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Biomedical subjects

S Webber

Publications and source records attributed to S Webber.

At least 55 records · Page 3Linked to original sources
PubMed1994

Biological activity of a novel rationally designed lipophilic thymidylate synthase inhibitor.

AG-331 (N6[4-(N-morpholinosulfonyl)benzyl]-N6-methyl-2,6-diamino- benz[cd]indole glucuronate) is a novel lipophilic thymidylate synthase (TS) inhibitor. The properties of this compound were investigated in H35 rat hepatoma cells and in three variant cell lines resistant to antifolates by differing mechanisms. There was no evidence for any intracellular effect of AG-331 on dihydrofolate reductase (DHFR); however, the low degree of cross-resistance found for the H35FF line, which has elevated TS levels, suggested that TS may not be the sole locus of action of AG-331 in hepatoma cells. TS-directed effects of AG-331 were suggested by the pattern of its inhibition of deoxyuridine incorporation into DNA and the lesser effects of purine incorporation. In addition, H35 cells treated with 10 microM AG-331 were shown to accumulate in the S phase of the cell cycle, and this effect could be reversed by coadministration of thymidine. However, when treatments were conducted at a 5-fold higher concentration of AG-331, no S-phase block was apparent, suggesting the loss of a TS-directed effect at high inhibitor concentrations. Thymidine and folinic acid also failed to protect cells against AG-331 cytotoxicity, suggesting an alternate mode of action. Similar results were also obtained in protection experiments with a human hepatoma cell line, HEPG2, although previous results obtained in colon- and breast-cancer cell lines have suggested TS specific effects for AG-331. The possibility that biotransformation of AG-331 to other toxic species may occur in liver-derived cell lines has yet to be investigated.

Animals↗
PubMed1994

Adsorption filtration of leukocyte-reduced single donor platelet units.

The effect of adsorption filtration of leukocyte-reduced single donor platelet (SDP) units collected on a CS-3000+ with a TNX chamber was compared to filtration of SDP units prepared via an isoradial chamber. The leukocyte enumeration technique employed a Nageotte chamber and had a nominal lower detection limit of 0.05 leukocytes/microliter (if no cells were seen, the concentration was recorded as 0.01 microliter). Although the prefiltration leukocyte content of the TNX units was unexpectedly low (2.4 +/- 2.8 x 10(4) per unit), filtration reduced the leukocyte content further to no greater than 1.7 +/- 0.07 x 10(3) (p < 0.05). (The full extent of the leukocyte removal could not be assessed as no leukocytes were seen in counting any of the 20 filtered TNX units). The 20 isoradial (control) SDP units achieved similar leukocyte depletion, with a postfiltration leukocyte content of 2.7 +/- 2.5 x 10(3) (p > 0.05, compared to filtered TNX units). Thus while significant leukocyte reduction occurred with adsorption filtration of both control and leukocyte-depleted SDP units, comparison of the relative degree of reduction awaits application of more sensitive leukocyte counting techniques. This study documented, however, that additional leukocyte reduction can be obtained by applying adsorption filtration to SDP units with an already low leukocyte content.

Adsorption↗
PubMed1994

Acute myocarditis. Rapid diagnosis by PCR in children.

BACKGROUND: The diagnosis of viral myocarditis remains difficult and generally depends on clinical and histological criteria. Viral cultures and serology are often unrewarding, with low yields. The purpose of this study was to analyze the usefulness of polymerase chain reaction (PCR) in the rapid diagnosis of acute myocarditis in children. METHODS AND RESULTS: PCR was used to analyze 38 myocardial tissue samples from 34 patients with suspected acute viral myocarditis and 17 control patients with congenital heart disease (14) or hypertrophic cardiomyopathy (3). Myocardial samples were obtained at the time of right ventricular biopsy (13 samples), from explanted hearts (18 samples) at transplantation, and from cardiac autopsy specimens (24 samples) and were evaluated for the presence of enterovirus, cytomegalovirus (CMV), adenovirus, and herpes simplex virus (HSV) using PCR primers designed to consensus and unique sequences of these viral genomes. Blood also was obtained at the time of biopsy (11) or transplant (18). In 26 of 38 myocardial samples (68%), viral genome was detected by PCR (15 adenoviral, 8 enteroviral, 2 HSV, 1 CMV), whereas all control myocardial samples and blood samples were negative. Four patients had positive viral cultures, and these matched the PCR findings. Disagreement with histopathology occurred in 13 of 26 PCR-positive specimens, usually associated with adenovirus. CONCLUSIONS: PCR offers a rapid, sensitive diagnostic method for myocardial viral infection. While enterovirus is an important etiological agent, adenovirus was more prevalent in this series and should be evaluated when etiology is sought. PCR used in conjunction with standard endomyocardial biopsy appears to enhance the likelihood of detecting viral genome in the myocardium of patients with clinical evidence of myocarditis.

