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PubMed1997

Effects of mutations within and adjacent to the terminal repeats of hepatitis B virus pregenomic RNA on viral DNA synthesis.

The viral polymerase and several cis-acting sequences are essential for hepadnaviral DNA replication, but additional host factors are likely to be involved in this process. We previously identified two sequences, UBS and DBS (upstream and downstream binding sites), present in multiple copies in and adjacent to the pregenomic RNA (pgRNA) terminal redundancy, that were specifically recognized by a 65-kDa host factor, p65. The possible roles of these two sequences in hepatitis B virus (HBV) replication were investigated in the context of the intact viral genome. UBS is contained within the terminal redundancy of pgRNA, and the 5' copy of this sequence is essential for viral replication. Mutations within the central core of UBS ablate p65 binding and selectively block synthesis of plus-strand DNA, without affecting RNA packaging or minus-strand synthesis. The DBS sequence, which is located downstream of the pgRNA polyadenylation site, overlaps the core (C) protein coding region. All mutations introduced into this site severely affected viral replication. However, these effects were shown to result from dominant negative effects of mutant core polypeptides rather than from cis-acting effects on RNA recognition. Thus, the 5' UBS but not DBS sites play important cis-acting roles in HBV DNA replication; however, the involvement of p65 in these roles remains a matter for investigation.

Cells, Cultured↗

PubMed1999

Requirements for RNA replication of a poliovirus replicon by coxsackievirus B3 RNA polymerase.

A chimeric poliovirus type 1 (PV1) genome was constructed in which the 3D RNA polymerase (3D(pol)) coding sequences were replaced with those from coxsackievirus B3 (CVB3). No infectious virus was produced from HeLa cells transfected with the chimeric RNA. Processing of the PV1 capsid protein precursor was incomplete, presumably due to inefficient recognition of the P1 protein substrate by the chimeric 3CD proteinase containing CVB3 3D sequences. The ability of the chimeric RNA to replicate in the absence of capsid formation was measured after replacement of the P1 region with a luciferase reporter gene. No RNA synthesis was detected, despite efficient production of enzymatically active 3D(pol) from the 3D portion of the chimeric 3CD. The chimeric 3CD protein was unable to efficiently bind to the cloverleaf-like structure (CL) at the 5' end of PV1 RNA, which has been demonstrated previously to be required for viral RNA synthesis. The CVB3 3CD protein bound the PV1 CL as well as PV1 3CD. An additional chimeric PV1 RNA that contained CVB3 3CD sequences also failed to produce virus after transfection. Since processing of PV1 capsid protein precursors by the CVB3 3CD was again incomplete, a luciferase-containing replicon was also analyzed for RNA replication. The 3CD chimera replicated at 33 degrees C, but not at 37 degrees C. Replacement of the PV1 5'-terminal CL with that of CVB3 did not rescue the temperature-sensitive phenotype. Thus, there is an essential interaction(s) between 3CD and other viral P2 or P3 protein products required for efficient RNA replication which is not fully achieved between proteins from the two different members of the same virus genus.

Blotting, Western↗

PubMed1996

Stochastic Resonance and time advance coding in chemical reactions.

Stochastic Resonance (SR) is a phenomenon which may be found in nonlinear systems close to an excitation threshold. SR is a means for enhancing a weak periodic subthreshold signal from its noisy background by adding stochastic fluctuations, i.e. in biological and physical systems. It has been proposed that SR is important for the ability of neural systems to detect weak periodic signals. In the present work we show experimentally that SR occurs in two nonlinear chemical reactions, namely in the enzymatic Peroxidase-Oxidase (PO) reaction and in the Belousov-Zhabotinskii (BZ) reaction. A small sinusoidal signal with increasing noise is imposed on the focal steady state near a subcritical Hopf bifurcation. When the threshold is crossed beyond a certain noise amplitude, the system responds with spikes. The resulting interspike histogram and the plot of the signal to noise ratio, which is evaluated from the respective Fourier spectra, pass through a maximum at an optimal external noise level. An alternative way to cross the excitation threshold without noise is the variation of the bias value of the sinusoidal signal. The variation of the bias value causes the spikes to appear earlier if the sinusoidal function is moved closer towards the threshold. This so-called time advance coding is shown experimentally for the first time in the BZ reaction by imposing sinusoidal flow rate variations using different bias values. The phenomenon has been proposed by Hopfield to be a means for analog pattern recognition.

Artifacts↗

PubMed2004

Archaea recruited D-Tyr-tRNATyr deacylase for editing in Thr-tRNA synthetase.

