Biomedical subjects

T L Madden

Publications and source records attributed to T L Madden.

15 recordsLinked to original sources

PubMed2001

Development of a simplified, sensitive high-performance liquid chromatographic method using fluorescence detection to determine the concentration of UCN-01 in human plasma.

UCN-01 is a naturally derived anticancer agent isolated in the culture broth of actinomyces streptomyces. We have developed a sensitive high-performance liquid chromatographic method for the determination of UCN-01 in human plasma. UCN-01 was isolated from human plasma after intravenous administration, by using 100% ice-cold acetonitrile liquid-liquid phase extraction. Liquid chromatographic separation was achieved by isocratic elution on a phenyl analytical column. The mobile phase consisted of acetonitrile-0.5 M ammonium acetate (45:55) with 0.2% triethylamine added as a modifier. The UCN-01 peak was identified from other peaks using fluorescence excitation energy and emission energy wavelengths of 310 and 410 nm, respectively. Retention time for UCN-01 was 4.2 +/- 0.5 min. The UCN-01 peak was baseline resolved, with nearest peak at 2.6 min distance. No interfering peaks were observed at the retention time of UCN-01. Peak area amounts from extracted samples were proportional over the dynamic concentration range used: 0.2 to 30 microg/ml. Mean recoveries of UCN-01 at concentrations of 0.5 and 25 microg/ml were 89 and 90.2%, respectively. Relative standard deviations for UCN-01 calibration standards ranged from 1.89 to 2.31%, with relative errors ranging from 0.3 to 11.6%. Assay precision for UCN-01 based on quality control samples of 0.50 microg/ml was +/- 4.86% with an accuracy of +/-5.7%. For drug extracted from plasma the lowest limit of detection was 0.1 microg/ml, with the lowest limit of quantitation being 0.2 microg/ml. This method is suitable for routine analysis of UCN-01 in human plasma at concentration from 0.2 to 30 microg/ml.

Alkaloids↗

PubMed2001

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin, a novel highly lipophilic camptothecin derivative, in human plasma and urine.

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2 +/- 0.5 min and 8.0 +/- 0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at -70 degrees C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.

Antineoplastic Agents↗

PubMed2001

Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements.

PSI-BLAST is an iterative program to search a database for proteins with distant similarity to a query sequence. We investigated over a dozen modifications to the methods used in PSI-BLAST, with the goal of improving accuracy in finding true positive matches. To evaluate performance we used a set of 103 queries for which the true positives in yeast had been annotated by human experts, and a popular measure of retrieval accuracy (ROC) that can be normalized to take on values between 0 (worst) and 1 (best). The modifications we consider novel improve the ROC score from 0.758 +/- 0.005 to 0.895 +/- 0.003. This does not include the benefits from four modifications we included in the 'baseline' version, even though they were not implemented in PSI-BLAST version 2.0. The improvement in accuracy was confirmed on a small second test set. This test involved analyzing three protein families with curated lists of true positives from the non-redundant protein database. The modification that accounts for the majority of the improvement is the use, for each database sequence, of a position-specific scoring system tuned to that sequence's amino acid composition. The use of composition-based statistics is particularly beneficial for large-scale automated applications of PSI-BLAST.

Algorithms↗

PubMed2001

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap'99, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri-tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov.

Animals↗

PubMed2000

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval and resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing pages, GeneMap'99, Davis Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP) pages, Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP) pages, SAGEmap, Online Mendelian Inheritance in Man (OMIM) and the Molecular Modeling Database (MMDB). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov

Animals↗

PubMed1999

BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences.

'BLAST 2 Sequences', a new BLAST-based tool for aligning two protein or nucleotide sequences, is described. While the standard BLAST program is widely used to search for homologous sequences in nucleotide and protein databases, one often needs to compare only two sequences that are already known to be homologous, coming from related species or, e.g. different isolates of the same virus. In such cases searching the entire database would be unnecessarily time-consuming. 'BLAST 2 Sequences' utilizes the BLAST algorithm for pairwise DNA-DNA or protein-protein sequence comparison. A World Wide Web version of the program can be used interactively at the NCBI WWW site (http://www.ncbi.nlm.nih.gov/gorf/bl2.++ +html). The resulting alignments are presented in both graphical and text form. The variants of the program for PC (Windows), Mac and several UNIX-based platforms can be downloaded from the NCBI FTP site (ftp://ncbi.nlm.nih.gov).

Algorithms↗

PubMed1998

Protein sequence similarity searches using patterns as seeds.

