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Spatial patterning of response to odors in the peripheral olfactory system.

The low odor specificities of the olfactory receptors suggest that odor recognition depends on the simultaneous activity in an ensemble of receptor neurons. This ensemble could conceivably code quality without reference to the point of origin of each input on the receptor sheet. However, the nose-to-bulb projection appears sufficiently precise to provide the bulb with a topographical map of the receptor sheet although it is poorly delineated in the anteroposterior plane. (It is also known that the morphological changes that follow prolonged exposure to odors are more differentiated in the coronal than in the anteroposterior plane.) Furthermore, it is clear from work at both epithelial and bulbar levels that a spatiotemporal pattern of excitation is generated by odor stimulation of the receptor sheet and that this pattern differs for different odors. This evidence, then, supports the view that there is a spatial component to odor quality coding. This spatial pattern has two elements. One is imposed and depends (at least in part) on differences in the retentivity of different odorants by the mucous sheet, which has powerful sorptive properties. It effectiveness seems particularly weak for odorants with relatively long retention times. The second component is inherent and depends on the tendency of receptors with similar peak odor specificities to aggregate in the same region (or regions) of the epithelium. Different odors or groups of odors maximally excite different regions, which may overlap. The imposed component could not, in itself, provide an adequate mechanism for odor recognition, partly because many compounds have comparable or even identical mean retention times (e.g., enantiomeric isomers). The inherent component, on the other hand, possesses this potential. However, either or both forms of patterning may cooperate with a third nonspatial mechanism (based on differential responsiveness of receptors to different odors) in coding odor quality.

Animals↗

Transient phonemic codes and immunity to proactive interference.

Empirical data indicate that when memory for subspan lists of taxonomically related material is tested immediately after study, prior experience with lists involving the same material has no effect upon recall or recognition. In six experiments, we explored the possibility that immunity to proactive interference (PI) is related to discriminative information that is provided by transient phonemic codes. In these experiments, we manipulated the strength of phonemic codes as well as their presence or absence. Immunity to PI was found only when it was presumed that a phonemic representation of the target items existed and that information provided discriminative information. In all other cases, PI was observed. The finding that PI effects correspond with the manipulation of phonemic information in a principled fashion provides strong evidence for the role of phonemic codes in producing short-term PI effects.

Adult↗

DNA sequence specificity of the v-erb A oncoprotein/thyroid hormone receptor: role of the P-box and its interaction with more N-terminal determinants of DNA recognition.

The viral erb A oncogene is a mutated allele of a normal cell gene for a thyroid hormone receptor. The DNA recognition properties of the v-erb A protein are altered from those of the thyroid hormone receptor, due in part to a point mutation in the P-box of the zinc-finger domain of the viral allele. We report here the effects of systematically varying this P-box codon; our results suggest that this P-box amino acid contributes to DNA specificity not by promoting recognition of the appropriate response elements, but rather by excluding binding of the erb A protein to inappropriate half-sites. In this manner, DNA recognition by the v-erb A protein appears to differ from that by the glucocorticoid receptor. A variety of P-box amino acids were compatible with recognition of the prototypic AGGTCA half-site; intriguingly, several of these mutant erb A proteins could also recognize a variety of alternative half-site sequences. Recognition of these alternative half-sites required a compatible amino acid sequence in the N terminus of the erb A protein. Our results begin to define a code by which the identity of the amino acids in the zinc-finger and N-terminal domains is reflected in the DNA recognition properties of the receptor.

Amino Acid Sequence↗

Differential amplification of pheromone genes of the ciliate Euplotes raikovi.

In hypotrich ciliates, the entire silent chromosomal genome of the germinal nucleus (micronucleus) undergoes extensive DNA rearrangements that, during the development of the somatic nucleus (macronucleus) at the beginning a new cell life cycle, eventually result in the production of linear DNA molecules. These molecules represent functional genes, each one consisting of a central coding region flanked by two shorter regions, which apparently lack canonical elements for regulation of replication and transcription. These are amplified to thousands of copies in the "adult" macronucleus of the vegetative cell. We defined the extent of this amplification for allelic codominant genes which, in the macronucleus of Euplotes raikovi, encode polypeptide cell recognition factors (pheromones). This amplification was shown to be allele-specific. The copy numbers of genes coding for pheromones Er-1, Er-2, and Er-10 were determined to be 2.5-2.9 x 10(4), 0.9-1.2 x 10(4), 1.6-1.85 x 10(4) respectively, and these numbers did not appreciably vary during the vegetative cell proliferation. This differential amplification of pheromone genes was (i) independent of whether two genes coexisted in the same heterozygous cell or were separated in the corresponding homozygotes, and (ii) directly correlated with quantitative variations in mRNA synthesis and pheromone secretion. On the basis of these results, it is suggested that a mechanism of gene-specific amplification may be used by hypotrich ciliates to modulate gene expression.

