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At least 649 records · Page 36Linked to original sources

PubMed1980

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma.

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.

Animals↗

PubMedDate not supplied

Patient cues and medical interviewing in general practice: examples of the application of sequential analysis.

AIMS: To illustrate how sequence analysis may be applied to the medical interview to: 1. explore how physicians without formal training in communication skills elicit and respond to patient cues and expression of expectations and opinions; and 2. test the hypothesis that physicians' closed ended questions determine the use of subsequent closed ended questions. METHODS: 238 consultations in primary care, coded with the Verona Medical Interview Classification System, were analysed. Lag 1 analysis was applied to study which physician behaviour precedes and follows patient cues. Pattern recognition analysis for five lag sequences was performed to test the occurrence of predefined specific code chains, where a closed and an open ended question were followed either by two closed-ended questions or by two patient facilitating interventions RESULTS: Patients' cue offers were most likely after facilitative interventions, but not after open-ended questions; physicians were most likely to respond to these expressions with facilitation. Physicians' tendency to use closed ended questions increased after previous closed questions and decreased after an open-ended question. CONCLUSIONS: Lag sequential analysis and pattern recognition analysis are useful methods to study exploratory and theory driven hypotheses and allow an initial approach to validate the supposed appropriateness of specific physician interventions.

Communication↗

PubMed2005

Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis.

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.

Arabidopsis↗

PubMed1983

The ethically trained physician: myth or reality?

Through a questionnaire distributed to 300 physicians in the Toronto area, three aspects of their ethical awareness were examined: the formal codes, the need for consultation in making decisions and the need for training in medical ethics. Most of the physicians (81%) felt that they were facing ethical problems in their daily practice. A majority of these would try to solve the problems either themselves (30%) or through discussion with a colleague (43%). When they turned outside the profession it was sometimes to a lawyer (12%), which suggests concern with the legalities of some situations. Only a small proportion of the respondents were found to be familiar with two of the established codes of ethics, yet 13% would still turn to the code of the Canadian Medical Association for guidance. Finally, there was widespread recognition of the need for proper training in medical ethics and for the establishment of a specialty in this field.

Attitude of Health Personnel↗

PubMed2001

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

Amino Acid Motifs↗

PubMed2001

Class I release factors in ciliates with variant genetic codes.

In eukaryotes with the universal genetic code a single class I release factor (eRF1) most probably recognizes all stop codons (UAA, UAG and UGA) and is essential for termination of nascent peptide synthesis. It is well established that stop codons have been reassigned to amino acid codons at least three times among ciliates. The codon specificities of ciliate eRF1s must have been modified to accommodate the variant codes. In this study we have amplified, cloned and sequenced eRF1 genes of two hypotrichous ciliates, Oxytricha trifallax (UAA and UAG for Gln) and Euplotes aediculatus (UGA for Cys). We also sequenced/identified three protist and two archaeal class I RF genes to enlarge the database of eRF1/aRF1s with the universal code. Extensive comparisons between universal code eRF1s and those of Oxytricha, Euplotes, and Tetrahymena which represent three lineages that acquired variant codes independently, provide important clues to identify stop codon-binding regions in eRF1. Domain 1 in the five ciliate eRF1s, particularly the TASNIKS heptapeptide and its adjacent region, differs significantly from domain 1 in universal code eRF1s. This observation suggests that domain 1 contains the codon recognition site, but that the mechanism of eRF1 codon recognition may be more complex than proposed by Nakamura et al. or Knight and Landweber.

Amino Acid Sequence↗

PubMed2005

Human serotonin transporter variants display altered sensitivity to protein kinase G and p38 mitogen-activated protein kinase.

Human serotonin [5-hydroxytryptamine (5-HT)] transporters (hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are prominent targets for therapeutic intervention in mood, anxiety, and obsessive-compulsive disorders. Multiple hSERT coding variants have been identified, although to date no comprehensive functional analysis of these variants has been reported. We transfected hSERT or 10 hSERT coding variants and examined total and surface protein expression, antagonist recognition, and transporter modulation by posttranslational, regulatory pathways. Two variants, Pro339Leu and Ile425Val, demonstrated significant changes in surface expression supporting alterations in 5-HT transport capacity (V(max)). Regardless of basal transport activity, all SERT variants displayed a capacity for rapid, phorbol ester-triggered down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala, Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity for 5-HT uptake stimulation after acute protein kinase G (PKG)/p38 mitogen-activated protein kinase (MAPK) activation. Epstein-Barr virus (EBV)-transformed lymphocytes natively expressing the most common of these variants (Gly56Ala) exhibited a similar loss of 5-HT uptake stimulation by PKG/p38 MAPK activators. HeLa cells transfected with the Gly56Ala variant demonstrated elevated basal phosphorylation and, unlike hSERT, could not be further phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These studies reveal cellular phenotypes associated with naturally occurring human SERT coding variants and suggest that altered transporter regulation by means of PKG/p38 MAPK-linked pathways may influence risk for disorders attributed to compromised 5-HT signaling.

