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Biomedical subjects

W R Taylor

Publications and source records attributed to W R Taylor.

At least 127 records · Page 7Linked to original sources
PubMed1994

Comparison of glyceraldehyde-3-phosphate dehydrogenase and 28S-ribosomal RNA gene expression as RNA loading controls for northern blot analysis of cell lines of varying malignant potential.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been a gene of choice in Northern blot analyses as an internal RNA loading control. We have investigated the expression of GAPDH and 28S-ribosomal RNA (28S rRNA) genes in mouse 10T1/2 cells and in a variety of tumorigenic and highly malignant metastatic cell lines derived from the 10T1/2 cell line. We observed that GAPDH mRNA levels varied markedly among the tumorigenic and highly malignant cell lines and were elevated in these cell lines when compared to the normal mouse 10T1/2 cells. In contrast, the levels of 28S rRNA did not significantly vary among the tumorigenic and highly malignant cell lines and were approximately at the same level as that found in the normal parental mouse 10T1/2 cell line. These observations indicate that much caution should be taken when using GAPDH gene expression as an RNA loading control for Northern blots. Based upon these observations, we recommend the use of 28S rRNA gene expression as a preferred RNA loading control for Northern blot analysis in which total RNA is used.

Animals↗
PubMed1994

Sequence conservation and correlation measures in protein structure prediction.

The rapid elucidation of protein sequences has allowed multiple sequence alignments to be calculated for a wide variety of proteins. Such alignments reveal positions that exhibit amino acid conservation--either of specific chemical groups in active and binding sites or of the more chemically inert hydrophobic residues that contribute to the protein core. The latter can provide constraints on the position of the protein chain and any local periodicity can suggest the type of secondary structure. Conservation measures, however, cannot provide specific pairwise packing information (each conserved hydrophobic position might pack against any other). However, if correlated changes between positions were observed then specific pairs of residue could be identified as interacting and therefore probably spatially adjacent. Most 'observations' of correlated changes have been anecdotal and of the few systematic studies that have been made, most have mistakenly incorporated a strong bias towards selecting conserved positions. When the conservation effect is separated (as best as possible) then little correlation signal remains to help identify adjacent positions.

Animals↗
PubMed1994

Remotely related sequences and structures: analysis and predictive modelling.

Through the comprehensive analysis of protein sequence and structural data, relationships can be established that suggest, with varying degrees of success, structural models for a protein for which only the sequence is known. The certainty with which a model can be proposed depends on the degree of similarity between the sequence of unknown structure and the sequence of a protein of known structure. Methods are being developed to detect remote similarities between sequences or structures, and to predict protein structure based on such small levels of similarity.

Amino Acid Sequence↗
PubMed1994

Motif-biased protein sequence alignment.

A method was developed for pairwise protein sequence alignment to emulate the effect of structural knowledge or multiple sequences. Runs of matches of the preferred length were emphasized through the use of a product-bias allowing short motifs to influence the alignment to a degree that was a realistic reflection of their infrequency of occurrence. This gave motifs a locally high scoring match, making their alignment relatively less sensitive to the value of the gap penalty. This property should be a great advantage when a large number of sequence comparisons are made with a fixed set of parameter values, as typically occurs in the scan of a sequence databank with a probe or in the development of a multiple alignment.

Algorithms↗
PubMed1994

Compensating changes in protein multiple sequence alignments.

A method was developed to identify compensating changes between residues at positions in a multiple sequence alignment. (For example, one position might always contain a positively charged residue when the other is negatively charged and vice versa.) A correlation-based method was used to measure the compensation found in the four residues at a pair of positions in any two sequences in a multiple alignment. All possible sequence pairings were measured at the pair of positions and the resulting matrix analysed to give a measure of cooperativity among the pairs. The basic method was sufficiently flexible to consider a number of amino acid relatedness models based both on scalar and vectorial properties. Pairs of compensating positions were selected by the method and their mean separation (in a protein of known structure) was compared to both the mean pairwise separation over all residues and the pairwise separation over an equivalent sample of pairs of residues selected on the basis of their conservation alone. The latter is an important control that has been omitted from previous studies. The results indicated that, at best, there was a slight effect (of marginal significance) leading to the selection of closer pairs by the compensation measure when compared to the mean of all pairs. However, this was never as good as the simpler measure based on conservation alone, which always found a significant majority of proteins with a sample mean less than the overall mean.

Amino Acid Sequence↗
PubMed1994

Secondary structure formation in model polypeptide chains.