Acute Disease↗
PubMed1993

Design of thymidylate synthase inhibitors using protein crystal structures: the synthesis and biological evaluation of a novel class of 5-substituted quinazolinones.

The design, synthesis, and biological evaluation of a new class of inhibitors of thymidylate synthase (TS) is described. The molecular design was carried out by a repetitive crystallographic analysis of protein-ligand structures. At the onset of this project, we focused on the folate cofactor binding site of a high-resolution ternary crystal complex of Escherichia coli TS, 5'-fluorodeoxyuridylate (5-FdUMP) and a classical glutamate-containing folic acid analog. A preliminary ternary crystal structure of a novel compound was successfully solved. Upon analysis of this initial complex, further structural elaborations were made, and a series of active 5-(arylthio)quinazolinones was developed. The synthetic strategy was based on the displacement of a halogen at the 5-position of a quinazolinone by various aryl thioanions. The compounds were tested for inhibition of purified E. coli and/or human TS, and were assayed for cytotoxicity against three tumor cell lines in vitro. Significant thymidine protection effects were observed with several of the inhibitors, indicating that TS was the intracellular locus of activity.

Crystallography↗
PubMed1993

Charging for library and information services in medical libraries: a review of the literature and a survey of current practice.

A summary of some of the issues concerned with charging for library services (particularly information services and services provided by medical libraries) is given. The results of a survey carried out in July 1992 are presented. The survey asked respondents to indicate which library services they charged for, the level of charges and the extent to which charges to those outside the organization and those within it differed. Twenty-five libraries (response rate of 69%) replied to the survey. Some further areas for research and discussion are indicated.

Decision Making, Organizational↗
PubMed1992

Design and synthesis of novel 6,7-imidazotetrahydroquinoline inhibitors of thymidylate synthase using iterative protein crystal structure analysis.

Antifolate inhibitors of thymidylate synthase (TS) have primarily been based on the structure of folic acid. This paper describes the identification and development of novel 6,7-imidazotetrahydroquinoline TS inhibitors by iterative ligand design, synthesis, and crystallographic analysis of protein-inhibitor complexes. Beginning with a high-resolution crystal structure of E. coli TS (TS, EC 2.1.1.45), an imidazotetrahydroquinoline inhibitor was designed de novo to occupy the folate binding pocket. Structural modifications of the initial compound 1h (Ki approximately 5 microM human/E. coli TS) were then made on the basis of feedback from additional cocrystal structures and activity data. An amino group in the 2-position of the imidazole was found to increase the potency of the series by 1-2 orders of magnitude. Other substitutions on the imidazole ring (1-CH3, 2-CH3, 2-NHCH3, 2-SCH3) generally led to weaker inhibition. Additional improvements in activity were obtained by modification of the substituents on the tetrahydroquinoline nitrogen, bringing the Ki of three of the compounds below 15 nM against the human TS enzyme. The compounds were tested for cytotoxicity and were shown to inhibit the growth of three tumor cell lines in vitro.

Adenocarcinoma↗
PubMed1992

Crystal-structure-based design and synthesis of benz[cd]indole-containing inhibitors of thymidylate synthase.

