Biomedical subjects

D R Phillips

Publications and source records attributed to D R Phillips.

At least 145 records · Page 8Linked to original sources

PubMed1991

Problems and potential of researching epidemiological transition: examples from Southeast Asia.

The concept of epidemiological transition is now quite widely recognized, if not so widely accepted. The transition appears to progress at varying speeds and to different extents spatially; it seems that there can be considerable international, regional and local variations in its progress. The paper examines this contention in the case of a number of countries in Southeast Asia, principally Hong Kong, Malaysia and Thailand. Drawing on evidence from this region, the paper highlights the importance when researching epidemiological transition of the time period under consideration; socio-cultural variations; the nature and quality of data, and spatial scale. It makes some suggestions as to the potential of the concept of epidemiological transition in health care planning and development studies.

Asia, Southeastern↗

PubMed1991

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells.

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗

PubMed1991

Subcellular localization of porphyrins using confocal laser scanning microscopy.

The in vitro subcellular distribution patterns of 10 porphyrins, varying in hydrophobicity and charge, were studied using confocal laser scanning microscopy on two cell lines (V79 and C6 glioma cells) for incubation times up to 24 h. All of the porphyrins were taken up rapidly by both cell lines and distinct classes of subcellular distribution patterns were observed: general cytoplasmic staining; localization in lysosomes (usually associated with general cytoplasmic staining); localization in mitochondria (and general cytoplasmic staining); localization in mitochondria with subsequent uptake into lysosomes. Structure-localization relationships which have emerged are that porphyrins with dominantly cationic side chains localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.

Animals↗

PubMed1991

Isolation and characterization of phosmidosine. A new antifungal nucleotide antibiotic.

A new nucleotide antibiotic, phosmidosine was isolated from a culture filtrate of a newly isolated streptomycete identified as Streptomyces sp. RK-16. HRFAB-MS and elemental analysis established the molecular formula of C16H24N7O8P. 1H, 13C and 31P NMR indicated the presence of a methyl phosphate group and UV spectra were similar to those of 8-hydroxyadenosine. The antibiotic inhibited spore formation of Botrytis cinerea at the concentration of 0.25 micrograms/ml.

Antifungal Agents↗

PubMed1990

Characterization of the human platelet glycoprotein IIIa gene. Comparison with the fibronectin receptor beta-subunit gene.

This study was designed to determine the structure of the gene for glycoprotein (GP) GPIIIa, the beta-subunit of the platelet membrane GPIIb-IIIa complex. The complexity of the gene was determined after Southern analysis of human chromosomal DNA. Overlapping genomic clones were isolated from cosmid and phage lambda libraries that contained the entire coding unit of the human gene for the mature GPIIIa protein. The genomic clones spanned approximately 60 kilobase pairs of human DNA sequence. The exon containing segments of the clones was mapped and the exons, including the exonintron junctions, were sequenced. The GPIIIa protein is divided into 14 exons ranging in size from 87 to 430 nucleotides separated by introns, which were 0.3 to 9 kilobase pairs in size. The 3' exon was larger than 1700 nucleotides and contained the 3'-untranslated region. Several structural domains of the GPIIIa protein were contained within individual exons. These included (i) the transmembrane spanning segment, (ii) the cytoplasmic region containing the potential phosphorylation sites, and (iii) the six domains in the NH2-terminal half of GPIIIa that are highly conserved between two other integrin beta-subunits. In contrast, other domains such as the four cysteine-rich repeats were interrupted by introns. Genomic clones for the beta-subunit of the fibronectin receptor (beta 1) were also isolated, partially mapped, and sequenced. Of the eight splice sites identified in beta 1, six occurred at the same amino acid residue in GPIIIa. These results provide genetic evidence that GPIIIa and beta 1 have a common evolutionary origin within the integrin family.

Amino Acid Sequence↗

PubMed1990

Induction of stable transcriptional blockage sites by adriamycin: GpC specificity of apparent adriamycin-DNA adducts and dependence on iron(III) ions.

