Biomedical subjects

W R Taylor

Publications and source records attributed to W R Taylor.

At least 109 records · Page 6Linked to original sources

PubMed1995

Receptive field properties of starburst cholinergic amacrine cells in the rabbit retina.

Patch-clamp recordings were made from ON starburst cholinergic amacrine cells with somas located in the ganglion cell layer of an isolated, dark-adapted rabbit retina preparation. Light responses were analysed and cell identify was confirmed anatomically. The centre light response had a linear current-voltage relation with a reversal potential close to 0 mV. The receptive field size was similar to the dendritic field size. Cholinergic amacrine cells displayed significant surround inhibition. The receptive field profile consisted of a central excitatory region flanked by an inhibitory surround. The surround attenuated the central response to 36% of the maximum. The surround was probably mediated by a combination of presynaptic and postsynaptic inhibition. Starburst amacrine cells did not display action potentials and the presence of a large, voltage-dependent outward current limited depolarizing responses to a maximum potential of about -40 mV. Light responses were completely suppressed during application of 100 microM D,L-2-amino-4-phosphonobutyric acid (APB), consistent with activation exclusively through rod bipolar cells (on) and ON-cone bipolar cells. In darkness the cells displayed a tonic inward current that could be blocked by 100 microM APB and 2 microM CNQX.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1995

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

Cell Cycle↗

PubMed1995

Increased phosphorylation of histone H1 in mouse fibroblasts transformed with oncogenes or constitutively active mitogen-activated protein kinase kinase.

We compared the nucleosomal organization, histone H1 subtypes, and histone H1 phosphorylated isoforms of ras-transformed and parental 10T1/2 mouse fibroblasts. In agreement with previous studies, we found that ras-transformed mouse fibroblasts have a less condensed chromatin structure than normal fibroblasts. ras-transformed and parental 10T1/2 cells had similar amounts of H1 subtypes, proteins that have a key role in the compaction of chromatin. However, labeling studies with 32P and Western blot experiments with an antiphosphorylated H1 antibody show that interphase ras-transformed cells have higher levels of phosphorylated H1 isoforms than parental cells. G1/S phase-arrested ras-transformed cells had higher amounts of phosphorylated H1 than G1/S phase-arrested parental cells. Mouse fibroblasts transformed with fes, mos, raf, myc, or constitutively active mitogen-activated protein (MAP) kinase kinase had increased levels of phosphorylated H1. These observations suggest that increased phosphorylation of H1 is one of the consequences of the persistent activation of the mitogen-activated protein kinase signal transduction pathway. Indirect immunofluorescent studies show that phosphorylated H1b is localized in centers of RNA splicing in the nucleus, suggesting that this modified H1 subtype is complexed to transcriptionally active chromatin.

3T3 Cells↗

PubMed1995

Global fold determination from a small number of distance restraints.

We have designed a distance geometry-based method for obtaining the tertiary fold of a protein from a limited number of structure-specific distance restraints and the secondary structure assignment. Interresidue distances were predicted from patterns of conserved hydrophobic amino acids deduced from multiple alignments. A simple model chain representing the protein was then folded by projecting its distance matrix into Euclidean spaces with gradually decreasing dimensionality until a final three-dimensional embedding was achieved. Tangled conformations produced by the projection steps were eliminated using a novel filtering algorithm. Information on various aspects of protein structure such as accessibility and chirality was incorporated into the conformation refinement, increasing the robustness of the algorithm. The method successfully identified the correct folds of three small proteins from a small number of restraints, indicating that it could serve as a useful computational tool in protein structure determination from NMR data.

Algorithms↗

PubMed1995

Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.

1. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded under voltage-clamp conditions. Consistent with activation of non-NMDA-type glutamate receptors, the sEPSCs reversed at potentials above 0 mV, were blocked by 1 microM CNQX and prolonged by 2 mM aniracetam. 2. The peak conductance of the averaged sEPSCs (n = 70-400) was 130 +/- 60 pS (mean +/- S.D.; 17 cells, ranging from 70 to 290 pS). Amplitude distributions were skewed towards larger amplitudes. 3. The decay of individual and mean sEPSCs was exponential with a mean time constant (tau d) of 3.75 +/- 0.84 ms (n = 13), which was voltage independent. The 10-90% rise time of the sEPSCs was 1.30 +/- 0.44 ms (n = 13). There was no correlation between sEPSC rise time and tau d suggesting that dendritic filtering alone did not shape the time course of sEPSCs. 4. Light-evoked EPSCs in these retinal ganglion cells are mediated by concomitant activation of NMDA and non-NMDA receptors; however, no NMDA component was discerned in the sEPSCs, even when recording at -96 mV in Mg(2+)-free solutions. The decay time course was not altered by 20 microM AP7, an NMDA antagonist, nor was an NMDA component unmasked by adding glycine or D-serine. These results suggest that NMDA and non-NMDA receptors are not coactivated by a single vesicle of transmitter during spontaneous release, and thus are probably not colocalized in the postsynaptic membrane at the sites of spontaneous release. 5. The sEPSCs were an order of magnitude faster than the non-NMDA receptor-mediated EPSCs evoked by light stimuli, and it is proposed that the EPSC time course is determined largely by the extended time course of release of synaptic vesicles from bipolar cells. The quantal content of a light-evoked non-NMDA receptor-mediated EPSC in an on-off cell is about 200 quanta.

Ambystoma↗

PubMed1995

Transesophageal dobutamine stress echocardiography in the evaluation of coronary artery disease.

OBJECTIVES: The goal of this study was to determine the feasibility, safety, sensitivity and specificity of transesophageal dobutamine stress echocardiography for the detection of coronary artery disease. BACKGROUND: Dobutamine stress echocardiography has been shown to be an extremely sensitive and specific noninvasive technique for the detection of myocardial ischemia. However, inadequate transthoracic images preclude the use of dobutamine stress echocardiography in a small but significant group of patients. Transesophageal echocardiography provides better resolution than that obtained with routine transthoracic imaging. METHODS: Patients scheduled for routine cardiac catheterization underwent transesophageal dobutamine stress echocardiography. All patients underwent coronary arteriography within 48 h of the study, and lesion severity was determined by quantitative coronary angiography. Significant coronary obstruction was defined as stenosis > 50%. RESULTS: Fifty-one male patients were enrolled in the study; six were excluded for technical reasons. There were no adverse outcomes or complications. Of 27 patients with significant coronary artery disease, 22 had positive study results (sensitivity 82%). Of 13 patients without significant obstructive coronary disease, 1 had a false positive study result (specificity 93%). In patients with a minimal lumen diameter < 1.25 mm, sensitivity was > 80%, and in patients with a minimal lumen diameter > 1.5 mm, sensitivity was < 70%, suggesting that lesions with a minimal lumen diameter < 1.25 mm are more likely to be physiologically significant. CONCLUSIONS: Transesophageal dobutamine stress echocardiography is a feasible, safe and accurate technique for the detection of myocardial ischemia. There are inherent limitations to this technique in that transesophageal echocardiography must be performed. Transesophageal dobutamine stress echocardiography may allow extension of dobutamine stress testing to patients with inadequate transthoracic echocardiographic imaging and may provide an opportunity for further research applications.

Coronary Angiography↗

PubMed1995

Conductance and kinetics of single cGMP-activated channels in salamander rod outer segments.