Model polypeptide chains were folded into 3-D compact conformations using distance geometry techniques. Interresidue distances were predicted from the hydrophobicity of the monomers and were refined by repeated projections into lower-dimensional spaces. Main-chain hydrogen bond networks were constructed and propagated through the structure by adjusting local conformations to comply with ideal distance constraints around hydrogen bonds. The resulting folds were compact globules with distinct hydrophobic cores and contained secondary structure elements like real protein molecules. Apart from similarity in appearance, several properties of the model chains were also very close to those of native folded polypeptides. The method in its present form can serve as a starting point for the development of a novel structure prediction algorithm.

Chemical Phenomena↗
PubMed1993

A local alignment method for protein structure motifs.

A method for the comparison of protein three-dimensional substructures was developed. The method employs the double dynamic programming method of Taylor & Orengo but identifies multiple local alignments rather than a single global alignment. A modification based on the Smith Waterman algorithm for sequence alignment enables the automatic identification and growth of the most structurally similar local alignments irrespective of length and composition. The method can also be used in a search mode to match substructures. Roughly five minutes is required to find all Greek-key motifs in a protein of 100 residues. Two novel globin folds have been detected using the method, the antibiotic protein colicin A and diphtheria toxin, neither of which have any functional relationship to the globins.

Amino Acid Sequence↗
PubMed1993

Deriving an amino acid distance matrix.

Various methods were investigated to convert an amino acid similarity matrix into a low-dimensional, metric distance matrix. Using projection techniques, no unique transformation was found and of the many inversion forms investigated, simple negation normalized by the diagonal elements produced a good fit to the original data. An inter-row distance also gave a comparable fit and when evaluated by weighted least-squares minimization was found to be preferable. A rank-ordered form of the matrices was also derived by constraining neighbours to be equidistant in 3D-space. This produced a network configuration not unlike that produced in a previous analysis of amino acid physicochemical properties. The derived forms might find applications in sequence alignment, including pattern-matching algorithms, and the construction of phylogenetic trees.

Amino Acid Sequence↗
PubMed1993

Polarized secretion of IGF-I and IGF-I binding protein activity by cultured aortic endothelial cells.

Insulin-like growth factor-I (IGF-I) secretion by the vascular endothelium has been proposed to play a role in the regulation of vascular smooth muscle cell proliferation. Because vascular smooth muscle cells are adjacent to the abluminal surface of the endothelium, we tested the hypothesis that secretion of IGF-I by endothelial cells is polarized. Porcine aortic endothelial cells were cultured on permeable membranes and IGF-I measured by radioimmunoassay. Basal secretion exceeded apical secretion by a ratio of 2.3 +/- 0.2:1.0 (P < 0.05). We also identified 35 kDa IGF-I binding protein activity that is preferentially secreted on the basal surface of endothelial cells. We conclude that both IGF-I and IGF-I binding protein activity secretion by endothelial cells is polarized towards the basal surface of the endothelium. A polarized secretion mechanism for IGF-I may be of importance in the normal growth and differentiation of the vasculature as well as in the development of vascular pathology.

Animals↗
PubMed1993

A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.

Amino Acid Sequence↗
PubMed1993

Cell motility, invasion, and malignancy induced by overexpression of K-FGF or bFGF.

We have tested the hypothesis that fibroblast growth factors contribute to cell locomotion and invasion, by investigating the properties of NIH-3T3 fibroblasts that secrete K-FGF following transfection with the K-fgf proto-oncogene and NIH-3T3 fibroblasts transfected with either the normal basic fibroblast growth factor (bFGF) coding sequence that lacks a known secretory signal sequence, or a chimeric bFGF sequence fused to an immunoglobulin signal sequence that targets the growth factor to the secretory pathway. The cell lines were tested for altered invasive characteristics on a physiologically relevant collagen substratum, and their motility rates were measured in cell locomotion assays using time-lapse video microscopy in the absence or presence of suramin, an inhibitor of growth factor-receptor interaction. The data were analyzed by comparing the motility rates of all K-fgf- and bFGF-transfected cell lines as a function of malignant potential. These studies show for the first time that K-fgf gene expression potently increases cell motility characteristics. Further, the bFGF transfectants generally exhibited increased rates of cell locomotion which were not dependent upon the presence of a signal sequence for secretion. These results support models of cell locomotion in which stimulation of this process can occur through both autocrine and intracrine pathways. In addition, a significant correlation was found to exist between cell locomotion and malignant potential, supporting the view that growth factor-induced motility, although not sufficient on its own to induce metastasis, is important in the promotion of tumor cell dissemination.

3T3 Cells↗
PubMed1993

Recurring structural motifs in proteins with different functions.