Animals↗

Speech recognition for 40 patients receiving multichannel cochlear implants.

We collected data on 40 patients who received the Nucleus multichannel cochlear implant. Results were reviewed to determine if the coding strategy is effective in transmitting the intended speech features and to assess patient benefit in terms of communication skills. All patients demonstrated significant improvement over preoperative results with a hearing aid for both lipreading enhancement and speech recognition without lipreading. Of the patients, 50% demonstrated ability to understand connected discourse with auditory input only. For the 23 patients who were tested 12 months postoperatively, there was substantial improvement in open-set speech recognition.

Adult↗

Identification of HLA class I dependent immunogenic peptides from clonotypic TCRbeta expressed in cutaneous T-cell lymphoma.

The clonotypic T-cell receptor (TCR) is a potential target antigen for specific immunotherapy of cutaneous T-cell lymphoma (CTCL). We identified T-cell epitopes from the rearranged TCR beta chain of the malignant T-cell population by the "reverse immunology" approach. Peptide-specific T-cell lines were generated against predicted epitopes and tested for the recognition of tumor cells and cells transfected with the full-length DNA coding for TCRV beta chain. Two peptides derived from the clonotypic TCRVbeta of a HLA-A2 positive patient could induce peptide-specific T cells from peripheral blood mononuclear cells of healthy donors and the patient as assessed by IFN-gamma ELISpot assay. Furthermore, the reactive CTLs efficiently recognized autologous Sézary tumor cells, as well as HLA-A2 positive 293 cells transfected with recombinant plasmid expressing the corresponding TCRVbeta29 protein. Similar results were obtained in a HLA-A3+ patient for TCRVbeta7-Jbeta2.7. In conclusion, our experiments show that the TCR beta chain harbors epitopes suitable as targets for specific vaccination which might be a promising approach for the specific immunotherapy of cutaneous T-cell lymphoma patients.

Epitopes↗

Lhx9 and Lhx9alpha LIM-homeodomain factors: genomic structure, expression patterns, chromosomal localization, and phylogenetic analysis.

Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9alpha, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3'-coding and 3'-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9alpha are coded by six exons spanning 10 kb of genomic sequence and that Lhx9alpha is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9alpha, the expression patterns of Lhx9 and Lhx9alpha were compared during embryogenesis. Lhx9alpha was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9alpha could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code.

3' Untranslated Regions↗

Naturally occurring nucleosome positioning signals in human exons and introns.

We describe the structural implications of a periodic pattern found in human exons and introns by hidden Markov models. We show that exons (besides the reading frame) have a specific sequential structure in the form of a pattern with triplet consensus non-T(A/T)G, and a minimal periodicity of roughly ten nucleotides. The periodic pattern is also present in intron sequences, although the strength per nucleotide is weaker. Using two independent profile methods based on triplet bendability parameters from DNase I experiments and nucleosome positioning data, we show that the pattern in multiple alignments of internal exon and intron sequences corresponds to a periodic "in phase" bending potential towards the major groove of the DNA. The nucleosome positioning data show that the consensus triplets (and their complements) have a preference for locations on a bent double helix where the major groove faces inward and is compressed. The in-phase triplets are located adjacent to GCC/GGC triplets known to have the strongest bias in their positioning on the nuclesome. Analysis of mRNA sequences encoding proteins with known tertiary structure exclude the possibility that the pattern is a consequence of the previously well-known periodicity caused by the encoding of alpha-helices in proteins. Finally, we discuss the relation between the bending potential of coding and non-coding regions and its impact on the translational positioning of nucleosomes and the recognition of genes by the transcriptional machinery.

Base Sequence↗

A novel method to develop highly specific models for regulatory units detects a new LTR in GenBank which contains a functional promoter.

