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Biomedical subjects

B Ward

Publications and source records attributed to B Ward.

At least 109 records · Page 6Linked to original sources
PubMed1988

Determination of netropsin-DNA binding constants from footprinting data.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

Base Composition↗
PubMed1988

The treatment of intraperitoneal malignant disease with monoclonal antibody guided 131I radiotherapy.

Seven patients with small volume ovarian carcinoma, remaining after conventional therapy with surgery and a platinum containing chemotherapy regimen, were treated with intraperitoneal monoclonal antibody guided radiotherapy. 100 mCi131I conjugated to 10 mg of monoclonal antibody were injected i.p. in 2,000 ml peritoneal dialysis fluid. Patients were evaluated 3 months later; 3 had clinical progressive disease while third look laparotomy demonstrated progressive disease in 3 of the remaining 4 patients. The seventh patient did not have a third look laparotomy and is currently inevaluable for response. Five patients with recurrent malignant ascites not controlled by diuretics or repeated paracentesis were similarly treated with 75-170 mCi131I conjugated to 10 mg monoclonal antibody. In three patients the ascites was controlled for a mean of 4 months. One patient died too early to assess the control of his ascites but tumour cells disappeared from the ascitic fluid after therapy. In the patient whose ascites were not controlled, a subpopulation of antigen-negative tumour cells was demonstrated. This study was unable to demonstrate a therapeutic benefit for i.p. injected monoclonal antibody guided radiotherapy for solid intraperitoneal tumour but suggests that it may be capable of controlling the accumulation of antigen positive malignant ascites.

Adult↗
PubMed1987

Cytogenetic analysis of four human ovarian carcinoma cell lines.

Four cell lines derived from adenocarcinomas of the ovary, including three recently established cell lines, have been karyotyped. Chromosomes #1, #3, and #6 were found to be frequently involved in translocations with various other chromosomes, in agreement with results of other investigators, strongly implicating genes on these chromosomes in ovarian tumorigenesis.

Adenocarcinoma↗
PubMed1987

Quantitative footprinting analysis of the netropsin-DNA interaction.

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.

Antiviral Agents↗
PubMed1986

DNA binding specificity of a series of cationic metalloporphyrin complexes.

The sequence specificities of a series of cationic metalloporphyrins toward a 139 base pair restriction fragment of pBR-322 DNA have been studied by DNase I footprinting methodology. Analysis using controlled digests and quantitative autoradiography/microdensitometry revealed that the 5- and 6-coordinate complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine, MT4MPyP, where M is Mn, Fe, Co, and Zn, were found to bind to AT regions of DNA. Footprinting analysis involving the radiolabel on the opposing strand of restriction fragment showed site skewing in the direction of the 3' end of the fragment, indicating that the porphyrins bind in the minor groove of DNA. The significant increase in DNase I catalyzed hydrolysis observed in various regions of the fragment appeared to be primarily due to a decrease in available substrate DNA upon porphyrin binding with possible contributions from structural changes in DNA caused by ligand binding. The complexes NiT4MPyP and CuT4MPyP were found to bind to both AT and GC regions of the fragment, producing different degrees of inhibition in the two regions. Since the outside-binding porphyrins can neither intercalate or effectively hydrogen bond to DNA, they appear to read sequence by responding to steric and/or electrostatic potential effects located in the minor groove of DNA.

Base Sequence↗
PubMed1986

Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity.

Oligopeptides have been synthesized that are structurally related to the antiviral antitumor antibiotic netropsin, but in which each of the pyrrole units is successively replaced by an imidazole moiety, as well as their di- and triimidazole-containing counterparts. These compounds bind to duplex DNA with constants in the range (1.06-1.98) X 10(6) M-1 but not to single-stranded DNA. Since they bind to T4 DNA, it is inferred that, like the parent antibiotic netropsin, they are also minor groove selective. This series of compounds exhibits a progressively decreasing preference for AT sites in binding studies with both native DNAs and synthetic oligonucleotides and a corresponding increasing acceptance of GC base pairs. Footprinting experiments utilizing a 139 base pair HindIII/NciI restriction fragment from pBR 322 DNA revealed that these lexitropsins, or information-reading oligopeptides, recognize more sites than the parent netropsin. In addition, some regions of enhanced nuclease action as the result of drug binding to the fragment were identified. The diimidazole compound in particular recognizes GC-rich sites, implying the formation of new hydrogen bonds between G-C(2)NH2 in the minor groove and the additional N3 imidazole nitrogens. It is clear however that, since the lexitropsins appear to tolerate the original (AT)4 site, an N-methylimidazole group on the ligand will permit either a GC or AT base pair in the binding sequence. Another factor that may be significant in molecular recognition is the high negative electrostatic potential of A X T regions of the minor groove, which is likely to strongly influence binding of these cationic species to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

DNA cleavage specificity of a group of cationic metalloporphyrins.

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and discussed.

Base Sequence↗
PubMed1986

A prospective study of 123I-labeled monoclonal antibody imaging in ovarian cancer.