BACKGROUND: Structures that have diverged from a common ancestor often retain functional and sequence similarity, although the latter may be very reduced. Even so, the overall fold of the structure is generally highly conserved. Now however, several have been identified of proteins that have been identified that have different functions but which have converged to a similar fold. These proteins will also have low sequence identities. RESULTS: By comparing the complete structure databank against itself, using sequence and structure alignment techniques, we have been able to identify six new examples of structurally related folds that have no apparent sequence or functional similarity. These related proteins include a family of crambin-like folds and a family of ferredoxin II folds. We found that all the similarities between structures are present in small proteins and occur as motifs within the core of a larger protein. CONCLUSION: The low sequence similarity and the lack of any obvious functional relationship between proteins with similar structures suggest that the proteins have diverged from independent ancestors. The similarities may therefore be of interest for understanding the various stereochemical and physical criteria that operate to generate a favourable fold.

Journal Article↗
PubMed1993

Connection topology of proteins.

One-dimensional amino acid sequences and three-dimensional folded polypeptide chains were modelled as non-directed graphs in which nodes corresponded to amino acids and arcs represented connections between them. In the case of folded chains, non-backbone connections were assigned to amino acid pairs if their distance was lower than a threshold. Two topological indices, the connectedness number and the effective chain length were devised to compare folding topologies. Loops created by non-backbone connections in the structure graphs were studied by simple graphical representations, revealing the hierarchy in native protein structures.

Amino Acid Sequence↗
PubMed1993

Mortality and use of health services surveys in rural Zaire.

The Combatting Childhood Communicable Disease (CCCD) project is a comprehensive public health programme designed to reduce child mortality by 25% through the use of the following strategies: vaccination, oral rehydration therapy, and prompt treatment for malaria. To evaluate this programme, cross-sectional surveys were conducted in neighbouring health zones in Zaire in 1984 to determine the use of selected medical services by the population and to estimate the child mortality rate before the CCCD programme began. A reinterview survey was conducted on a subsample of women previously interviewed to determine the reliability of the mortality estimates. In both health zones 84-85% of women used antenatal services, 45% of children under age 6 who had had fewer were treated with an anti-malarial drug, 19-22% of children age 12-23 months had been vaccinated against measles, and virtually no children who had had diarrhoea were treated with oral rehydration therapy. Women's underreporting of births and deaths resulted in low estimates of mortality in both surveys. The reinterview survey provided more accurate estimates of mortality and led to a better understanding of the factors influencing underreporting. The estimated infant mortality rate was 74 deaths per 1000 livebirths; and the probability of dying before age 5 was 191 per 1000. Because births and deaths reported with incomplete dates were excluded from analysis, the mortality rates from the reinterview survey are underestimates. Given the difficulty in obtaining accurate estimates of mortality, primary importance should be given to developing and improving routine health information systems that measure changes in health status and provide information to evaluate programmes.

Adolescent↗
PubMed1993

Changes in use of health services in a rural health zone in Zaire.

As part of the Combatting Childhood Communicable Diseases (CCCD) project funded by the US Agency for International Development (USAID), the Zairian CCCD programme conducted surveys in the rural health zones of Kingandu and Pai-Kongila, Zaire, in 1984-1985 and 1988-1989 to determine whether a strategy of selective primary health care would affect childhood mortality. This paper describes the changes in the medical care infrastructure and the increasing coverage of selected services. The strategies evaluated were vaccination, oral rehydration therapy, and treatment of febrile episodes with antimalarial drugs for children; and tetanus vaccination and malaria prophylaxis for pregnant women. The health infrastructure in the Kingandu and Pai-Kongila Health Zones expanded considerably from 1984 to 1989, with health centres increasing from 7 to 18. During this period, economic conditions deteriorated moderately, with the nation experiencing nearly 700% inflation. Medical care costs remained stable because of external subsidies. Use of health services was assessed in 1984, 1988, and 1989. Between 1984 and 1989, the proportion of children aged 12-23 months vaccinated against measles increased from 22% to 71%. Coverage with other vaccine antigens increased similarly. Women's knowledge of the correct recipe for the preparation of sugar-salt solution increased from 0% to 61%. Reported treatment at home with sugar-salt or oral rehydration solution increased from 6% to 53%. The proportion of children with febrile episodes who were treated presumptively for malaria with chloroquine remained unchanged (47% in 1984; 44% in 1988). We conclude that, despite a moderate deterioration in economic conditions, Kingandu and Pai-Kongila Health Zones achieved remarkable increases in use of selected health services between 1984 and 1989, especially in vaccination coverage.

Adolescent↗