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Biomedical subjects

W R Taylor

Publications and source records attributed to W R Taylor.

At least 73 records · Page 4Linked to original sources
PubMed1998

A Plasmodium chabaudi protein contains a repetitive region with a predicted spectrin-like structure.

cDNA and genomic DNA clones covering the entire open reading frame (ORF) for a Plasmodium chabaudi 96V protein were isolated. From the first ATG codon the intronless gene codes for a 229-kDa protein. Antisera raised against recombinant polypeptides coded by two different regions of the gene reacted with a 240/225-kDa doublet on Western blots of parasite extracts. In immunofluorescence studies the same sera detected the antigen at the apical end of the merozoite, possibly in rhoptry organelles. In Western blotting experiments the recombinant polypeptides were recognised by antibodies induced by natural infection. A 364-amino acid residue repetitive region, based on 32 11-mer repeats divided by two 6-mer repeats into three blocks, is located in the centre of the protein. Analysis of this repetitive region led us to propose a model in which each of the three units forms an alpha-helical coiled-coil triple-helix containing a possible leucine-histidine zipper. Each unit resembles in structure the units present in spectrin. The repeat region is flanked by predicted heptad based alpha-helical coiled-coil regions, and we propose that the protein forms a dimer. The 229-kDa protein has the overall character of a cytoskeletal protein. We have named the 229-kDa protein repetitive organellar protein (ROPE) and suggest that ROPE may be involved in the process of invasion, possibly by interacting with the erythrocyte cytoskeleton, and that the leucine histidine-zipper may be involved in molecular mimicry of spectrin.

Amino Acid Sequence↗
PubMed1998

Dynamic sequence databank searching with templates and multiple alignment.

Sequence databank searches are often performed iteratively, taking the results of a search to form a probe (either a pattern or profile) for a subsequent scan of the databank. The advantage of this approach is that, as more sequences are drawn into the probe, it should, in principle be possible to detect increasingly distant members of the family. This approach works well when supervised by an "expert" who has a good "eye" for the quality of the sequence alignment and whether novel matches should be rejected or incorporated into the probe. However, all attempts to automate the process have proved difficult, as the process is inherently unstable. Errors in the alignment, or the misalignment of a non-family member, lead to a deterioration of the probe specificity, so allowing further incorrect sequences to be identified. Here, a combination of two methods is used to provide a check on such instability. A pattern matching (template) search method is used (with a BLAST-like pre-filter for speed) to return sequence segments for alignment in a standard multiple alignment program (MULTAL). Sequences are aligned only to a fixed limit of similarity and any sequences or sub-families that have not joined the original "seed" family are rejected. The remaining core family then provides the basis for a subsequent pattern derivation and databank search. The constant check by the multiple alignment phase allows the search phase to be pushed continually towards the boundary of similarity. This is maintained by lowering the cutoff on the scores of acceptable sequences each time the family remains the same over successive search cycles. The procedure was observed to be stable under misalignments and to have an ability to recognise distantly related family members across super-families that was comparable to Psi-BLAST. The method is applied to the analysis of the hormone-binding domains of the insulin and related growth-factor receptors.

Azurin↗
PubMed1998

Calcium extrusion from mammalian photoreceptor terminals.

Ribbon synapses of vertebrate photoreceptors constantly release glutamate in darkness. Transmitter release is maintained by a steady influx of calcium through voltage-dependent calcium channels, implying the presence of a mechanism that is able to extrude calcium at an equal rate. The two predominant mechanisms of intracellular calcium extrusion are the plasma membrane calcium ATPase (PMCA) and the Na+/Ca2+-exchanger. Immunohistochemical staining of retina sections revealed strong immunoreactivity for the PMCA in rod and cone terminals, whereas staining for the Na+/Ca2+-exchanger was very weak. The PMCA was localized to the plasma membrane along the sides of the photoreceptor terminals and was excluded from the base of the terminals where the active zones are located. The amplitude of a calcium-activated chloride current was used to monitor the intracellular calcium concentration. An upper limit for the time required to remove intracellular free calcium is obtained from two time constants measured for the calcium-activated chloride current tail currents: one of 50 msec and a second of 190 msec. Calcium extrusion was inhibited in the absence of intracellular ATP or in the presence of the PMCA inhibitor orthovanadate, but was unaffected by replacement of external Na+ with Li+. The data indicate that the PMCA, rather than the Na+/Ca2+-exchanger, is the predominant mechanism for calcium extrusion from photoreceptor synaptic terminals.

Animals↗
PubMed1998

The p53 network.