1. The conductance and kinetics of single 3',5'-cyclic guanosine monophosphate (cGMP)-activated channels of retinal rod outer segments were studied in inside-out membrane patches. The size of the single channel currents was increased by using low concentrations of divalent cations. 2. At saturating cGMP concentration, the current flickered at high frequency. Occasionally, the current was interrupted by closures lasting tens or hundreds of milliseconds. At +50 mV the maximum current during an opening was slightly more than 1 pA, but the open channel level was poorly resolved due to the speed of the gating transitions. 3. Amplitude histograms confirmed the presence of a sublevel of current, roughly a quarter the size of the peak current, at low cGMP concentrations. The fraction of time in the sublevel decreased with increasing cGMP concentration, suggesting that the sublevel may be due to opening by the partially liganded channel. 4. Consistent with previous macroscopic current recordings, single channel activation by cGMP had an apparent dissociation constant of 8.6 microM, and a Hill coefficient of 2.8. 5. At saturating cGMP concentrations, the channel was modelled as a two-state system with the following parameters. The open channel conductance was 25 pS. The opening rate constant, beta, was 1.5 x 10(4) s-1 at 0 mV, and had a voltage sensitivity equivalent to the movement of 0.23 electronic charges outward through the membrane electric field. The closing rate constant, alpha, was 2.1 x 10(4) s-1 and was voltage insensitive. Assuming that the open-state chord conductance was voltage independent, the inferred voltage dependence of beta largely accounted for the outward rectification in the steady-state macroscopic current-voltage relation of multichannel patches, at saturating cGMP concentration.

Animals↗

PubMed1995

Acute mass burns caused by o-chlorobenzylidene malononitrile (CS) tear gas.

The use of tear gas in controlling riots has been an accepted practice in many countries for the past four decades. In a recent event, a large quantity of tear-gas canisters were used during a situation of unrest in a Hong Kong Refugees' Detention Centre. We report 96 cases of acute burn injury as an unpredicted side effect of o-chlorobenzylidene malononitrile (CS) tear gas. There were 47 females and 49 males with an age ranging between < 1 to 51 years. These burns were categorized as minor burns, with the total body surface area (TBSA) ranging from 1 to 8 per cent with mean percentage of 3. Most of the patients sustained superficial or partial-skin thickness injuries. Only two patients were admitted to the Prince of Wales Hospital Burns Centre because of deeper burns; debridement and skin grafting was required in one of them. The mechanism of burn injury was due to the flame generated from the grenade explosion, direct contact between the hot canister and the victim's skin, and the effect of the chemical powder inside the canisters when it splashed onto the victim's body. We suggest that the noxious transient effects of tear gas are underestimated, furthermore varying cutaneous effects and deep burns may result from its uncontrolled use during riots. There is a continuing need to reassess the potential toxic effects of CS tear gas as a riot control agent and to debate whether its future use can be condoned under any circumstances.

Acute Disease↗

PubMed1995

The role of p53 in regulating genomic stability when DNA and RNA synthesis are inhibited.

In addition to its induction by DNA damage, p53 is induced by drugs that starve cells for DNA and RNA precursors, or by inhibitors of DNA or RNA polymerase. In normal cells, the induction of p53 by dNTP starvation serves a protective role, mediating rapid, reversible cell-cycle arrest without DNA damage. In most cell lines, this first line of defense is missing, so that starvation for dNTPs causes DNA to break, thus increasing the probability of genomic instability, chromosome deletions and gene amplification. The mechanism of how p53 is induced remains unclear.

Animals↗

PubMed1995

Effects of magnesium on nitric oxide synthase activity in endothelial cells.