The X-ray crystal-structure-based design, synthesis, and biological activity of a novel family of benz[cd]indole-containing inhibitors of thymidylate synthase (TS) are described. The structure-activity of the lead compound was studied by conceptually dividing the molecule into four regions and independently optimizing the substituents for each region. Combination of favored substituents for each region led to inhibitors with Ki's against the human enzyme in the range of 10-20 nM. Thymidine shift experiments suggested that the cytotoxic properties of the best enzyme inhibitors were due to TS targeting in cells. The inhibitors were synthesized from substituted 6-aminobenz[cd]indol-2(1H)-ones by alkylation with both a simple alkyl group and a substituted benzylic portion. The 2,6-diaminobenz[cd]indoles were prepared from the corresponding lactams by conversion to the thiolactam, alkylation to the methylated thiolactam, and then displacement with a substituted or unsubstituted amine.

Alkylation↗
PubMed1990

Neonatal pneumonia.

All babies admitted to the neonatal unit during a period of 41 months were prospectively studied to find out the incidence, aetiology, and outcome of neonatal pneumonia, and the value of routine cultures of endotracheal tubes. Pneumonia of early onset (before age 48 hours) occurred in 35 babies (incidence 1.79/1000 live births). In 20 (57%) it was caused by group B streptococci. Blood cultures showed the presence of organisms in 16 of the 35 (46%). There were 41 episodes of pneumonia of late onset in 39 babies. Thirty six of the 39 were preterm, and 34 were artificially ventilated (10% of all ventilated babies). Endotracheal tube colonisation had occurred in 94% of these, most commonly by Gram negative organisms and Staphylococcus epidermidis. In only one of seven cases with simultaneous bacteraemia was the same organism grown from cultures of the blood. After controlling for gestational age and duration of artificial ventilation there was no difference in the incidence or timing of endotracheal tube colonisation between babies who did and did not have pneumonia of late onset. Ten babies with pneumonia of early onset (29%) died; all were preterm infants. Only one death (2%) was associated with an episode of pneumonia of late onset. Routine surveillance cultures were not helpful in predicting and managing pneumonia of late onset.

Anti-Bacterial Agents↗
PubMed1989

The transport of pteridines in CCRF-CEM human lymphoblastic cells.

The transport routes used by CCRF-CEM human lymphoblastoid cells for the influx and efflux of unconjugated pteridines were analyzed using [3H]6-hydroxymethylpterin as a model compound. Influx proceeds by a mechanism that exhibits a Km of 66.7 microM and a Vmax of 0.077 nmol/min per mg cellular protein. The process is somewhat sensitive to metabolic inhibitors, particularly uncouplers of oxidative phosphorylation, and is significantly affected by the presence of other pteridines in the extracellular medium. The results suggest that pterins with either no 6-substituent (pterin) or those with methyl, hydroxyl, or formyl groups in this position, which exhibit Ki values between 25 and 77 microM, may share the same pathway for uptake. 6-Carboxypterin exhibits low affinity for the system (Ki greater than 500 microM), as do 7-substituted and 6,7-di-substituted derivatives and compounds with larger groups at the 6-position, such as neopterin and biopterin (Ki = 250-300 microM). Efflux of [3H]6-hydroxymethylpterin occurs rapidly and can proceed by at least two routes. The first, comprising approximately 50% of total efflux, is inhibited by extracellular pterins and exhibits similar properties to the uptake system in both its pattern of sensitivity to metabolic inhibitors and its specificity for pteridine structure. The route by which the remaining efflux occurs is relatively insensitive to metabolic inhibition. Adenine significantly inhibits 6-hydroxymethylpterin influx and efflux (Ki = 10.6 microM for uptake) but does not appear to share the same transport system. Similarly, methotrexate and folic acid exhibit little affinity for the unconjugated pteridine transport routes.

Biological Transport↗
PubMed1989

Synergy between 5,10-dideaza-5,6,7,8-tetrahydrofolic acid and methotrexate in mice bearing L1210 tumors.

In vivo studies with 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), an inhibitor of glycinamide ribonucleotide transformylase, indicate that at doses ranging from 2.5 to 10 mg/kg, it prolongs the survival of mice implanted with L1210 tumors. Lower doses of this agent have no effect. Parallel studies with methotrexate indicate that DDATHF is not as potent or as efficacious as methotrexate in this animal model. Low doses of DDATHF combined with low doses of methotrexate can cause a significant increase in the survival of L1210 tumor-bearing mice, suggesting synergism between these two antifolates.

