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PubMed1993

The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity.

The genome of flaviviruses consists of an infectious single-stranded RNA molecule which contains a type 1 cap structure at the 5'-terminus. The cap is synthesized by RNA triphosphatase, guanylyltransferase and methyltransferase. Since flaviviruses replicate in the cytoplasm, it can be assumed that these functions are performed by virus-coded proteins. We previously showed that subtilisin treatment of membranes isolated from cells infected with the West Nile flavivirus results in release of a 50 kDa molecular weight fragment of the viral nonstructural protein NS 3. This so-called p50-S protein contains the residue gly (168) of NS 3 at the amino-terminus and represents an RNA-stimulated NTPase. In the present report we present experimental evidence which indicates that the p50-S protein also contains the active site of an RNA triphosphatase. The activity specifically cleaves the beta,gamma-triphosphate bond at the 5'-terminus of RNA. The localization of NS 3 protein sequence elements with known functions indicates that this multifunctional protein contains a protease in the amino-terminal part, a helicase in the central region and the RNA triphosphatase in the carboxy-terminal domain. An amino acid sequence element which may be involved in recognition of the 5'-terminal RNA triphosphate is tentatively identified. A homologous element may be present in the vaccinia virus-coded RNA triphosphatase.

Acid Anhydride Hydrolases↗

PubMed1997

On wearing two hats: role conflict in serving as both psychotherapist and expert witness.

OBJECTIVE: This article explores the clinical, legal, and ethical problems that typically occur when a psychotherapist serves as both a treating clinician and forensic evaluator (or expert witness) in the same case. METHOD: The professional literature, ethics codes, opinion surveys, and the changing economic and institutional contexts of psychotherapy are reviewed in order to identify obstacles to widespread recognition of this straightforward ethical issue. The processes of psychotherapy and forensic evaluation are then analyzed so as to reveal fundamental incompatibilities between the psychotherapist's clinical and legal functions. RESULTS: Attempting to treat and evaluate the same person typically creates an irreconcilable role conflict. This role conflict manifests itself in different conceptions of truth and causation, different forms of alliance, different types of assessment, and different ethical guidelines. CONCLUSIONS: Although circumstances sometimes compel a practitioner to assume the dual role of treater and evaluator, the problems that surround this practice argue for its avoidance whenever possible.

Attitude of Health Personnel↗

PubMed2000

DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection.

Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.

Amino Acid Sequence↗

PubMed1999

Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA.

Previous studies have indicated that proopiomelanocortin (POMC) is translationally regulated. We proposed that the regulatory mechanism involves an interaction between trans-acting protein factors and a cis-acting stem-loop structure in the coding region of POMC mRNA. Functional interactions were tested by examining the translation of mouse POMC mRNA in a rabbit reticulocyte system. Specific binding was demonstrated with ultraviolet-crosslinking and RNA gel mobility shift assays. The evidence presented supports our hypothesis that the translational regulation of POMC gene expression involves recognition of the stem-loop by RNA-binding proteins. Furthermore, POMC stem-loop RNA-binding proteins specifically recognized a predicted stem-loop found in the coding region of corticotropin-releasing hormone, suggesting a novel mechanism of gene regulation that may extend to other neuropeptides as well.

Animals↗

PubMed2002

Does "whole-word shape" play a role in visual word recognition?

To analyze the impact of outline shape on visual word recognition, the visual pattern of the stimuli can be distorted by size alternation. Contrary to the predictions of models that rely on outline shape (Allen, Wallace, & Weber, 1995), the effect of size alternation was greater for low-frequency words than for high-frequency words in a lexical decision task (Experiment 1). In Experiment 2, the effect of case type (lowercase vs. UPPERCASE) occurred for low-frequency words, but not for high-frequency words. The effect of neighborhood size was remarkably similar in the two experiments. The results can be readily explained in the framework of a resonance model (Grossberg & Stone, 1986), in which a mismatch between the original sensory pattern and the abstract orthographic code slows down the formation of a stable percept.

Attention↗

PubMed1984

Recognition of HLA-A2 and -B7 antigens by cloned cytotoxic T lymphocytes after gene transfer into human and monkey, but not mouse, cells.

