55 results on '"Siegel MM"'
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2. Structural characterization of bisretinoid A2E photocleavage products and implications for age-related macular degeneration.
- Author
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Wu Y, Yanase E, Feng X, Siegel MM, and Sparrow JR
- Subjects
- Aldehydes chemistry, Carbohydrates chemistry, Humans, Light, Lipids chemistry, Lipofuscin chemistry, Oxygen chemistry, Photochemistry methods, Pyruvaldehyde chemistry, Spectrometry, Mass, Electrospray Ionization methods, Vitamin A chemistry, Macular Degeneration therapy, Pyridinium Compounds chemistry, Retinal Pigment Epithelium cytology, Retinoids chemistry
- Abstract
Fluorescent bisretinoids, such as A2E and all-trans-retinal dimer, form as a by-product of vitamin A cycling in retina and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin pigments. These pigments are implicated in pathological mechanisms involved in several vision-threatening diseases including age-related macular degeneration. Efforts to understand damaging events initiated by these bisretinoids have revealed that photoexcitation of A2E by wavelengths in the visible spectrum leads to singlet oxygen production and photooxidation of A2E. Here we have employed liquid chromatography coupled to electrospray ionization mass spectrometry together with tandem mass spectrometry (MS/MS), to demonstrate that A2E also undergoes photooxidation-induced degradation and we have elucidated the structures of some of the aldehyde-bearing cleavage products. Studies in which A2E was incubated with a singlet oxygen generator yielded results consistent with a mechanism involving bisretinoid photocleavage at sites of singlet molecular oxygen addition. We provide evidence that one of the products released by A2E photodegradation is methylglyoxal, a low molecular weight reactive dicarbonyl with the capacity to form advanced glycation end products. Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation; this is the first report of its production via bisretinoid photocleavage. It is significant that AGE-modified proteins are detected in deposits (drusen) that accumulate below RPE cells in vivo; drusen have been linked to age-related macular degeneration pathogenesis. Whereas various processes play a role in drusen formation, these findings are indicative of a contribution from lipofuscin photooxidation in RPE.
- Published
- 2010
- Full Text
- View/download PDF
3. Structure characterization of lipocyclopeptide antibiotics, aspartocins A, B & C, by ESI-MSMS and ESI-nozzle-skimmer-MSMS.
- Author
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Siegel MM, Kong F, Feng X, and Carter GT
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Fatty Acids chemistry, Magnetic Resonance Imaging, Molecular Structure, Oligopeptides chemistry, Peptides, Cyclic chemistry, Spectrometry, Mass, Electrospray Ionization methods, Tandem Mass Spectrometry methods
- Abstract
Three lipocyclopeptide antibiotics, aspartocins A (1), B (2), and C (3), were obtained from the aspartocin complex by HPLC separation methodology. Their structures were elucidated using previously published chemical degradation results coupled with spectroscopic studies including ESI-MS, ESI-Nozzle Skimmer-MSMS and NMR. All three aspartocin compounds share the same cyclic decapeptide core of cyclo [Dab2 (Asp1-FA)-Pip3-MeAsp4-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11]. They differ only in the fatty acid side chain moiety (FA) corresponding to (Z)-13-methyltetradec-3-ene-carbonyl, (+,Z)-12-methyltetradec-3-ene-carbonyl and (Z)-12-methyltridec-3-ene-carbonyl for aspartocins A (1), B (2), and C (3), respectively. All of the sequence ions were observed by ESI-MSMS of the doubly charged parent ions. However, a number of the sequence ions observed were of low abundance. To fully sequence the lipocyclopeptide antibiotic structures, these low abundance sequence ions together with complementary sequence ions were confirmed by ESI-Nozzle-Skimmer-MSMS of the singly charged linear peptide parent fragment ions H-Asp5-Gly6-Asp7-Gly8-Dab9-Val10-Pro11-Dab2(1+)-Asp1-FA. Cyclization of the aspartocins was demonstrated to occur via the beta-amino group of Dab2 from ions of moderate intensity in the ESI-MSMS spectra. As the fatty acid moieties do not undergo internal fragmentations under the experimental ESI mass spectral conditions used, the 14 Da mass difference between the fatty acid moieties of aspartocins A (1) and B (2) versus aspartocin C (3) was used as an internal mass tag to differentiate fragment ions containing fatty acid moieties and those not containing the fatty acid moieties. The most numerous and abundant fragment ions observed in the tandem mass spectra are due to the cleavage of the tertiary nitrogen amide of the pipecolic acid residue-3 (16 fragment ions) and the proline residue-11 (7 fragment ions). In addition, the neutral loss of ethanimine from alpha,beta-diaminobutyric acid residue 9 was observed for the parent molecular ion and for 7 fragment ions., (Copyright 2009 John Wiley & Sons, Ltd.)
