and single deviants at the second position of a tone pair elicited larger MMNs than corresponding deviants in the non-paired condition or first and single deviants at the first position of a tone pair. Results demonstrate that auditory objects generated by temporal proximity are treated as relatively encapsulated units and that automatic deviance-detection may rely on multiple hierarchically organized representations of sound sequences.

Adult↗

PubMed1991

Anterior and posterior association cortex contributions to the somatosensory P300.

A P300 (P3)-evoked response is generated in a variety of mammalian species upon detection of significant environmental events. The P3 component has been proposed to index a neural system involved in attention and memory capacity. We investigated the contribution of anterior and posterior association cortex to somatosensory P3 generation. Somatosensory event-related potentials (ERPs) were recorded in controls (n = 10) and patients with unilateral lesions in temporal-parietal junction (n = 8), lateral parietal cortex (n = 8), or dorsolateral frontal cortex (n = 10). Subjects pressed a button to mechanical taps of the fifth finger (targets; p = 0.12), randomly interposed in sequences of taps to the second (standards; p = 0.76) and the third or fourth finger (tactile novels; p = 0.06). Occasional shock stimuli were delivered to the wrist (shock novels; p = 0.06). The scalp-recorded P3 was differentially affected by anterior and posterior association cortex lesions. Subjects with temporal-parietal lesions showed markedly reduced P3s to all types of stimuli at all scalp locations. The reductions were largest at the parietal electrode site over the lesioned hemisphere. Parietal patients had normal P3s for all stimulus types except for contralateral shock novels, which generated reduced P3s. Frontal lesions had reductions of the novelty P3 over frontal sites with minimal changes in the target P3. The data support the existence of multiple intracranial P3 sources. The data further indicate that association cortex in the temporal-parietal junction is critical for generating the scalp-recorded target and novelty P3s, whereas dorsolateral frontal cortex contributes preferentially to novelty P3 generation. The N2 component was reduced by parietal and frontal lesions in patients who had intact target P3s, suggesting that different neural systems underlie N2 and P3 generation.

Aged↗

PubMed1994

Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting.

Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins. The first, InsA, is the product of insA. The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB. Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame. Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression. Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation. Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition. It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB. A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not. Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown. The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs.

Amino Acid Sequence↗

PubMed1998

Direct identification of a second distinct site of contact between cholecystokinin and its receptor.

We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.

Amino Acid Sequence↗

PubMed1991

Mutation and selection during the secondary response to 2-phenyloxazolone.

The most characteristic feature of the mouse antibody response to the hapten 2-phenyloxazolone is the recurrent expression of the light-chain variable region Igk-VO chi 1 gene in its germ-line or mutated configuration. The analysis of somatic mutants of the Igk-VO chi 1 gene reported here indicates that, as found during the primary response, hypermutation is also activated during the secondary response. Somatic mutations in the Igk-VO chi 1 gene increased in sequences obtained at day 14 and day 21 in the primary response and again in the secondary response at days 3, 5, and 7. The ratio of replacement to silent mutations also increased, particularly between days 5 and 7, suggesting that a stage of negative selection operates on new somatic mutants generated in the secondary response. Most Igk-VO chi 1 mutants isolated in the secondary response had the features of selected memory clones (i.e., they carried mutations known to increase binding affinity for the hapten). However, some clones had chain-termination codons, and others had mutations predicting a nonfunctional light chain. At least three and possibly five of these clones also expressed the mutation characteristic of the memory response to 2-phenyloxazolone (His-34----Asn-34/Gln-34). We conclude that after a second antigenic challenge, new somatic variants, including some leading to the loss of antigen binding, are generated by hypermutation of cells derived from the memory pool.

Amino Acid Sequence↗

PubMed1988

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.

Amino Acid Sequence↗

PubMed2001

Transient forces generated by projectiles on variable quality mouthguards monitored by instrumented impact testing.