Explore the source record for details and available documents.

Apoptosis↗
PubMed1998

High-affinity glutamate transporters in the rat retina: a major role of the glial glutamate transporter GLAST-1 in transmitter clearance.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1+/-0.4 microM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

ATP-Binding Cassette Transporters↗
PubMed1998

Mechanical deformation of the arterial wall in hypertension: a mechanism for vascular pathology.

Hypertension is an independent risk factor for atherosclerotic disease. It has been proposed that the atherogenic potential of hypertension is due to the development of a proinflammatory state within the arterial wall that is, at least in part, a result of the generation of reactive oxygen species. This article proposes that mechanical deformation of the arterial wall is a critical stimulus in this scheme. Data is reviewed that demonstrate that mechanical deformation of the arterial wall stimulates the generation of reactive oxygen species and subsequently results in the upregulation of redox-sensitive proinflammatory gene products.

Animals↗
PubMed1998

The study of the influence of flow on vascular endothelial biology.

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.

Animals↗
PubMed1998

Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy.

Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H2O2 production by studying the effects of Ang II on intracellular H2O2 generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H2O2 levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II-stimulated superoxide to H2O2, which accumulates more rapidly than it is degraded by catalase. This increase in H2O2 was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT1 receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II-induced production of H2O2 was almost completely inhibited, confirming that the source of Ang II-induced H2O2 was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H2O2 is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II-induced hypertrophy (62+/-2% inhibition). These data indicate that AT1 receptor-mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H2O2, suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.

Angiotensin II↗
PubMed1998

A comparison of efficacy of sargramostim (yeast-derived RhuGM-CSF) and filgrastim (bacteria-derived RhuG-CSF) in the therapeutic setting of chemotherapy-induced myelosuppression.

A randomized, double-blind, multicenter study in 181 afebrile cancer patients with ANC levels < 500/microL receiving myelosuppressive chemotherapy was undertaken to compare sargramostim (yeast-derived recombinant human granulocyte-macrophage colony-stimulating factor, RhuGM-CSF) and filgrastim (bacteria-derived recombinant human granulocyte colony-stimulating factor, RhuG-CSF) in the treatment of chemotherapy-induced myelosuppression. Patients received daily subcutaneous (SC) injections of either agent until ANC levels reached at least 1500/microL. There was no statistical difference between treatment groups in the mean number of days to reach an ANC of 500/microL, but the mean number of days to reach ANC levels of 1000/microL and 1500/microL was approximately one day less in patients receiving filgrastim. Fewer patients in the sargramostim arm were hospitalized, and they had a shorter mean length of hospitalization, mean duration of fever, and mean duration of i.v. antibiotic therapy compared with patients who received filgrastim. Both growth factors were well tolerated. No patient was readmitted to the hospital after growth factor was discontinued. Sargramostim and filgrastim have comparable efficacy and tolerability in the treatment of standard-dose chemotherapy-induced myelosuppression in community practice.

Adult↗
PubMed1998

Case of the month: January 1998--43 year old male with radicular pain.

A 43-year-old male presented with progressively worsening right lower extremity pain. MRI of the spine showed a discrete intradural, extramedullary, homogeneously enhancing, sausage-shaped mass at L1-L2, noted intraoperatively to expand the filum terminale. Gross, histological, and electron microscopic findings were those of a paraganglioma. The case is used to discuss the differential diagnosis for sausage-shaped tumors of the filum terminale.

Adult↗
PubMed1997

Ribonucleotide reductase R2 gene expression and changes in drug sensitivity and genome stability.

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.

3T3 Cells↗
PubMed1997

Defective induction but normal activation and function of p53 in mouse cells lacking poly-ADP-ribose polymerase.

Poly-ADP-ribose polymerase (PARP) and p53 are both induced by DNA damage and each has been proposed to mediate the normal cellular response to damage. We find that embryo fibroblasts from PARP-null mice have a approximately twofold lower basal level of p53 and that the induction of p53 in response to DNA damage or nucleotide depletion is more than twofold less than in normal mouse cells. These factors combine to decrease the induced level of the p53 protein in PARP-deficient cells by 4-5-fold, compared to normal cells. However, there is virtually no decrease in the induction of p53 activity in PARP-deficient cells, as assayed with a p53-responsive promoter. Furthermore, cells lacking PARP arrest normally in G1 after DNA damage, in contrast to cells lacking p53, where this checkpoint is absent. Other p53-dependent properties, such as the mitotic spindle checkpoint and permissivity for gene amplification, are also normal in PARP-deficient cells. We conclude that the induced level of the p53 protein is governed by a combination of PARP-dependent and PARP-independent pathways and that the activation of p53 is largely PARP-independent. The results are consistent with a model in which the regulation of gene expression by p53 involves both increases in the amount of the protein and activation of p53 as a transcription factor.