Thirty patients presenting with a pelvic mass were entered into a prospective study on the use of radioimmunoscintigraphy with the 123I-labeled monoclonal antibody HMFG2. The imaging data was obtained without knowledge of the clinical data and compared with subsequent surgical findings. A false-positive diagnosis of ovarian cancer was made in five of ten patients subsequently shown not to have ovarian cancer; thus the technique cannot be used as a screening test. A true-positive diagnosis was made in 19 out of 20 patients shown subsequently to have ovarian cancer. In 18 of these patients the distribution of uptake closely fitted the surgical findings. Methods of improving these results are described. In conclusion, radioimmunoscintigraphy is of no use in determining whether a pelvic mass is due to ovarian cancer, but has benefit in the evaluation of chemotherapy and may, in the future, prevent the need for second-look operations in some circumstances.

Antibodies, Monoclonal↗
PubMed1986

Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas.

We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors.

Antibodies, Monoclonal↗
PubMed1985

Prolonged outbreak of Norwalk gastroenteritis in an isolated guest house.

During November and December 1982, a persistent outbreak of gastroenteritis took place in an isolated guest house. An estimated 26% of the changing population of guests and staff developed symptoms. Laboratory studies implicated Norwalk agent as the cause of illness, although the original source of the outbreak could not be established. Information on clinical features of the illness, its mode of spread, and eventual control is presented.

Adolescent↗
PubMed1984

Kinetic study of CO and O2 binding to horse heart myoglobin reconstituted with synthetic hemes lacking methyl and vinyl side chains.

Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this view.

Animals↗
PubMed1984

A comparison of the short-term incorporation of erucic acid and oleic acid in the perfused guinea-pig heart.

A comparison was made of the incorporation of radioactive erucic acid and oleic acid in the isolated perfused guinea pig heart, 2, 15 and 30 min after a radioactive pulse. The complementary techniques of (a) freeze-clamping followed by lipid extraction and thin layer chromatography and (b) electron microscope autoradiography were used. The incorporation of 3H-erucic acid into esterified lipids was much slower than that of 3H-oleic acid. Less radioactive CO2 was produced by hearts perfused with 14C erucic acid then by hearts perfused with 14C oleic acid. There was no significant effect of erucic acid on the relative areas of subcellular organelles in the autoradiographs and, in particular, there was no increase in the volume of lipid droplets. However, the incorporation of radioactivity into lipid droplets was much greater with 3H-oleic acid than with 3H-erucic acid, consistent with the higher incorporation into tissue triacylglycerol. Although oxidized less than oleic acid, erucic acid was readily transported to the mitochondria. High levels of radioactivity in free fatty acid in the hearts perfused with erucic acid suggest that a low rate of activation of the fatty acid to acyl-CoA limits both oxidation and the formation of triacylglycerol. Electron microscopy of the hearts perfused with erucic acid revealed a widespread, and quantitatively demonstrable general movement of lipid droplets towards the surface of the cell. This was occasionally accompanied by a local rupturing of the sarcolemma, possibly prior to expulsion of the lipid droplet from the cell.

Animals↗
PubMed1984

Incorporation and distribution of 3H oleic acid in the isolated, perfused guinea-pig heart made hypoxic.

Improved methods of autoradiography and lipid extraction have been used to study the influence of hypoxia on the fate of radioactive fatty acids in the isolated guinea pig heart. Evidence is provided that hypoxia causes a shift of the rate-limiting step from transport into the cell to oxidation in the mitochondria. This leads to an increased radioactivity in myocardial free fatty acid and in the cytosol. Radioactivity in the mitochondria is decreased and disappears more slowly. At the same time, there is an increased radioactivity in lipid droplets and in triacylglycerol. There is no evidence of a specific location of radioactivity in the sacroplasmic reticulum.

Animals↗
PubMed1983

Resonance Raman detection of Fe-CO stretching and Fe-C-O bending vibrations in sterically hindered carbonmonoxy "strapped hemes". A structural probe of Fe-C-O distortion.

We report resonance Raman studies of the Fe-C-O distortion in sterically hindered heme-CO complexes. The steric hindrance is provided by a hydrocarbon chain strapped across one face of the heme. Increasing the steric hindrance (by decreasing the chain length), which reduces the CO binding affinity, is found to increase the Fe-CO stretching frequencies: heme 5 (unstrapped), 495 cm-1; FeSP-15, 509 cm-1; FeSP-14, 512 cm-1; FeSP-13, 514 cm-1. This is interpreted in terms of a decrease in the CO effective mass and increased interactions between the C atom of CO and the N atom(s) of the pyrrole ring(s). Resonance Raman enhancement of the Fe-C-O bending mode upon Soret excitation may be correlated with the overlap between the porphyrin (pi*) and CO (pi*) orbitals when the CO ligand is tilted. Its intensity relative to that of the Fe-CO stretching mode increases with increasing steric hindrance in these "strapped hemes". In addition, we have estimated the Fe-C-O angles from isotope data in various heme-CO complexes. It is inferred that the angles are 167 +/- 5 degrees (FeSP-15) and 175 +/- 5 degrees (FeSP-14, FeSP-13, Mb X CO, and Hb X CO).

Carbon Monoxide↗