OBJECTIVES: (a) To determine the force-time trace that occurs when a spring mounted simulated upper jaw is impacted; (b) to examine if mouthguards of variable quality have significant influence on such force-time traces; (c) to attempt to relate physical events to the profile of the force-time traces recorded. METHODS: A simulated jaw, consisting of ceramic teeth inserted into a hard rubber arch reinforced with a composite jawbone, was fitted with various mouthguards as part of a previous round robin study. A clinical assessment distinguished good, bad, and poor mouthguards, and these were each fitted to the jaw, which was then submitted to instrumental impact tests under conditions expected to produce tooth fractures. The force-time trace was recorded for such impact events. RESULTS: The spring mounting method caused two distinct peaks in the force-time trace. The initial one was related to inertia effects and showed an increase in magnitude with impactor velocity as expected. The second peak showed features that were related to the differences in the mouthguards selected. CONCLUSIONS: The use of a force washer within a conical ended impactor enabled force-time traces to be recorded during the impact of a spring mounted simulated jaw fitted with mouthguards of variable quality. The spring mounting system causes an initial inertial peak followed by a second peak once the spring mount has fully compressed. Good fitting guards, which keep most teeth intact, result in high stiffness targets that in turn generate high reaction forces in the impactor. If the spring mounting is omitted, the two peaks are combined to give even higher reaction forces. The force-time trace offers some potential for assessing both overall mouthguard performance and individual events during the impact sequence. Mouthguards with good retention to the jaw remained attached during the impact event and helped to preserve the structural integrity of the target. This in turn developed high forces in the second part of the force-time trace. Guards that detached during impact and allowed tooth fractures showed lower forces in the second part of the test. The force profile measured offered some quantitative support to, and agreement with, the observed clinical quality of the mouthguards.

Athletic Injuries↗

PubMed1988

Molecular cloning of two types of GAP complementary DNA from human placenta.

The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP. The messenger RNA of this GAP was detected in human lung, brain, liver, leukocytes, and placenta. The second type appeared to be generated by a differential splicing mechanism and encoded a novel form of GAP with a predicted molecular mass of 100,400 daltons. This protein lacks the hydrophobic amino terminus characteristic of the larger species, but retains GAP activity. The messenger RNA of this type was abundantly expressed in placenta and in several human cell lines, but not in adult tissues.

Amino Acid Sequence↗

PubMed2004

HIV-1 Vpr: genetic diversity and functional features from the perspective of structure.

RNA viruses are well known for the enormous genetic variation. Retroviruses share this feature with other RNA viruses, and human immunodeficiency virus type 1 (HIV-1) has been extensively investigated in this regard. Based on the DNA sequence analysis, HIV-1 has been classified into three groups; M, N, and O, with viral subtypes in each group. While the genetic variation between viral isolates has been documented throughout the genome, specifically, the env gene exhibits high variation. Analysis of the env gene from the sequential samples from HIV-1-infected patients reveals variation in the range of 1% per year. The variation observed in individual HIV-1 genes in the form of changes at the nucleotide level, as expected, should result in one of the possible scenarios: (1) no change in the amino acid, (2) conservative change in the amino acid, (3) nonconservative change in the amino acid, and (4) premature stop codon resulting in a truncated protein. Hence, it is likely that the variation may impact on the function of the protein, depending on the nature of the mutation. The goal of this review is to summarize the polymorphisms in Vpr using the available sequence information and discuss their effects on the functions of Vpr from the point of view of its structure. The data generated by several groups provide a base for understanding the consequences of natural polymorphisms in specific regions of the Vpr molecule. However, it is also clear that secondary changes (second site or compensatory mutations) may modify the effect of a specific mutation and a comprehensive analysis is needed to delineate the role of specific residues in Vpr molecule. This is an area which, we hope, will attract investigators for further studies, and may provide information for understanding the molecular basis of Vpr functions.

Acquired Immunodeficiency Syndrome↗