Cyclic GMP-Dependent Protein Kinases↗

PubMed1994

Cloning of a VLP-protein coding gene from a parasitoid wasp Venturia canescens.

Virus-like particles (VLPs) that are bound to the egg surface of a parasitic wasp Venturia canescens are void of nucleic acid and unable to infect host tissue but instead provide a passive protection against the host's immune recognition. To investigate evolutionary and functional properties of Venturia particles, we isolated cDNAs coding for a VLP-protein by screening an expression library of the wasp, using antibodies against purified VLPs. The corresponding coding DNA is present in the wasp genome as a single-copy gene.

Animals↗

PubMed1990

Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene.

Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors.

Amino Acid Sequence↗

PubMed1999

Interpolated markov chains for eukaryotic promoter recognition.

MOTIVATION: We describe a new content-based approach for the detection of promoter regions of eukaryotic protein encoding genes. Our system is based on three interpolated Markov chains (IMCs) of different order which are trained on coding, non-coding and promoter sequences. It was recently shown that the interpolation of Markov chains leads to stable parameters and improves on the results in microbial gene finding (Salzberg et al., Nucleic Acids Res., 26, 544-548, 1998). Here, we present new methods for an automated estimation of optimal interpolation parameters and show how the IMCs can be applied to detect promoters in contiguous DNA sequences. Our interpolation approach can also be employed to obtain a reliable scoring function for human coding DNA regions, and the trained models can easily be incorporated in the general framework for gene recognition systems. RESULTS: A 5-fold cross-validation evaluation of our IMC approach on a representative sequence set yielded a mean correlation coefficient of 0.84 (promoter versus coding sequences) and 0.53 (promoter versus non-coding sequences). Applied to the task of eukaryotic promoter region identification in genomic DNA sequences, our classifier identifies 50% of the promoter regions in the sequences used in the most recent review and comparison by Fickett and Hatzigeorgiou ( Genome Res., 7, 861-878, 1997), while having a false-positive rate of 1/849 bp.

Algorithms↗

PubMed2004

Temporal fine-structure cues to speech and pure tone modulation in observers with sensorineural hearing loss.

OBJECTIVE: The purpose of this study was to examine the effect of sensorineural hearing loss on the ability to make use of fine temporal information and to evaluate the relation between this ability and the ability to recognize speech. DESIGN: Fourteen observers with normal hearing and 12 observers with sensorineural hearing loss were tested on open-set word recognition and on psychophysical tasks thought to reflect use of fine-structure cues: the detection of 2 Hz frequency modulation (FM) and the discrimination of the rate of amplitude modulation (AM) and quasifrequency modulation (QFM). RESULTS: The results showed relatively poor performance for observers with sensorineural hearing loss on both the speech recognition and psychoacoustical tasks. Of particular interest was the finding of significant correlations within the hearing-loss group between speech recognition performance and the psychoacoustical tasks based on frequency modulation, which are thought to reflect the quality of the coding of temporal fine structure. CONCLUSIONS: These results suggest that sensorineural hearing loss may be associated with a reduced ability to use fine temporal information that is coded by neural phase-locking to stimulus fine-structure and that this may contribute to poor speech recognition performance and to poor performance on psychoacoustical tasks that depend on temporal fine structure.

Adult↗

PubMed1980

Isolation and sequence of the gene for iso-2-cytochrome c in Saccharomyces cerevisiae.

The two apocytochrome c proteins of yeast are coded for by separate genes. Iso-2-cytochrome c differs from the iso-1 protein at 17 positions within a homologous sequence of 108 amino acids. The previously cloned iso-1-cytochrome c coding sequence has been used to identify lambda-yeast recombinant phage containing the gene for iso-2-cytochrome c. The latter protein contains the dipeptide Ala-Ala which is coded for by the nucleic acid sequence G-C-N-G-C-N. The recognition specificity of restriction endonuclease Fnu4HI for G-C-N-G-C provided a rapid means of locating the region of the cloned fragment which codes for iso-2-cytochrome c. The DNA sequence of this gene has been determined and compared with that of the iso-1-cytochrome c locus. There is no intervening sequence within the gene for iso-2-cytochrome c. At 45 of the 91 positions for which iso-1- and iso-2-cytochrome c have the same amino acid, the codons differ. Such third position variation does not occur within the region coding for amino acids 70-80, the protein sequence that is also most conserved among all eukaryotic cytochromes c.