Aminoacyl-tRNA synthetases (AARSs) are key players in the maintenance of the genetic code through correct pairing of amino acids with their cognate tRNA molecules. To this end, some AARSs, as well as seeking to recognize the correct amino acid during synthesis of aminoacyl-tRNA, enhance specificity through recognition of mischarged aminoacyl-tRNA molecules in a separate editing reaction. Recently, an editing domain, of uncertain provenance, idiosyncratic to some archaeal ThrRSs has been characterized. Here, sequence analyses and molecular modeling are reported that clearly show a relationship of the archaea-specific ThrRS editing domains with d-Tyr-tRNATyr deacylases (DTDs). The model enables the identification of the catalytic site and other substrate binding residues, as well as the proposal of a likely catalytic mechanism. Interestingly, typical DTD sequences, common in bacteria and eukaryotes, are entirely absent in archaea, consistent with an evolutionary scheme in which DTD was co-opted to serve as a ThrRS editing domain in archaea soon after their divergence from eukaryotes. A group of present-day archaebacteria contain a ThrRS obtained from a bacterium by horizontal gene transfer. In some of these cases a vestigial version of the original archaeal ThrRS, of potentially novel function, is maintained.

Amino Acid Sequence↗

PubMedDate not supplied

Autism: developing a strategy for nursing to prevent discrimination.

The final phase of the Disability Discrimination Act (DDA) comes into force in October 2004. The DDA Code of Practice is a document to guide organizations in the implementation of and compliance with the Act. The DDA Code of Practice (2002) makes reference to specific impairments, such as hearing and visual impairments, and there are numerous examples of how 'reasonable adjustments' can be made to enable access to those so disabled. There is recognition that some impairments are hidden, e.g. learning disabilities and diabetes. However, no mention is made of those with autism. The impairments of autism including Asperger syndrome are also 'hidden impairments' as defined by the DDA and this will mean that it will not be immediately obvious as to how the Act will help nurses to amend their practice to enable people with autism full inclusion and access to health care. This article will explore practical implications and outline how nurses can develop their practice to be compliant with the Act and to ensure good 'health care' practice for people diagnosed within the autism spectrum.

Autistic Disorder↗

PubMed2002

LCD versus CRT displays: a comparison of visual search performance for colored symbols.

Visual search performance for tactical symbols was examined with liquid-crystal (LCD) and cathode-ray tube (CRT) displays. Twenty-four adult participants (19 men, 5 women; mean age 41 years) searched for navy tactical display symbols on a map background. LCD and CRT displays of similar size and resolution (52 cm diagonal, 1280 x 1024 pixels) were used. Viewing angle (0 degrees vs. 60 degrees of azimuth), set size, target color (blue, red, or white), target presence, and search type (feature vs. conjunction) were also manipulated. Participants showed reduced sensitivity for red and blue symbols viewed 60 degrees off axis with the LCD relative to on-axis LCD, or to the CRT on or off axis. Colored symbols viewed off axis on the LCD produced longer response times in feature search and lower search efficiency in conjunction search. The results argue against the use of current LCD technology when off-axis viewing is likely and color coding is used.

Adult↗

PubMed2006

[Speaker identification based on Mel frequency cepstrum coefficient and complexity measure].

In present, the most basically used parameters for speaker identification are linear predictive coding (LPC) parameter, Mel frequency cepstrum coefficient(MFCC), etc. First in this paper only MFCC was used as the parameter and then Lempel-Ziv Complexity was combined with MFCC as parameters. The text-dependent recognition rate of 50 speakers increased from 42% to 80% and the text-independent recognition rate of 50 speakers increased from 60% to 72%. This test shows that Lempel-Ziv complexity, as a new parameter, can be applied to speaker identification.

Algorithms↗

PubMed1992

The intracellular targeting and membrane topology of 3-hydroxy-3-methylglutaryl-CoA reductase.