Protein families often are characterized by conserved sequence patterns or motifs. A researcher frequently wishes to evaluate the significance of a specific pattern within a protein, or to exploit knowledge of known motifs to aid the recognition of greatly diverged but homologous family members. To assist in these efforts, the pattern-hit initiated BLAST (PHI-BLAST) program described here takes as input both a protein sequence and a pattern of interest that it contains. PHI-BLAST searches a protein database for other instances of the input pattern, and uses those found as seeds for the construction of local alignments to the query sequence. The random distribution of PHI-BLAST alignment scores is studied analytically and empirically. In many instances, the program is able to detect statistically significant similarity between homologous proteins that are not recognizably related using traditional single-pass database search methods. PHI-BLAST is applied to the analysis of CED4-like cell death regulators, HS90-type ATPase domains, archaeal tRNA nucleotidyltransferases and archaeal homologs of DnaG-type DNA primases.

Adenosine Triphosphatases↗

PubMed1998

Pharmaceutical properties of related calanolide compounds with activity against human immunodeficiency virus.

The present studies were undertaken to compare the relative pharmacokinetic parameters and bioavailability of two chemically related natural products which are nonnucleoside inhibitors of reverse transcriptase. Both (+)-calanolide A (Cal A; NSC 675451) and (+)-dihydrocalanolide A (DHCal A; NSC 678323) are currently under development for the treatment of HIV infections. HPLC-based analytical assays were developed for both compounds using modifications of a previously published procedure. The assays were used to compare the intravenous pharmacokinetics of the dihydro analogue relative to the parent compound, Cal A, and to determine the relative oral bioavailability of each drug in CD2F1 mice. Although the pharmacokinetic parameters of each drug were similar (Cal A, 25 mg/kg: AUC: 9.4 [microg/mL]. hr, t1/2beta: 0.25 h,, t1/2gamma: 1.8 h, clearance: 2.7 L/h/kg versus DHCal A, 25 mg/kg: AUC: 6.9 [microg/mL].hr, t1/2beta: 0.22 h,, t1/2gamma: 2.3 h, clearance: 3.6 L/h/kg), the oral bioavailability of DHCal A (F = 46. 8%) was markedly better than that obtained for Cal A (F = 13.2%). The relative ability of Cal A and DHCal A to change to their inactive epimer forms, (+)-calanolide B and (+)-dihydrocalanolide B, respectively, was also determined. While conversion of active to inactive forms of the drugs was noted to occur in vitro especially under acidic conditions, no epimer forms of either compound were noted in plasma of mice after administration of either CalA or DHCal A. Considered together with preliminary toxicology findings, the pharmacokinetic data obtained in the present series of experiments suggest that selection of the dihydro derivative of (+)-calanolide A may be a reasonable choice for further preclinical development and possible Phase I clinical evaluation.

Administration, Oral↗

PubMed1997

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.

The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

Algorithms↗

PubMed1997

A phase II and pharmacokinetic study of enloplatin in patients with platinum refractory advanced ovarian carcinoma.

This was a study of enloplatin in 18 evaluable patients with platinum refractory ovarian cancer. They received an i.v. infusion of enloplatin over 1.5 h without prehydration every 21 days. One patient had a partial response (6%; 95% CI 0-26%) lasting 2.8 months. The median survival was 9.4 months (95%; CI 5.1-19.7%). Neutropenia was the dose-limiting toxicity. Nephrotoxicity was manageable. Enloplatin is the major form of the free drug in plasma. However, 13.5 h after initiation of treatment, 85% of the drug in plasma is protein bound. Elimination of the drug is mainly renal. Enloplatin pharmacokinetics is similar to that of carboplatin. Thus, the plasma pharmacokinetics of enloplatin is dictated by the cyclobutanedicarboxylato (CBDCA) ligand and not the novel amino ligand.

Adult↗

PubMed1997

PowerBLAST: a new network BLAST application for interactive or automated sequence analysis and annotation.