Initiated transcription complexes were exposed to adriamycin for up to 48 h. Subsequent elongation of the transcription complex revealed the presence of a series of discrete long-lived blockage sites. The mole fraction of blocked transcripts increased linearly with reaction time, adriamycin concentration, and Fe(III) concentration. Optimal conditions for formation of the blocked transcript were 24-h reaction time, 10 microM adriamycin, and 75 microM Fe(III) ions. Nine high-intensity blocked transcripts were observed, and all correspond to transcription proceeding up to G of GpC sequences of the nontemplate strand. The presence of 75 microM Fe(III) ions enhanced the amount of transcriptional blockages by 12-15-fold. Two blocked transcripts decayed with a half-life of 0.32 and 1.9 h, and one of these exhibited 100% effective delayed termination 6 bp downstream of the original blockage site. All other blockages were unchanged after 3 h of elongation. Bidirectional transcription footprinting was used to define the physical size of the drug-induced blocking moiety as a maximum of 2 bp, and this was observed at all three GpC elements probed by RNA polymerase from both directions. The nature of the apparent covalent adducts has not yet been established but is probably a G-specific adduct deriving from a reduced form of the drug (quinone methide). Although the GpC specificity suggests an interstrand G-drug-G cross-link, these were not detected by heat denaturation and subsequent denaturing gel electrophoresis of the end-labeled promoter fragment.

Base Sequence↗

PubMed1990

Monte-Carlo simulation of multisite echinomycin-DNA interactions detected by in vitro transcription analysis.

The interaction of echinomycin with DNA was analyzed at 37 degrees C by in vitro transcription analysis using a 497 bp fragment of DNA containing the lac UV5 promoter. Sixteen discrete drug binding sites were detected. The mole fraction of blocked transcript at each site was monitored over 4 h, and the kinetic profile was analyzed by Monte-Carlo simulation. The time course for all 16 sites was fully described by this process. For each drug site, three parameters were resolved with the following variation between sites: relative drug occupancy (1-26), dissociation rate constant (0.06-0.70 min-1), and probability of termination of transcription (0-48%). Eight low-occupancy binding sites were at 5'-CA sequences (relative occupancy of 1.0-2.9). The eight major sites were all at 5'-CG sequences (relative occupancy of 6.3-26) and exhibited an average occupancy some 13-fold greater than the CA sites, corresponding to an average additional stability of approximately 1.6 kcal. The dissociation rates from apparent high-affinity sites were only partially correlated with relative occupancy. Ten binding sites exhibited a 3-48% probability of termination of transcription immediately adjacent to the 5'-CG central sequence. Termination probably arises from distortion of the DNA helix in regions flanking the binding site and was most dramatic (48% probability) where two adjacent CG sites were separated by only 1 bp. This termination phenomenon may well account for the observed effects of echinomycin in vivo.

Base Sequence↗

PubMed1990

Fluorinated anthracyclines: interactions with DNA.

Four fluorine containing derivatives of anthracyclines (daunomycin and adriamycin) were synthesised, and comprised C-13, 1,1,1-trifluoroethyl-hydrazones of each anthracycline, and C-14 4-F and 4-CF3-benzoate esters of adriamycin. All four derivatives intercalated into DNA in a manner similar to their parent anthracycline. The ester derivatives exhibited 3-4-fold higher binding affinity to DNA, and slower DNA dissociation kinetics than adriamycin. This stabilisation derives from additional contacts of the C-14 side chain to the DNA minor groove. The hydrazone derivatives showed lower binding affinity to DNA, and dissociated from DNA 3-4 times faster than the parent compound. The 19F resonance of the bound drug was broadened to 120 Hz and shifted 60 Hz downfield (0.32 ppm) relative to the sharp (7.5 Hz) peak of the free drug. These values imply a rapid exchange between the free and DNA bound forms (DNA lifetime greater than 5 ms), with the fluorine group residing in a hydrophobic region in close contact with the DNA minor groove. The 4-8 fold lesser specific potency of the ester derivatives supports the concept that DNA binding is an important factor, but not sole determinant of biological activity of these analogues.

Animals↗

PubMed1990

Fluorinated anthracyclines: synthesis and biological activity.

A number of fluorine containing derivatives of daunomycin and Adriamycin have been synthesised with a fluorine substituent located on the C-9 side chain. They included the p-trifluoromethyl-(4) and p-fluorobenzoate (3) esters of Adriamycin and the trifluoro-ethyl-hydrazones of Adriamycin (2) and daunomycin (1). The less polar derivatives (esters 3 and 4) appear to enter HeLa cells more readily than the polar derivatives (hydrazones 1 and 2) as indicated by the rates and extent of cellular uptake and the uptake was by facilitated diffusion. All four fluorinated derivatives were less active than their parent anthracyclines, but the difference was less pronounced when considering specific potency. The fluorinated derivatives (1-4) behave sufficiently similar to daunomycin and Adriamycin to enable their use as fluorine probes in 19F NMR physicochemical and cellular studies of anthracyclines.

Antibiotics, Antineoplastic↗

PubMed1990

Spatial patterns of use of health services in the Kingston metropolitan area, Jamaica.