We present evidence that the amino-terminal 39 residue region of 3-hydroxy-3-methylglutaryl- (HMG) CoA reductase, which includes the putative first transmembrane span, is a signal sequence for targeting HMG-CoA reductase to the endoplasmic reticulum. This evidence is based upon fractionation, endoglycosidase-H sensitivity and protease protection assays on an in vitro transcription/translocation system programmed with a mutant cDNA of HMG-CoA reductase that is deleted for sequences coding for all of the putative transmembrane spans except the first. We show that the protein product of this mutant cDNA is associated with microsomes, glycosylated, or protected from proteolysis only in the presence of Signal Recognition Particle. Also, we present evidence for a topological model of HMG-CoA reductase that consists of eight transmembrane spans. This evidence is based upon a concanavalin A binding assay for in vivo glycosylation of an engineered glycosylation site in each of a series of mutants of the fusion protein, HMGal (Skalnik, D. G., Narita, H., Kent, C., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6836-6841). This series of mutants was designed such that for each linker segment between transmembrane spans, a mutant was constructed with an engineered glycosylation site introduced into that linker segment. We show that only the mutants with glycosylation sites in the linker segments between transmembrane spans 1 and 2, 3 and 4, and 5 and 6 are glycosylated. These results support an eight transmembrane span model for the topology of HMG-CoA reductase and are inconsistent with a seven-transmembrane span model.

Amino Acid Sequence↗

PubMed1996

Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products.

The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual's immune response, and may also contribute to susceptibility to diseases such as autoimmunity.

Amino Acid Sequence↗

PubMed1997

Gene cloning of rat and mouse platelet glycoprotein V: identification of megakaryocyte-specific promoters and demonstration of functional thrombin cleavage.

Platelet glycoprotein (GP) V is a major surface protein cleaved during thrombin-induced platelet activation. GPV associates noncovalently with the GPIb-IX complex to form GPIb-V-IX, a receptor for von Willebrand factor and thrombin. We describe the cloning of the genes coding for rat and mouse GPV and compare them with the human gene. The two rodent genes have a similar structure and resemble the human GPV gene with a coding sequence (approximately 1,700 nucleotides) entirely contained in one exon and a single intron (approximately 900 nucleotides) in the 5' untranslated region. Both genes have megakaryocyte-type promoters with conserved tandem Ets and GATA recognition motifs and lack a TATA box. The mature rat and mouse proteins comprise 551 amino acids, have 70% sequence identity, and contain an additional 8-amino acid intracellular segment as compared with the human protein. As in human GPV, there is an NH2-terminal leucine-rich region of 15 repeats and a thrombin cleavage recognition sequence. Whereas the rat and human thrombin cleavage sites are similar, the mouse cleavage site resembles that of the human thrombin receptor. Functionality of these sites was demonstrated by thrombin cleavage of synthetic peptides and analysis by high-performance liquid chromatography (HPLC) or mass spectrometry. Cleavage of native rat GPV was confirmed by means of a polyclonal antibody directed against the new NH2-terminal peptide exposed after thrombin cleavage. This antibody specifically recognized thrombin-activated rat platelets by fluorescence-activated cell sorting (FACS) analysis. In addition, we raised monoclonal antibodies specific for rat GPV (88 kD), which recognized the NH2-terminal soluble fragment (70 kD) liberated after thrombin cleavage. Knowledge of these rodent GPV genes and availability of species-specific peptides and antibodies will be essential to further studies aiming to define the exact in vivo function of platelet GPV using animal models of thrombosis and gene inactivation experiments.

Amino Acid Sequence↗

PubMed1988

Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation.

The cytolytic potential of T effector cells appears to be intimately related to the presence of proteins stored in specialized cytoplasmic granules. A striking biological property of isolated granules is their lytic activity for a variety of target cells in a nonrestricted manner. Proteins contained within these granules of CTLs are specifically released upon target cell recognition. We have isolated and characterized six granule-associated proteins in two murine CTL lines in addition to the pore-forming and target membrane-disrupting perforin. Six full length cDNA clones have been identified in a CTL-specific cDNA expression library which code for the granule-associated serine esterases, designated as granzymes A to F. Granzymes A and B represent the genuine proteins encoded by the H factor/CTLA-3 cDNA and the CTLA-1/CCPI cDNA, respectively. The covalent amino acid structures of all six granzymes show the hallmarks for serine proteases and are highly related to that of rat mast cell protease I and II and cathepsin G, which have been found in granules of mast cells and neutrophilic granulocytes, respectively. The primary translation products are processed by removal of a hydrophobic signal peptide and a two residue-long propeptide at the amino-terminus. Immuno-electron microscopy shows that granzymes and perforin are stored together within secretory granules of CTLs. Simultaneous release of at least two of these granzymes has been observed during degranulation of a murine CTL line by anti-T3 antibodies. The biological role, particularly the proteolytic events elicited by granzyme A and other granzymes in the context of target cell recognition, are not known at present. It is unlikely that they form a proteolytic activation cascade together with pore-forming proteins analogous to the complement system. The strictly regulated secretion of granzymes and the lack of measurable enzymatic activity in the case of granzymes B, C, E and F towards a variety of synthetic substrates suggest a highly specific function for each of them.