The genes that code for the human major histocompatibility class I antigens, HLA-A2 and HLA-B7, were introduced into human, monkey, and mouse cell lines by cotransfection with suitable biochemical markers and the fluorescence-activated cell sorter was used to identify and/or select stable cell populations expressing high surface levels of these antigens. Levels of expression obtained were similar to those observed for endogenous HLA antigens on various human cell lines and were 25-80% of those observed on the human B-lymphoblastoid cell line JY. Serologically defined HLA-A2 and HLA-B7 polymorphic determinants remained intact on all transfected recipient cells analyzed. Cloned human allospecific cytotoxic T lymphocytes (CTL) specific for HLA-A2 or HLA-B7 were capable of lysing appropriate HLA-transfected human cells with comparable efficiency to JY cell lysis. Two of 10 CTL clones lysed appropriate monkey cell transfectants with approximately equal to 20% the efficiency of human cell transfectants. No specific lysis of any HLA-transfected mouse cell lines, including a B cell lymphoma, was observed despite comparable levels of surface antigen expression or after induction of higher levels by mouse gamma-interferon. Furthermore, L cells expressing human beta 2-microglobulin in addition to HLA-A2 or -B7 were not lysed by these CTL. Thus, an additional species-specific component may be involved in lysis by allogeneic CTL--possibly related to the function(s) of other surface proteins on target cells.

Animals↗

PubMed1985

A model of the neural mechanisms responsible for pattern recognition and stimulus specific habituation in toads.

A neural model of the mechanisms possibly responsible for stimulus-specific habituation in toads is proposed. The model follows the hypothesis that prey-predator recognition is performed by command units as a result of retina-tectum-pretectum interaction. The model allow us to study the possible coding that the nervous system of toads uses for different prey stimuli, the neural mechanisms of habituation and dishabituation, and the dynamic changes that the command units may have during these processes. The model proposes specific hypothesis and experiments to clarify the nature of these processes and to test the validity of the command unit hypothesis.

Animals↗

PubMed1991

The sequence of a 6.3 kb segment of yeast chromosome III reveals an open reading frame coding for a putative mismatch binding protein.

We report the sequence of a 6.3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified. The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.

Amino Acid Sequence↗

PubMed1997

Bridging the gap between monkey neurophysiology and human perception: an ambiguity resolution theory of visual selective attention.

When the visual system must process multiple objects simultaneously, as in the visual search paradigm, the neural coding of individual objects can become ambiguous due to the visual system's extensive use of coarse coding and distributed representations. Here we propose that the primary role of visual selective attention within the ventral object recognition pathway is to resolve these ambiguities. We begin by reviewing previous studies of the effects of attention on neural responses in monkeys, which provide the basis for this hypothesis, and then describe a new set of experiments showing that similar attentional mechanisms operate in the human brain. In these new experiments, event-related potentials (ERPs) were recorded from normal human observers while they performed tasks analogous to those used previously in monkeys. The central finding was that an attention-related ERP wave called the "N2pc component" was present under the same conditions that led to attentional modulations of neural responses in monkey visual cortex. These human electrophysiological results provide a bridge between cognitive-level theories of visual attention and the behavior of individual neurons in visual cortex.

Adolescent↗

PubMed2006

Illusory conjunctions in simultanagnosia: coarse coding of visual feature location?

Simultanagnosia is a disorder characterized by an inability to see more than one object at a time. We report a simultanagnosic patient (ED) with bilateral posterior infarctions who produced frequent illusory conjunctions on tasks involving form and surface features (e.g., a red T) and form alone. ED also produced "blend" errors in which features of one familiar perceptual unit appeared to migrate to another familiar perceptual unit (e.g., "RO" read as "PQ"). ED often misread scrambled letter strings as a familiar word (e.g., "hmoe" read as "home"). Finally, ED's success in reporting two letters in an array was inversely related to the distance between the letters. These findings are consistent with the hypothesis that ED's illusory reflect coarse coding of visual feature location that is ameliorated in part by top-down information from object and word recognition systems; the findings are also consistent, however, with Treisman's Feature Integration Theory. Finally, the data provide additional support for the claim that the dorsal parieto-occipital cortex is implicated in the binding of visual feature information.

Aged↗

PubMed1994

Conformational change in antithrombin induced by heparin probed with a monoclonal antibody against the 1C/4B region.

A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2003

Construction of a new tumour necrosis factor fusion-protein expression vector for high-level expression of heterologous genes in Escherichia coli.