This study focuses on the influence of distance, transport and accessibility on the use of health services in Kingston, Jamaica. It reviews various factors affecting the use of health care with particular reference to Third World cities and presents results from a case study of utilization in the Kingston Metropolitan Area. Three pairs of sites of contrasting social status were selected and 50 respondents questioned in each about spatial patterns of primary health care (PHC) attendance. Types of facility attended, mode of transport, travel times, distance and frequency of utilization were discussed. Some distinctive differences appeared between low and high status site respondents with regard to distances to facilities and travel times, which were almost always higher for the low status respondents. Most respondents were not using their nearest facilities, for varying reasons which included, for poorer respondents, need to attend frequently distant public facilities and, for wealthier respondents, loyalty to old family doctors and use of company-related doctors. The mobility of the higher status respondents afforded them considerable choice of locations used for health care and their attendance was much more convenient than that of lower status respondents. Utilization rates were somewhat higher in the high status sites although not to the extent found by some earlier studies in similar settings.

Developing Countries↗

PubMed1990

Activism for medical geographers: American, British and Canadian viewpoints.

This paper describes some of our personal efforts to launch research projects that address public health issues of interest to geographers in the United States, Canada and Britain. In pressing these agendas we have found through our experiences that there are personal and disciplinary costs associated with activism. We describe the loss of identity with geography; the frustration of trying to persuade bench scientists, corporate representatives, and government officials of the importance of our work; the loss of research time and contact with both our academic colleagues and students.

Adolescent↗

PubMed1990

Platelet membrane glycoproteins: functions in cellular interactions.

The understanding of the structure and function of platelet membrane glycoproteins has been facilitated by studies showing that they belong to larger gene families of cell surface receptors involved in cellular interactions. In some instances (e.g. GP IIb-IIIa and GP Ib-IX) the study of the platelet proteins has served as a prototype for relatively newly described gene families (e.g. integrins and LRG proteins, respectively). In other instances, e.g. PECAM-1, the background of information on immunoglobulin domain-containing proteins has served to indicate functions. Receptor-ligand interactions have been characterized at the molecular level, and studies of genetic defects affecting platelet receptors have contributed significantly to understanding structure-function relationships. Gene transfection studies provide encouraging results that might lead to gene therapy. The knowledge about platelet ligand-receptor processes contributes not only to our understanding of normal platelet function, but also to a more generalized understanding of adhesive mechanisms used by many cells to interact with their environment.

Amino Acid Sequence↗

PubMed1990

The synthesis, stability and biological activity of bis-intercalating bis-daunomycin hydrazones.

The synthesis of a series of bis-daunomycin hydrazones (5a-g)--all moderately stable at 37 degrees C, pH 6.8, with a half-life of approximately 30 h--is reported. Under a pulse exposure of 2 h they exhibited growth inhibition of mouse L1210 cells, and were 2-3 fold more active than daunomycin. Under continuous exposure growth inhibition conditions with human colon cell lines (HT-29 and HCT-8) they hydrolysed to daunomycin and a partially hydrolysed mono-derivative of daunomycin, and there was no apparent increase in activity over that of the parent anthracycline. Their rate of hydrolysis was observed to increase rapidly with decreasing pH.

Animals↗

PubMed1990

New insight into drug-DNA interactions at individual drug binding sites probed by RNA polymerase during active transcription of the DNA.

An in vitro transcription assay has been used to probe drug-DNA interactions during active transcription of the DNA. The method relies upon the formation of a stable, synchronized population of initiated transcripts comprising a short length of RNA, achieved by the absence of one nucleotide in the initiation mixture. Subsequent equilibration of the transcription complex with drug, followed by elongation of the initiated transcripts, yields lengths of RNA determined by transcription up to each drug-occupied site. A variety of site-specific phenomena have been observed, including delayed termination of transcription 5-10 bp downstream of actinomycin binding sites; 10-20% probability of termination at most echinomycin sites; drug residence-time-dependent termination of bacteriophage polymerases; enhanced residence time compared to physicochemical measurement of drug-DNA dissociation rates. The use of two counter-directed promoters separated by 100 bp results in a sensitive bidirectional transcription footprinting procedure able to resolve adjacent drug sites separated by only 1 bp. The significance of the method of in vitro transcription is the ability to quantitate a range of parameters describing individual drug sites in situations where multiple drug-DNA interactions exist.

Base Sequence↗

PubMed1989

Bidirectional transcription footprinting of DNA binding ligands.