Magnesium modulates endothelium-dependent vasodilation in intact blood vessels. Therefore, the effects of magnesium on nitric oxide (NO) release by isolated endothelial cells and nitric oxide synthase (NOS) activity in endothelial cell homogenates were studied. Unstimulated and bradykinin-stimulated NO release by porcine aortic endothelial cell (PAEC) monolayers were unaffected by 30 min of exposure to magnesium concentrations varying from 0.010 to 10.0 mM. In contrast, when A-23187-stimulated cells were exposed to 0.01, 3.16, and 10.0 mM MgCl2, NO release was decreased by 11.3 +/- 1.8, 11.7 +/- 3.0, and 20.3 +/- 7.2%, respectively, compared with cells exposed to 1.0 mM MgCl2 (P < 0.01). These data suggested that a change in the intracellular magnesium concentration had an effect on NO release, in contrast to a change in the extracellular concentration, which did not have an effect. To further assess this possibility, crude NOS extracts were prepared from PAEC and exposed to MgCl2. NOS activity was measured via the conversion of L-[3H]arginine to L-[3H]citrulline. Increasing the concentration of MgCl2 by 1.0, 3.16, and 10.0 mM caused a 16.0 +/- 6.8, 17.1 +/- 1.7, and 38.6 +/- 5.3% decrease in citrulline formation, respectively (P < 0.05), suggesting a direct inhibition of NOS by MgCl2. No significant difference in the degree of inhibition of NOS activity was found between MgSO4 and MgCl2, thus ruling out a nonspecific chloride effect. In addition, increasing the concentration of NaCl to 15 mM had no effect on NOS activity, ruling out a nonspecific osmotic effect [101.6 +/- 10.5% of control activity, P = not significant (NS)].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Antisense oligodeoxyribonucleotide inhibition of TGF-beta 1 gene expression and alterations in the growth and malignant properties of mouse fibrosarcoma cells.

Transforming growth factor (TGF-beta) is a family of multifunctional signalling molecules that play a fundamental role in both normal and malignant cell behavior. Procedures that alter mouse TGF-beta 1 gene expression provide an important approach for analyzing the complex regulatory processes associated with this member of the growth factor family. Therefore, we have designed oligodeoxyribonucleotides (oligos) in an antisense orientation, which are complementary to regions of the TGF-beta 1 message, in an attempt to obtain an oligo sequence that specifically reduces TGF-beta 1 synthesis. We observed that oligos containing a mixture of phosphorothioate and phosphodiester linkages were less toxic and more specific when compared to those only containing phosphorothioate. A non-toxic sequence was identified that markedly reduced the levels of TGF-beta 1 in oligo-treated malignant mouse fibrosarcoma cells. The invasive and metastatic properties of these fibrosarcoma cells were also significantly decreased following treatment with the antisense oligo. The results indicate an important role for altered TGF-beta 1 expression in the regulation of malignant cell proliferation, invasion and metastasis. These results also indicate that this oligo sequence is a useful tool for studies directed towards understanding the complex relationships between TGF-beta 1 and cellular regulation.

Animals↗

PubMed1994

Protein structure modelling from remote sequence similarity.

Many methods exist for taking a sequence that exhibits similarity to another of known structure and building a molecular model. However, when the sequence similarity is very remote and fragmentary, this 'modelling-by-homology' approach is less reliable. Current methods that tackle this problem are reviewed below, taking as an example the construction of a predicted model for the retroviral protease. This earlier work, which was only partially automatic, identified many of the outstanding difficulties that have subsequently been automated in computer programs, developed both by the author and many others. Because of the rapid proliferation of methods and their variants, an exhaustive review of the literature has not been possible and the following survey concentrates on the developments of the author and colleagues to explain the basic methods.

Algorithms↗

PubMed1994

Structure, transmembrane topology and helix packing of P-type ion pumps.

Electron microscopy has recently provided improved structures for P-type ion pumps. In the case of Ca(2+)-ATPase, the use of unstained specimens revealed the structure of the transmembrane domain. The composition of this domain has been controversial due to the variety of methods used to study the number and exact locations of transmembrane crossings within the sequence. After reviewing the results from several members of the family, we found a consensus for 10 transmembrane segments, and also that 10 helices fitted well into the structure of Ca(2+)-ATPase. Thus, we present the most detailed model for transmembrane structure so far, in the hope of stimulating more precise experimental strategies.

Calcium-Transporting ATPases↗

PubMed1994

A model recognition approach to the prediction of all-helical membrane protein structure and topology.

This paper describes a new method for the prediction of the secondary structure and topology of integral membrane proteins based on the recognition of topological models. The method employs a set of statistical tables (log likelihoods) complied from well-characterized membrane protein data, and a novel dynamic programming algorithm to recognize membrane topology models by expectation maximization. The statistical tables show definite biases toward certain amino acid species on the inside, middle, and outside of a cellular membrane. Using a set of 83 integral membrane protein sequences taken from a variety of bacterial, plant, and animal species, and a strict jackknifing procedure, where each protein (along with any detectable homologues) is removed from the training set used to calculate the tables before prediction, the method successfully predicted 64 of the 83 topologies, and of the 37 complex multispanning topologies 34 were predicted correctly.