We report the construction and application of a new fusion-protein expression plasmid (TNFHis) for Escherichia coli. The plasmid contains both P(R) and P(L) promoters and is optimized to allow a higher level of expression of mature coding sequences. It also contains a six-histidine tag for convenient purification as well as thrombin and hydroxylamine recognition sites for cleaving heterologous protein. The potential use of this expression vector is demonstrated by comparing the expression levels of human tumour necrosis factor (TNF), interferon, interleukin 11, colony-forming factor, osteoprotegrin and interleukin 2 in E. coli. Furthermore, all expressed TNF fusion proteins can be detected by anti-TNF alpha antibody or by specific antibodies and purified by Ni(2+)-nitrilotriacetate beads. The expressed TNF fusion proteins can be cleaved by hydroxylamine.

Escherichia coli↗

PubMed1986

Previous experience as a confounding factor in comparing cochlear-implant processing schemes.

It is of great importance to compare the relative merits of different cochlear-implant speech-processing strategies. Some groups have compared different strategies within single subjects, but usually the subject has prior experience with one strategy, and no allowance is made for this prior experience. We show in the present study that this is inappropriate. We tested one subject using the Melbourne (Cochlear Corp.) multichannel implant with the device set to process sounds in two different ways. In the first processing scheme, the device functioned normally, extracting information about voicing frequency, amplitude and second-formant frequency. This information activated the 21-channel device, determining pulse rate, pulse amplitude and electrode position (respectively). In the second processing scheme, a single electrode (with the largest dynamic range) was activated. This electrode coded overall amplitude and voicing frequency. The subject was tested on an audiovisual test of a 14-choice consonant recognition in the form /iCi/ over a period of over 4 months. During this time the subject used the 21-channel processor outside of the laboratory. Upon initial connection, there was little difference between the results obtained with the two schemes when tested in sound alone or in sound plus vision. However, after about 4 months, scores obtained with the 21-channel processor in sound plus vision were superior to the scores obtained with the one channel. This advantage came from a superiority in the features of voicing and nasality, but not place. Scores for sound-alone conditions between the two processing schemes remained similar for the 4-month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Auditory Threshold↗

PubMed1995

A rapid access motif database (RAMdb) with a search algorithm for the retrieval patterns in nucleic acids or protein databanks.

We present here a codification structure, entirely interfaced with the main packages for biomolecule database management, associated with a new search algorithm to retrieve quickly a sequence in a database. This system is derived from a method previously proposed for homology search in databanks with a preprocessed codification of an entire database in which all the overlapping subsequences of a specific length in a sequence were converted into a code and stored in a hash-coding file. This new algorithm is designed for an improved use of the codification. It is based on the recognition of the rarest strings which characterize the query sequence and the intersection of sorted lists read in the codification structure. The system is applicable to both nucleic acid and protein sequences and is used to find patterns in databanks or large sets of sequences. A few examples of applications are given. In addition, the comparison of our method with existing ones shows that this new approach speeds up the search for query patterns in large data sets.

Algorithms↗

PubMed1999

Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements.

U6 spliceosomal RNA has a complex secondary structure that includes a highly conserved stemloop near the 3' end. The 3' stem is unwound when U6 RNA base-pairs with U4 RNA during spliceosome assembly, but likely reforms when U4 RNA leaves the spliceosome prior to the catalysis of splicing. A mutation in yeast U6 RNA that hyperstabilizes the 3' stem confers cold sensitivity and inhibits U4/U6 assembly as well as a later step in splicing. Here we show that extragenic suppressors of the 3' stem mutation map to the gene coding for splicing factor Prp24. The suppressor mutations are located in the second and third of three RNA-recognition motifs (RRMs) in Prp24 and are predicted to disrupt RNA binding. Mutations in U6 RNA predicted to destabilize a novel helix adjacent to the 3' stem also suppress the 3' stem mutation and enhance the growth defect of a suppressor mutation in RRM2 of Prp24. Both phenotypes are reverted by a compensatory mutation that restores pairing in the novel helix. These results are best explained by a model in which RRMs 2 and 3 of Prp24 stabilize an extended intramolecular structure in U6 RNA that competes with the U4/U6 RNA interaction, and thus influence both association and dissociation of U4 and U6 RNAs during the splicing cycle.

Amino Acid Sequence↗

PubMed2005

NKp30 (NCR3) is a pseudogene in 12 inbred and wild mouse strains, but an expressed gene in Mus caroli.