An in vitro transcription assay has been developed to define the exact location of DNA binding ligands. The method employs two counterdirected Escherichia coli promoters separated by approximately 100 bp. Selective transcription from each promoter yields transcripts up to each ligand site. Nonsaturating levels of ligands result in fractional occupancy of ligand at each site, and hence a range of RNA transcript lengths. The bidirectional promoter system results in a transcription footprint which was derived from transcription from both promoters up to the 5' side of each occupied ligand site and defines the sequence specificity and binding site size of the DNA-bound ligand. The transcriptional footprint is precise to +/- 1 bp from the 5' and 3' ends of each binding site. Multiple ligand sites can be ranked in terms of relative fractional occupancy at each site, and the ranking is comparable from either transcription direction. The method was compared to classical DNase I footprinting with a series of DNA binding drugs [actinomycin D, echinomycin, bis(thiadaunomycin), mithramycin, nogalamycin, and an acridine-tripyrrole]. In all cases, specific binding sites were resolved more clearly by transcription footprinting than by DNase I footprinting. Because of the nature of the transcription assay, all occupied ligand sites were detected by this method, in contrast to DNase I footprinting where many sequences are not probed, and where ligand sites are often not accurately defined.

Acridines↗

PubMed1989

In vitro transcription analysis of the role of flanking sequence on the DNA sequence specificity of adriamycin.

An in vitro transcription assay has been used to define twelve high occupancy transcriptional blockage sites in 260 bp of heterogenous DNA on three promoter-containing DNA fragments. Transcription proceeded to immediately upstream of CpA sequences in nine of these sites, and this defines the most preferred intercalation site as CpA. In almost all cases, this sequence was flanked by T on the 5' end. The consensus sequence for the highest affinity Adriamycin site is therefore 5'-TCA, with some evidence for preference of AT base pairs flanking both ends of this trinucleotide [i.e., (t)TCA(a.t)(a.t)]. In contrast, lower occupancy CpA sites were flanked on the 5' end by a GC base pair, with a preference for up to three GC base pairs on the 3' end. The low affinity consensus sequence is therefore (G.C)CA(g.c)(g.c)(g.c).

Base Sequence↗

PubMed1989

Sequence-dependent termination of bacteriophage T7 transcription in vitro by DNA-binding drugs.

An in vitro T7 bacteriophage transcription system has been utilized in which the RNA was initiated to a specific length (defined by the absence of the appropriate nucleoside triphosphate). When the DNA-RNA-RNA polymerase ternary complex was exposed to nonsaturating levels of DNA-binding ligands (i.e., a small fractional occupancy at each site), and the RNA transcript then allowed to elongate in the presence of all four nucleoside triphosphates, there was a synchronous increase of RNA lengths up to sites occupied by ligands. A unique characteristic is that bacteriophage transcription was completely terminated at every ligand site, in contrast to bacterial RNA polymerases where "read-through" past drug sites occurs and results merely in a delay of transcription at each site due primarily to dissociation of drug from the DNA. Similar termination of transcription at each drug site was observed with T3 and SP6 RNA polymerases. The termination at drug sites in the bacteriophage system results in RNA of specific lengths which define the location of ligand sites, and the RNA concentration provides a measure of relative ligand occupancy at that site. Termination of transcription was observed with four drugs with relatively long DNA residence times (half-life greater than or equal to 300 s at 20 degrees C for nogalamycin, actinomycin, mithramycin, and echinomycin) but to a lesser extent with drugs of intermediate residence times [a bis(thiadaunomycin) and an acridine-tripyrrole, with half-lives of 230 and 7 s, respectively, at 20 degrees C]

Anti-Bacterial Agents↗

PubMed1989

DNA sequence-specific adducts of adriamycin and mitomycin C.

Adriamycin and mitomycin C were reduced by xanthine oxidase/NADH in the presence of a DNA template comprising a stable initiated ternary transcription complex derived from the lac UV5 promoter. Subsequent elongation of the transcription complex treated with mitomycin C revealed high levels of terminated transcripts one nucleotide prior to G residues on the coding strand (i.e. at X of XpC sequences of the non-coding strand). Lower levels of termination occurred with adriamycin, and these were also one nucleotide prior to G residues of the coding strand, but with greater sequence specificity since they were observed mainly at G of GpC sequences of the non-coding strand. The same sites were also observed with adriamycin in the absence of reducing conditions and the level of termination at these sites was enhanced up to 10-fold by Fe2+ and Fe3+, but not by Cu2+, Zn2+, Co2+ or Ni2+. These results suggest that an iron-adriamycin complex with DNA is highly sequence-specific and results in adducts, similar to those of mitomycin C, which can terminate the transcription process. Such a mechanism offers new insights into the possible mode of action of anthracyclines.

Base Sequence↗