Functional promoters are composed of individual modules (e.g. transcription factor binding sites, secondary structure elements, repeats) arranged in distinct patterns. Recognition of such patterns is essential for identification of promoters in non-coding sequences. However, this is difficult due to the absence of overall sequence similarity in promoters even if they are regulated in a similar way. We implemented simple formal representations of general features of regulatory regions into an algorithm capable of developing complex models reflecting both the element composition and the functional organization of individual elements (ModelGenerator). Though ModelGenerator requires a very simple initial model (e.g. two modules and their relative order) it will generate a much more sophisticated model by analysis of the training set of at least ten sequences. We show ModelGenerator to successfully model different retroviral long terminal repeat (LTR) classes (Lentivirus as well as avian and mammalian C-type) which contain functional promoters. Database searches with the program ModelInspector demonstrated the high specificity of these models and no apparent false negatives were detected. We also verified one match from GenBank to the mammalian C-type LTR model experimentally and showed this sequence to contain an active promoter. Thus, the concept of modular organization of functional regulatory DNA regions (e.g. promoters) could be successfully implemented into a set of computer tools which might be flexible and specific enough to be suitable for prospective analysis of new genomic DNA sequences.

Algorithms↗

Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli.

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.

Alkaline Phosphatase↗

Genetic epidemiology of MODY in the Czech republic: new mutations in the MODY genes HNF-4alpha, GCK and HNF-1alpha.

AIMS/HYPOTHESIS: The aim of this study was to examine the prevalence and nature of mutations in HNF4alpha/MODY1, GCK/MODY2 and HNF-1alpha/MODY3 genes in Czech subjects with clinical diagnosis of MODY. METHODS: We studied 61 unrelated index probands of Czech origin (28 males, 33 females) with a clinical diagnosis of MODY and 202 family members. The mean age of probands was 22.7+/-12.0 years (range, 6-62) and the mean age at the first recognition of hyperglycaemia was 14.7+/-6.0 years (range, 1-25). The promotor and coding regions inclusive intron exon boundaries of the HNF-4alpha, GCK and HNF-1alpha genes were examined by PCR-dHPLC (HNF-1alpha and GCK) and direct sequencing. RESULTS: We identified 20 different mutations in the HNF-4alpha, GCK and HNF-1alpha in 29 families (48% of all families studied), giving a relative prevalence of 5% of MODY1, 31% of MODY2 and 11.5% of MODY3 among the Czech kindred with MODY. Three of 3, 10 of 11 and 1 of 6 of the mutations identified in HNF-4alpha, GCK and HNF-1alpha respectively, were new. CONCLUSION/INTERPRETATION: Of the families 48% carried mutations in the MODY1-3 genes and of the identified mutations 70% were new. In 52% of Czech families with clinical characteristics of MODY, no mutations were found in the analysed genes. This finding shows that the majority of MODY mutations in a central European population are local and that other MODY genes could be responsible for autosomal dominant transmission of diabetes mellitus.

Adolescent↗

A description of the Mei2-like protein family; structure, phylogenetic distribution and biological context.

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.

Amino Acid Sequence↗

Enterococcus faecalis GP1764 induces an early differential gene expression in the intestine on key pathways related to cellular immune response and gut barrier function in chickens.

The aim of the present study was to elucidate the mode of action of Enterococcus faecalis GP1764 in improving performance traits during the starter phase by analyzing genome-wide gene expression and its interaction with microbial populations in the intestine of chickens challenged with an NSP-rich diet. At day 7, microbiota populations from ileal and cecal contents and transcriptomics from jejunal and cecal mucosa were analyzed between Control (Ctrl) and Enterococcus faecalis GP1764 (EntF) groups. Results from microbiota analysis demonstrated that EntF shifted β-diversity indices in ileum (neutral (p= 0.006) and phylogenetic (p= 0.006)) and caecum (phylogenetic (p= 0.017)). Transcriptomics revealed 43 differentially expressed genes for EntF vs. Ctrl in the jejunal mucosa. Of these, MHCY-36 (MHC-I-Related), RAG2 and MUC19-like genes were upregulated in EntF vs. Ctrl, protein-coding genes with immunomodulatory capacities as supported by GSEA and Cytoscape-ClueGo pathway analyses. Results suggest an intestinal immunomodulation induced through presentation of B vitamins metabolites, synthetized by EntF, to an undescribed subset of innate-like unconventional T lymphocytes in chickens, similar to MAIT cells in mammals. These cells could contribute to antibacterial responses and repair of damaged barrier tissue after inflammatory processes. The upregulation of the MUC19-like gene expression observed in the jejunal mucosa can protect gut integrity via the promotion of mucus production by goblet cells. Finally, RAG2, involved in V(D)J coding segments recombination in B- and T-cells may provide a greater recognition of foreign invaders, allowing the animals to efficiently fight against pathogenic infections. Collectively, these results suggest an important role of EntF in promoting the capacity of animals to rapidly act against pathogenic challenges, herein, inducing resilience towards dietary ingredients with anti-nutritional activity that impart moderate inflammation in chickens.