Animals↗
PubMed1997

Multiple sequence threading: an analysis of alignment quality and stability.

Methods that compare a protein sequence directly to a structure can be divided into those that construct a molecular model (threading methods) and those that perform a sequence alignment with the structure encoded as a sequence of structural states (one-dimensional/three-dimensional (1D/3D) matching). The former take into account the internal packing of the molecule but the latter do not. On the other hand, it is simple to include multiple sequence data in a 1D/3D comparison but difficult in a threading method. Here, a protein sequence/structure alignment method is described that uses a combination of matching predicted and observed residue exposure, predicted and observed secondary structure (1D/3D) together with pairwise packing interactions in the core (threading). Using a variety of distantly related and analogous protein structures, the multiple sequence threading (MST) method was compared to a single sequence threading (SST) method (that uses complex potentials of mean-force) and also to a multiple sequence alignment (MSA) program. It was found that the MST method produced alignments that were better than the best that could be obtained with either the SST or MSA method. The method was found to be stable to error in both secondary structure prediction and predicted exposure and also under variation of the key parameters (fully described in an Appendix). The contribution of the pairwise term was found to be small but without it, the correct alignments were less stable and structurally unreasonable deletions were observed when matching against larger structures. Using the parameters derived for alignment, the method was able to recognise related folds in the structure databank with a specificity comparable to other methods.

Algorithms↗
PubMed1997

Protein modeling by multiple sequence threading and distance geometry.

The application of homology modeling is often limited by the lack of known structures with sufficiently high sequence similarity to the target protein. The recent development of threading methods now enable the identification of likely folding patterns in a number of cases where the structural relatedness between target and template(s) is not detectable at the sequence level. We devised a hybrid method in which fold recognition was performed using the Multiple Sequence Threading (MST) method. The structural equivalences deduced from the threading output were used to guide the distance geometry program DRAGON in the construction of low-resolution C alpha/C beta models. The initial structures were converted to full-atom representation and refined using the general-purpose molecular modeling package QUANTA. The performance of the approach is illustrated on the CASP2 target T0004 (polyribonucleotide nucleotidyl-transferase S1 motif (PNS1) from Escherichia coli, PDB code: 1SRO) for which no obvious homologues with known structure were available. The correct fold of PNS1 was successfully identified, and the model was found to be more similar to the experimental PNS1 structure than the scaffold (C alpha RMSD of 6.2 A compared with 6.4 A). Our results indicate that a sensitive fold recognition algorithm coupled with a distance geometry program capable of rapidly generating initial structures can successfully complement high-resolution homology modeling methods in cases where sequential similarity is low.

Escherichia coli↗
PubMed1997

Pressure regulates osteoclast formation and MCSF expression in marrow culture.

One of the forces generated during skeletal loading is hydrostatic pressure. In the work presented here, the ability of increased pressure to influence recruitment of osteoclasts was evaluated. Murine marrow cultures, with pO2 and pCO2 kept constant, were subjected to either control (1.0 atm) or elevated (1.37 or 2.0 atm) hydrostatic pressure. As compared to control, cultures pressurized for 6 days at 1.37 atm formed less osteoclast-like cells (OCLC) (71 +/- 6% of control, P < 0.0001). A similar degree of inhibition occurred in cultures exposed to pressure during days 2-4 only (62 +/- 6%), while treatment during days 5-7 failed to inhibit the OCLC number relative to control (99 +/- 5%). Delivery of 2.0 atm pressure on days 2-4 generated 52 +/- 4% OCLC compared to control. Since macrophage colony stimulating factor (MCSF)-dependent proliferation of osteoclast precursors occurs during the pressure-sensitive period, semiquantitative RT-PCR for MCSF mRNA was performed after 3 days in 1.37 atm (days 2-4). As compared to controls, pressure caused a decrease in mRNA coding for the membrane bound form of MCSF (71.2 +/- 4% (n = 25, P < or = 0.05), while the MCSF RT-PCR product representing the secreted form showed no consistent change. This lack of response of the soluble MCSF RT-PCR product was expected, as levels of bioassayable MCSF were not altered by pressure. Extrapolating these data to in vivo conditions suggests that load-bearing will inhibit the formation of osteoclasts.

Animals↗