Amino Acid Sequence↗

PubMed1977

Cytotoxic T lymphocytes specific for I region determinants do not require interactions with H-2K or D gene products.

Gene products coded for by the major hisocompatibility complex (MHC) can serve as target antigens for cytotoxic T lymphocytes (CTL) (1). A variety of test systems are available which have yielded information consistently reinforcing the importance of this complex of genes in the generation and effector phases of the cytotoxic immune response. Originally, it was shown that allogeneically-induced CTL had specificity primarily for the products of the K and D loci of the mouse H-2 complex (2). More recently this has also been found to be the case for xenogeneic immunizations (3,4). Additional examples of T cell-mediated lysis have been reported involving viral-infected or chemically- modified syngeneic stimulating and target cells in which homology at H-2K or H-2D was required between the responding and target cells for appreciable lysis to occur (5-7). Moreover, CTL specific for minor histocompatability antigens are able to lyse only target cells bearing these membrane antigens and sharing a common H-2K or H2-D gene product with the effector (8,9). Two hypotheses have been proposed to explain the requirement for H-2 identity between effector and targets in these systems. CTL may recognize new antigenic determinants created by the interaction of the modifier with syngeneic K and D gene products. Alternately, a dual recognition system my exist, requiring an antigen-specific receptor as well as a second receptor with specificity for homologous H-2K or H-2D determinants (5). Neither model can be excluded at this time. The I region also contains genes coding for histocompatibility loci since animals differing at the I-A or I-C regions of the H-2 complex reject skin grafts (10-12), though less rapidly than mice differing at the H-2K or H-2D regions, Also CTL can be generated to I region determinants but less efficiently than CTL specific for H-2K or H-2D gene products (12-14). The question can therefore be raised, whether the I region minor histocompatibility loci function independently from the H-2K or H-2D loci or whether I region-specific cytolysis requires the participation of H-2K or H-2D gene products of the target cell. This communication illustrates the generation of CTL showing specificity for I region determinants in primary mixed lymphocyte cultures. Further, we demonstrate by genetic analysis and byt eh use of speficit alloantisera that CTL directed to Ia determinants (a) do not see these antigens as modifications of H-2K or H-2D gene products but as independent gene products coded for by the I region, and (b) they do not require interaction with target cells bearing the same H-2K or H-2D gene product as the effect CTL.

Animals↗

PubMedDate not supplied

[Computer program recognition of a cDNA sequence specifying signal peptides].

An application of a computational analysis of cDNA sequences is presented in this paper. The goal is the identification of functional domains on sequence data. The results show the capability of this technique to identify a zone of DNA associated with the signal peptide coding region, whose biological function at DNA or RNA level is still unknown.

Base Sequence↗

PubMed2004

Color and size interactions in a real 3D object similarity task.

In the natural world, objects are characterized by a variety of attributes, including color and shape. The contributions of these two attributes to object recognition are typically studied independently of each other, yet they are likely to interact in natural tasks. Here we examine whether color and size (a component of shape) interact in a real three-dimensional (3D) object similarity task, using solid domelike objects whose distinct apparent surface colors are independently controlled via spatially restricted illumination from a data projector hidden to the observer. The novel experimental setup preserves natural cues to 3D shape from shading, binocular disparity, motion parallax, and surface texture cues, while also providing the flexibility and ease of computer control. Observers performed three distinct tasks: two unimodal discrimination tasks, and an object similarity task. Depending on the task, the observer was instructed to select the indicated alternative object which was "bigger than," "the same color as," or "most similar to" the designated reference object, all of which varied in both size and color between trials. For both unimodal discrimination tasks, discrimination thresholds for the tested attribute (e.g., color) were increased by differences in the secondary attribute (e.g., size), although this effect was more robust in the color task. For the unimodal size-discrimination task, the strongest effects of the secondary attribute (color) occurred as a perceptual bias, which we call the "saturation-size effect": Objects with more saturated colors appear larger than objects with less saturated colors. In the object similarity task, discrimination thresholds for color or size differences were significantly larger than in the unimodal discrimination tasks. We conclude that color and size interact in determining object similarity, and are effectively analyzed on a coarser scale, due to noise in the similarity estimates of the individual attributes, inter-attribute attentional interactions, or coarser coding of attributes at a "higher" level of object representation.

Adult↗

PubMed2004

Tracking repeats using significance and transitivity.