Animals↗

PubMed1994

A mutation data matrix for transmembrane proteins.

The widely used Mutation Data Matrix (MDM), is an amino acid comparison matrix calculated from a study of the exchange probabilities (or odds) derived from an analysis of the evolutionary changes seen in groups of very similar proteins. In this work, a mutation data matrix is calculated for membrane spanning segments. This new mutation data matrix is found to be very different from matrices calculated from general sequence sets which are biased towards water-soluble globular proteins, and the differences are discussed in the context of specific structural requirements of membrane spanning segments. This new matrix will help improve the accuracy of integral membrane protein sequence alignments, and could also be of use in the rational design of site directed mutagenesis experiments for this class of proteins.

Amino Acid Sequence↗

PubMed1994

A role for myn in Ha-ras transformation of mouse 10T1/2 fibroblasts.

The human max protein has been shown to form heterodimers with myc family proteins. The ability of myc+max heterodimers to act as sequence-specific transcriptional activators appears to be essential for the oncogenic activity of myc. max (also called myn in murine cells) can homodimerize to form a transcriptionally inactive complex. We previously showed that in mouse 10T1/2 cells, a combination of activated ras, c-myc, and a mutant form of p53 can cooperate in the induction of cellular transformation and metastasis. In the study presented here, we tested the hypothesis that expression of the myn gene may play a role in this cooperative process. Analysis of myn mRNA in these cell lines revealed the presence of a major 2.0-kb RNA species. This message and the 21- and 22-kDa myn polypeptides it encodes were significantly overexpressed in cells transformed by activated ras alone, by ras in combination with c-myc or mutant p53, or by ras plus myc plus mutant p53, in comparison with untransformed parental 10T1/2 cells. We also found that induction of ras expression in a cell line harboring an inducible ras gene was accompanied by an increase in myn mRNA expression. Interestingly, cotransfection of 10T1/2 cells with ras and myn inhibited cellular transformation in a focus-forming assay when compared with transfection with ras alone. These results suggest a role for ras in the regulation of myn gene expression and suggest a model of oncogene cooperativity in which the relative levels of myc and myn gene expression can influence ras transformation of mouse 10T1/2 cells.

Animals↗

PubMed1994

Multiple protein structure alignment.

A method was developed to compare protein structures and to combine them into a multiple structure consensus. Previous methods of multiple structure comparison have only concatenated pairwise alignments or produced a consensus structure by averaging coordinate sets. The current method is a fusion of the fast structure comparison program SSAP and the multiple sequence alignment program MULTAL. As in MULTAL, structures are progressively combined, producing intermediate consensus structures that are compared directly to each other and all remaining single structures. This leads to a hierarchic "condensation," continually evaluated in the light of the emerging conserved core regions. Following the SSAP approach, all interatomic vectors were retained with well-conserved regions distinguished by coherent vector bundles (the structural equivalent of a conserved sequence position). Each bundle of vectors is summarized by a resultant, whereas vector coherence is captured in an error term, which is the only distinction between conserved and variable positions. Resultant vectors are used directly in the comparison, which is weighted by their error values, giving greater importance to the matching of conserved positions. The resultant vectors and their errors can also be used directly in molecular modeling. Applications of the method were assessed by the quality of the resulting sequence alignments, phylogenetic tree construction, and databank scanning with the consensus. Visual assessment of the structural superpositions and consensus structure for various well-characterized families confirmed that the consensus had identified a reasonable core.

Amino Acid Sequence↗

PubMed1994

A method for alpha-helical integral membrane protein fold prediction.

Integral membrane proteins (of the alpha-helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable-hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid-exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three-dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult.

Amino Acid Sequence↗