Apoproteins↗

PubMed2000

Medicinal chemistry based on the sugar code: fundamentals of lectinology and experimental strategies with lectins as targets.

Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.

Animals↗

PubMed1991

Construction of cloning cartridges for development of expression vectors in gram-negative bacteria.

A cloning cartridge was constructed that can be inserted into a plasmid of choice to form an expression vector in which gene expression is inducible with an inexpensive inducer, sodium salicylate, at low concentrations. This cartridge consists of a 3.6-kb restriction fragment which contains the positive regulatory gene nahR from plasmid NAH7, a promoter, PG, that nahR regulates, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance. Within promoter PG of the cloning cartridge, a sequence of three nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an NdeI recognition site (CATATG) for cloning of the 5' end of a gene without affecting the distance between the transcription start site and the gene coding region. In addition, the 5' end of a gene can be converted into an NdeI recognition site without altering the amino acid sequence it encodes and then cloned into this cartridge for regulated expression. Several other synthetic restriction sites were also inserted downstream of the NdeI site for accepting the 3' end of a cloned gene. A derivative of this cloning cartridge lacking the NdeI sequence was also constructed for cloning and expression of a restriction fragment containing a gene(s) of unknown sequence. Use of the cloning cartridges in a broad-host-range plasmid has allowed successful cloning and inducible expression of several genes in all of the gram-negative bacterial tested to date. Protein production to at least 10% of the total soluble cell proteins was observed from a cloned gene expressed in Pseudomonas putida.

Bacterial Proteins↗

PubMed1995

Highly efficient eukaryotic gene expression vectors for peptide secretion.

Recently, we constructed a series of highly efficient universal eukaryotic gene expression vectors (Sheay et al., BioTechniques 15:856-862, 1993). Such vectors contain a viral promoter and enhancer followed by the adenovirus tripartite leader sequence, a multiple cloning site for the insertion of the gene of interest and a polyadenylation sequence. To enable expression of peptides to be secreted into the tissue culture medium or to be incorporated into the cell membrane, several modifications have been introduced into such vectors: stop codons in all three reading frames were inserted at the end of the multiple cloning site and a DNA sequence coding for a signal peptide for transport through the endoplasmatic reticulum (ER) was introduced downstream of the adenovirus tripartite leader sequence followed by two unique restriction enzyme recognition sites. A protocol is described that allows fast and easy cloning of peptide-coding regions, i.e., PCR products, for expression and secretion. The transport of a genetically engineered chimeric transmembrane protein connected to this ER leader sequence was as efficient as that of the original protein from which the ER sequence has been derived. These universal vectors can also be used for the easy construction of any chimeric transmembrane or secretion proteins.

Amino Acid Sequence↗

PubMedDate not supplied

Highly efficient eukaryotic gene expression vectors for peptide secretion.

Amino Acid Sequence↗

PubMed2026

VIPR RNA-guided DNA recognition by noncontiguous geometric triplex formation.

Viral interference programmable repeat (VIPR) systems use a noncontiguous code for RNA-guided transcriptional silencing. How the Vipr protein and a VIPR RNA (vrRNA) comprising alternating GGY and NN segments achieve precise DNA targeting is unknown. Here, we present 21 cryo-electron microscopy structures that help explain the mechanism of target engagement. Vipr protomers oligomerize along the vrRNA to form a right-handed helical filament, sequestering each GGY motif and positioning the adjacent NN bases for target base pairing. DNA binding, in which every third nucleotide is skipped, results in a gapped vrRNA-DNA hybrid helix that encircles the nontarget DNA strand to form a geometric triplex. These findings suggest that triplex-mediated target-strand handoff could enable noncontiguous and programmable RNA-guided DNA recognition in VIPR systems.

DNA↗

PubMed1987

Recognition of hierarchically encoded images by technical and biological systems.

All the contours and regions of objects can be mapped to code-trees of the Hierarchical Structure Code (HSC). Invariant features like structure classes, shape descriptions, or relations between structures and components may be easily extracted from the HSC. HSC-based pattern recognition provides a straightforward transition between the signal-space of the image and the space of its symbolic representation. Physiological data are well predicted and do not exclude an implementation of an HSC-based system within the visual cortex.

Cybernetics↗