As the rate of DNA sequencing increases, analysis by sequence similarity search will need to become much more efficient in terms of sensitivity, specificity, automation potential, and consistency in annotation. PowerBLAST was developed, in part, to address these problems. PowerBLAST includes a number of options for masking repetitive elements and low complexity subsequences. It also has the capacity to restrict the search to any level of NCBI's taxonomy index, thus supporting "comparative genomics" applications. Postprocessing of the BLAST output using the SIM series of algorithms produces optimal, gapped alignments, and multiple alignments when a region of the query sequence matches multiple database sequences. PowerBLAST is capable of processing sequences of any length because it divides long query sequences into overlapping fragments and then merges the results after searching. The results may be viewed graphically, as a textual representation, or as an HTML page with links to GenBank and Entrez. For matching database sequences, annotated features are superimposed on the aligned query sequence in the output, thus greatly increasing the ease of interpretation. Such features may be used for automated annotation of new sequence because PowerBLAST output in ASN.1 form may be "dragged and dropped" into NCBI's Sequin program for sequence annotation and submission. PowerBLAST is capable of analyzing and annotating a 100-kb query in 60 min on NCBI's BLAST server.

Amino Acid Sequence↗

PubMed1996

Applications of network BLAST server.

The sequence databases continue to grow at an extraordinary rate. Contributions come from both small laboratories and large-scale projects, such as the Merck EST project. This growth has placed new demands on computational sequence comparison tools such as BLAST. Even now it is no longer practical to evaluate some BLAST reports manually; it is necessary to filter the output by, for example, organism, source, or degree of annotation. The new network BLAST service makes such tools possible. It is also possible to present BLAST output in different formats, such as BLANCE. Perhaps most important of all, it becomes simple to call BLAST from another application, making it one step within an integrated system. This makes the automated preparation of sequence evaluations that include BLAST runs possible. In the near future we expect to see a number of applications that use the network BLAST interface to help molecular biologists search against a database that is growing not only in size but in biological richness.

Algorithms↗

PubMed1994

Crowding-induced organization of cytoskeletal elements: II. Dissolution of spontaneously formed filament bundles by capping proteins.

Through calculations of molecular packing constraints in crowded solutions, we have previously shown that dispersions of filament forming proteins and soluble proteins can be unstable at physiological concentrations, such that tight bundles of filaments are formed spontaneously, in the absence of any accessory binding proteins. Here we consider the modulation of this phenomenon by capping proteins. The theory predicts that, by shortening the average filament length, capping alleviates the packing problem. As a result, the dispersed isotropic solution is stable over an expanded range of compositions.

Actin Depolymerizing Factors↗

PubMed1993

Crowding-induced organization of cytoskeletal elements: I. Spontaneous demixing of cytosolic proteins and model filaments to form filament bundles.

The theory for the effects of crowding on the behavior of reversibly self-assembling solutes is extended to mixtures containing nonassembling solutes. The theory predicts that excluded volume will cause dramatic demixing into domains of long, tightly packed, highly aligned fibers coexisting with an isotropic solution of unaggregated species. It suggests that the bundling of fibers in cells is entropically driven and that accessory binding proteins in the cytoplasm serve to modulate the process rather than create it.

Biophysical Phenomena↗

PubMedDate not supplied

Arglabin-DMA, a plant derived sesquiterpene, inhibits farnesyltransferase.

Arglabin [1(R),10(S)-epoxy-5(S),5(S),7(S)-guaia-3(4),11(13)-dien-6, 12-olide], a sesquiterpene gamma-lactone is isolated from Artemisia glabella, a species of wormwood endemic to the Karaganda region of Kazakstan. The compound has been modified to render it water-soluble through addition of a dimethylaminohydrochloride group to the C(13) carbohydride moiety to yield Arglabin-DMA. Arglabin-DMA is a registered antitumor substance in the Republic of Kazakstan. Previously, we have shown that this compound prevents protein farnesylation without altering geranylgeranylation. We now report that Arglabin-DMA inhibits the incorporation of [(3)H]farnesylpyrophosphate into human H-ras protein by FTase with an IC(50) of no greater than 25 microM. Kinetic studies show that the phosphorylated form of this compound competitively inhibits the binding of farnesyl diphosphate to FTase. This mechanism of action is different from other reported peptidomimetic FTIs which lower the affinity of ras protein to FTase. Our in vitro studies confirm that Arglabin-DMA inhibits post-translational modification of ras protein in cells. Arglabin-DMA inhibits anchorage-dependent proliferation of NB cells (IC50=10 microg/ml) and inhibits anchorage-independent growth of NB and KNRK cells with about the same IC(50). Soft-agar colony formation assay of H-ras and K-ras transformed cells show IC(50)s to be 2 and 5 microg/ml, respectively. In primary cultures of human tumor cells, Arglabin-DMA inhibits cell proliferation of a variety of tumor types with IC(90)s in the range of 0.85 to 5.0 microg/ml. Because of these pharmacologic properties, we propose that Arglabin-DMA is suitable for the treatment of ras related malignancies.

3T3 Cells↗