Enterococcus faecalis↗

Structures of protein domains that create or recognize histone modifications.

DNA is packed together with histone proteins in cell nuclei to form a compact structure called chromatin. Chromatin represents a scaffold for many genetic events and shows varying degrees of condensation, including a relatively open form (euchromatin) and a highly condensed form (heterochromatin). Enzymes such as histone acetyltransferases (HATs) and methylases covalently label the amino-termini of histones, thereby creating a 'histone code' of modifications that is interpreted by the recruitment of other proteins through recognition domains. Ultimately, this network of interacting proteins is thought to control the degree of chromatin condensation so that DNA is available when it is required for genomic processes. Reviewed here are the structures of HAT and SET domains, which mediate the acetylation and methylation of histones, respectively, and bromodomains and chromodomains, which recognize the modified histones. How these structures have increased our understanding of DNA regulation is also discussed.

Acetylation↗

High-frequency deletional rearrangement of immunoglobulin kappa gene segments introduced into a pre-B-cell line.

We describe an immunoglobulin gene recombination indicator in which a specific rearrangement via deletion results in the acquisition of a dominant phenotype. The indicator consists of the Escherichia coli xanthine/guanine phosphoribosyltransferase (gpt) gene, whose translation is prevented by the presence of an upstream initiation codon out of frame with respect to the gpt coding sequence. Flanking this barrier initiation codon are the heptamer-spacer-nonamer recognition sequences from a kappa chain variable region (V kappa) and from a kappa chain joining region (J kappa). A proper V-J joint results in the deletion of the translational barrier and allows expression of the selectable marker. When tested by transfection into fibroblasts, no rearrangements were detected and the presence of the barrier initiation codon was sufficient to completely abolish gpt expression in these cells. Similarly, no rearrangements were detected after transfer of the test gene into myeloma cells. However, when the construct was introduced into the pre-B-cell line 38B9, greater than 80% of the transfected cells showed evidence of a specific rearrangement. These rearrangements were associated with the translation of gpt, although no selection for its expression was needed. DNA sequence analysis of six different V-J joints revealed that the rearrangement proceeded with a high degree of accuracy. These results indicate that only very minimal DNA sequences (21 base pairs 5' of the V heptamer and 4 base pairs 3' of its nonamer; less than 45 base pairs 5' of the J nonamer and 3' of its heptamer) are required for efficient rearrangement and provide formal proof that kappa gene segments can rearrange by a deletional mechanism.

Antibody Diversity↗

New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study.

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (lambdas = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5.10(-5)) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22-23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0. 001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

Arthritis, Rheumatoid↗

Identification of differentially expressed genes in Malus domestica after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere.

Biological control of plant diseases by the application of antagonistic micro-organisms to the plant phyllosphere is only marginally understood. Suppression subtractive hybridization (SSH) was used for the identification of genes expressed after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the phyllosphere of the apple scab-susceptible cultivar Malus domestica cv. Holsteiner Cox. In total, 157 expressed sequence tag (EST) clones were obtained. The sequencing of 113 ESTs which have a significantly elevated transcript level and the comparison of the obtained sequences with databases revealed similarities to different classes of pathogenesis-related proteins, for example, RNase-like PR10 protein and endochitinase, or similarities to proteins expressed under stress conditions that could have a protective function, for example, a germin-like protein, glutathione S-transferase, thioredoxin-like proteins, and heat shock proteins. In addition, several transcripts were identified that code for proteins which have a crucial role at different stages of pathogen recognition and in signalling pathways or an as yet unknown function in plant defence. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated after pathogen infection are also up-regulated after the application of a non-pathogenic bacterium to a M. domestica cultivar. The expression of these proteins might increase the plant resistance towards pathogen infection and damage.

Blotting, Northern↗

Identification of synthetic vowels by patients using the Symbion multichannel cochlear implant.

In this report we describe the vowel identification ability of eight patients who scored above 70% correct on a test of spondee word identification. The stimuli were 12 synthetic vowels in "bVt" format. The vowels differed in the frequency of F1, F2, and F3. The mean identification accuracy was 60% correct. Front vowels and dipthongs, (3), and (u) were relatively well identified. The vowels in "but," "bought," and "bout," which were characterized by high F1s and low F2s were not well identified. The results are consistent with a model of recognition in which F1 is specified, with relatively good accuracy, by a rate code and in which extreme values of F2 are specified by a rate/place code.

Adolescent↗