MOTIVATION: Internal repeats in coding sequences correspond to structural and functional units of proteins. Moreover, duplication of fragments of coding sequences is known to be a mechanism to facilitate evolution. Identification of repeats is crucial to shed light on the function and structure of proteins, and explain their evolutionary past. The task is difficult because during the course of evolution many repeats diverged beyond recognition. RESULTS: We introduce a new method TRUST, for ab initio determination of internal repeats in proteins. It provides an improvement in prediction quality as compared to alternative state-of-the-art methods. The increased sensitivity and accuracy of the method is achieved by exploiting the concept of transitivity of alignments. Starting from significant local suboptimal alignments, the application of transitivity allows us to (1) identify distant repeat homologues for which no alignments were found; (2) gain confidence about consistently well-aligned regions; and (3) recognize and reduce the contribution of non-homologous repeats. This re-assessment step enables us to derive a virtually noise-free profile representing a generalized repeat with high fidelity. We also obtained superior specificity by employing rigid statistical testing for self-sequence and profile-sequence alignments. Assessment was done using a database of repeat annotations based on structural superpositioning. The results show that TRUST is a useful and reliable tool for mining tandem and non-tandem repeats in protein sequence databases, capable of predicting multiple repeat types with varying intervening segments within a single sequence. AVAILABILITY: The TRUST server (together with the source code) is available at http://ibivu.cs.vu.nl/programs/trustwww

Algorithms↗

PubMed1994

Disorders of verbal and pictorial memory in right and left brain-damaged patients.

It is still controversial whether verbal and pictorial stimuli are independently processed and stored in memory, as assumed by the dual code hypothesis, or a single code is used both for verbal and for pictorial stimuli, as assumed by the verbal loop hypothesis and by the propositional code hypothesis. According to the first hypothesis, verbal and pictorial memory are independently disrupted by brain damage, whereas according to the second hypothesis a co-occurrence of verbal and pictorial memory disorders are usually observed. To test these contrasting predictions, we constructed a verbal and a pictorial memory task very similar with respect to testing procedures and to material to be memorized and we administered them to 33 left and 27 right brain-damaged patients and to 21 normal controls. The following results were obtained: 1) Disorders of verbal and of pictorial memory were dissociated by brain injury in about one-third of our patients; 2) A consistent relationship was observed between laterality of lesion and type of selective memory impairment, since word recognition was selectively impaired by left and picture recognition by right brain injury; 3) However, only on the test of verbal memory was a significant difference between right and left brain-damaged patients obtained, whereas on the test of pictorial memory only a nonsignificant trend in the opposite direction was observed. These data are in favor of the dual code hypothesis and suggest that the links between left hemisphere and verbal code may be stronger than the relationship between right hemisphere and pictorial code.

Adult↗

PubMed1985

Two genes encoding the bovine mitochondrial ATP synthase proteolipid specify precursors with different import sequences and are expressed in a tissue-specific manner.

Two cDNAs encoding different precursor proteins of the same mature proteolipid subunit of mitochondrial ATP synthase have been cloned from a bovine cDNA library. The hybridisation probe was a mixture of 17-mer oligonucleotides containing 256 discrete sequences. The coding sequences of the two cDNAs differ in 25 silent positions of codons and the 3' non-coding sequences are only weakly related. The precursor sequences, which direct the import of the proteolipid into the mitochondrion, are 61 and 68 amino acids long. They are related to each other in regions which probably are recognition signals for the processing protease. The corresponding genes are expressed differently in various tissues in a way that reflects their embryonic origin.

Animals↗

PubMed1988

Delirium in liver transplantation candidates: discriminant analysis of multiple test variables.

We report the results of electroencephalograms, Mini-Mental State exam, Trailmaking Tests A and B, and serum albumin levels in 108 consecutive liver transplantation candidates. We compared test results to a clinical DSM-III diagnosis of delirium. Although each variable could differentiate between the two groups (delirium n = 18; nondelirium n = 90) at a statistically significant level, a discriminant analysis involving either all variables or only three particular variables (Trailmaking B, EEG code, and albumin) resulted in the highest specificity (97.8%) and sensitivity (83.3%), with a correct classification of 95.4% of subjects. The analysis also generates an equation that can be applied to clinical situations to enhance the accurate recognition of delirium. In addition, to explain abnormal Trailmaking B scores and/or EEGs in subjects who did not otherwise meet DSM-III criteria for delirium, we suggest the presence of a "subclinical delirium."

Adolescent↗