Animals↗
PubMed1989

Methotrexate 5-aminoallyl-2'-deoxyuridine 5'-monophosphate: a potential bifunctional inhibitor of thymidylate synthase.

Mercuration of 2'-deoxyuridine 5'-phosphate (dUMP) followed by alkylation with allylamine in the presence of K2PdCl4 afforded the 5-aminoallyl deoxynucleotide, which was isolated by sequential Dowex 50 H+ and DEAE-Sephadex chromatography. Further reaction of the product with the N-hydroxysuccinimide ester of methotrexate (MTX) in dry dimethyl sulfoxide gave an MTX-aminoallyl-dUMP covalent complex separable by DEAE-Sephadex chromatography. Reprecipitation with acid from basic solution offered further purification and the structure was confirmed by elemental analysis, NMR and absorbance spectra. The product was an inhibitor of rat liver dihydrofolate reductase (I50 approximately 250 nM, cf. MTX I50 approximately 60 nM) and Lactobacillus casei thymidylate synthase. With the latter enzyme, inhibition was competitive with both nucleotide and folate substrates (Ki = 2.6 and 3.5 microM, respectively) and partial enzyme-inhibitor binary complex could be detected by gel electrophoresis. Large fluorescence changes were observed on titration of the synthase with MTX-aminoallyl-dUMP and alterations in the UV difference spectra similar to those seen on titration of the enzyme with MTX were also noted. The compound was a poor growth inhibitor for cultured murine L1210 and human CCRF-CEM cell lines, which probably reflects low cellular uptake.

Animals↗
PubMed1988

Synthesis and biological properties of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis of the antifolate 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) has been modified. It is prepared from 2-acetamido-6-formyl-4(3H)-pyrido[2,3-b]pyrimidone and [P-(N-[1,3-bis(ethoxycarbonyl)propan-1-yl]aminocarbonyl)] phenylmethyl]-triphenylphosphonium bromide. The synthesis proceeds via a sodium hydride promoted Wittig condensation in 1-methyl-2-pyrrolidone followed by catalytic reduction, mild base hydrolysis, and acid precipitation of the product. Synthesis of DDATHF is achieved in a total of seven steps from commercially available reagents. DDATHF is transported effectively into CCRF-CEM cells and inhibits growth of both human (CEM) and murine (L1210) cells in culture. Studies reported here support the view that methotrexate and DDATHF are transported via a shared transport mechanism.

Animals↗
PubMed1988

The estimation of dihydropteridine reductase in human blood cells.

Significant homology between dihydropteridine reductase (DHPR) from rat and human sources has been established by the ability of polyclonal antibodies raised to the rat-liver enzyme to detect the human protein in Western blots. The antibody also reacted with a single protein in bovine, dog and porcine kidney extracts, however, only trace reactivity was detected in rabbit. Quantitation of Western blots by soft laser densitometry showed that the response was proportional to total protein present in analyses of both pure rat-liver enzyme samples and crude extracts of rat and human liver. The DHPR contents of human blood cells were analysed by this method and the results compared to levels determined in enzymatic assays. Extracts of platelets and lymphocytes showed good correlation between these two methods, however, granulocytes exhibited high apparent enzyme activity but no DHPR protein detectable in blots. Erythrocyte extracts showed approximately 50% lower DHPR protein levels than predicted by activity measurements. These results are discussed in relation to the accuracy of detecting DHPR deficiencies in humans by enzymatic assay of whole blood samples.

Animals↗
PubMed1987

Structural studies and isolation of cDNA clones providing the complete sequence of rat liver dihydropteridine reductase.

The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.

Amino Acid Sequence↗
PubMed1987

Preliminary studies on the primary structure of rat liver dihydropteridine reductase.

Cyanogen bromide cleavage of reductively alkylated homogeneous rat liver dihydropteridine reductase afforded several peptide fragments identifiable by polyacrylamide electrophoresis of which 6 (CB-1 to CB-6) could be individually isolated by C8 reverse phase HPLC. Each was characterised by N-terminal amino acid analysis and sequence information was derived for CB-1, CB-4 and CB-6. The blocked N-terminal of the holoenzyme was identified as pyroglutamate and the C-terminal sequence was obtained by sequential degradation.

Amino Acid Sequence↗