Ancient duplications and rearrangements of protein-coding segments have resulted in complex gene family relationships. As a result, gene products may acquire new specificities, altered recognition properties, modified functions, and even loss of functionality. The natural cytotoxicity receptor (NCR) family are natural killer (NK)-activating receptors whose members are NKp46 (NCR1), NKp44 (NCR2), and NKp30 (NCR3). The NCR proteins are putative immunoglobulin superfamily members whose ligands are unknown. The NKp46 gene is present and expressed in human and mouse, NKp44 is only present and expressed in human, and NKp30 is present and expressed in human but is a nonexpressed pseudogene in mouse. By searching databases we have detected alternatively spliced forms of the three NCR members. In addition, we have shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis that the human NKp30 gene presents differential expression patterns in tissues. However, no expressed sequence tags (ESTs) are detected for mouse NKp30, and the genomic sequence contains two premature stop codons, which would encode a severely truncated nonfunctional protein. We have sequenced genomic DNA from 13 mouse inbred and wild strains and discovered that NKp30 is a pseudogene in every mouse strain sequenced except Mus caroli where two single nucleotide polymorphisms (SNPs) abolished the premature stop codons. We observed that the laboratory-inbred strains are, for the exonic sequences, genetically identical, except Mus m. musculus C3H. The Mus musculus strains only have a few SNPs, but the rest of the Mus strains have accumulated gradually several SNPs, mainly in the functional immunoglobulin and intracellular domains. RT-PCR analysis performed on RNA from M. caroli tissue samples identified two transcripts, one of which would encode a putative soluble NKp30 protein, also detected in rat but not in human. We have observed that the intracellular domains of NKp30 (and NKp46) are not conserved among the different species, with the most striking difference when comparing human against mouse and rat. The NKp44 gene is only found in human and shows three different splice forms varying in their "stalk" and intracellular domains. Searching for NKp44 orthologs, we found similarity to ESTs from a novel rodent TREM family member, which we termed TREM6, and not to any possible NKp44 ortholog.

Alternative Splicing↗

PubMed2003

A sensitive transcriptome analysis method that can detect unknown transcripts.

We have developed an AFLP-based gene expression profiling method called 'high coverage expression profiling' (HiCEP) analysis. By making improvements to the selective PCR technique we have reduced the rate of false positive peaks to approximately 4% and consequently the number of peaks, including overlapping peaks, has been markedly decreased. As a result we can determine the relationship between peaks and original transcripts unequivocally. This will make it practical to prepare a database of all peaks, allowing gene assignment without having to isolate individual peaks. This precise selection also enables us to easily clone peaks of interest and predict the corresponding gene for each peak in some species. The procedure is highly reproducible and sensitive enough to detect even a 1.2-fold difference in gene expression. Most importantly, the low false positive rate enables us to analyze gene expression with wide coverage by means of four instead of six nucleotide recognition site restriction enzymes for fingerprinting mRNAs. Therefore, the method detects 70-80% of all transcripts, including non-coding transcripts, unknown and known genes. Moreover, the method requires no sequence information and so is applicable even to eukaryotes for which there is no genome information available.

Animals↗

PubMed2001

Mutational analysis of the Arabidopsis RPS2 disease resistance gene and the corresponding pseudomonas syringae avrRpt2 avirulence gene.

Plants have evolved a large number of disease resistance genes that encode proteins containing conserved structural motifs that function to recognize pathogen signals and to initiate defense responses. The Arabidopsis RPS2 gene encodes a protein representative of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of plant resistance proteins. RPS2 specifically recognizes Pseudomonas syringae pv. tomato strains expressing the avrRpt2 gene and initiates defense responses to bacteria carrying avrRpt2, including a hypersensitive cell death response (HR). We present an in planta mutagenesis experiment that resulted in the isolation of a series of rps2 and avrRpt2 alleles that disrupt the RPS2-avrRpt2 gene-for-gene interaction. Seven novel avrRpt2 alleles incapable of eliciting an RPS2-dependent HR all encode proteins with lesions in the C-terminal portion of AvrRpt2 previously shown to be sufficient for RPS2 recognition. Ten novel rps2 alleles were characterized with mutations in the NBS and the LRR. Several of these alleles code for point mutations in motifs that are conserved among NBS-LRR resistance genes, including the third LRR, which suggests the importance of these motifs for resistance gene function.

Alleles↗