- Published
- 2009
- Full Text
- View/download PDF
4. Activation loop phosphorylation modulates Bruton's tyrosine kinase (Btk) kinase domain activity.
- Author
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Lin L, Czerwinski R, Kelleher K, Siegel MM, Wu P, Kriz R, Aulabaugh A, and Stahl M
- Subjects
- Adenosine Triphosphate metabolism, Adenylyl Imidodiphosphate pharmacology, Agammaglobulinaemia Tyrosine Kinase, Amino Acid Sequence, Animals, Binding Sites, Blotting, Western, Catalysis drug effects, Cell Line, Chromatography, High Pressure Liquid, Enzyme Activation, Enzyme Inhibitors pharmacology, Humans, Kinetics, Peptide Fragments metabolism, Phosphorylation drug effects, Protein-Tyrosine Kinases chemistry, Protein-Tyrosine Kinases genetics, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Spectrometry, Mass, Electrospray Ionization, Spodoptera, Substrate Specificity, Protein-Tyrosine Kinases metabolism, Tyrosine metabolism
- Abstract
Bruton's tyrosine kinase (Btk) plays a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. A number of cell signaling studies clearly show that Btk is activated by Lyn, a Src family kinase, through phosphorylation on activation loop tyrosine 551 (Y(551)). However, the detailed molecular mechanism regulating Btk activation remains unclear. In particular, we do not fully understand the correlation of kinase activity with Y(551) phosphorylation, and the role of the noncatalytic domains of Btk in the activation process. Insect cell expressed full-length Btk is enzymatically active, but a truncated version of Btk, composed of only the kinase catalytic domain, is largely inactive. Further characterization of both forms of Btk by mass spectrometry showed partial phosphorylation of Y(551) of the full-length enzyme and none of the truncated kinase domain. To determine whether the lack of activity of the kinase domain was due to the absence of Y(551) phosphorylation, we developed an in vitro method to generate Y(551) monophosphorylated Btk kinase domain fragment using the Src family kinase Lyn. Detailed kinetic analyses demonstrated that the in vitro phosphorylated Btk kinase domain has a similar activity as the full-length enzyme while the unphosphorylated kinase domain has a very low k(cat) and is largely inactive. A divalent magnesium metal dependence study established that Btk requires a second magnesium ion for activity. Furthermore, our analysis revealed significant differences in the second metal-binding site among the kinase domain and the full-length enzyme that likely account for the difference in their catalytic profile. Taken together, our study provides important mechanistic insights into Btk kinase activity and phosphorylation-mediated regulation.
- Published
- 2009
- Full Text
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5. GPC spin column HPLC-ESI-MS methods for screening drugs noncovalently bound to proteins.
- Author
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Siegel MM
- Subjects
- Protein Binding, Chromatography, Gel methods, Chromatography, High Pressure Liquid methods, Drug Evaluation, Preclinical methods, Pharmaceutical Preparations metabolism, Proteins metabolism, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
Secondary drug screening methods are described for determining the relative degree of non-covalent binding between drug candidates and a protein of therapeutic interest by gel centrifugation chromatography using GPC spin columns for isolating the protein-drug complexes, under native conditions, and reversed-phase HPLC coupled with ESI-MS for highly resolved and sensitive detection of the drug in the complex, under denaturing conditions. The necessary control samples and limitations of this work are fully described. The GPC spin column HPLC-ESI-MS methodology for screening of drugs non-covalently bound to proteins is illustrated for the non-covalent binding of geldanamycin with Hsp90cat protein.
- Published
- 2009
- Full Text
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6. FTICR-MS applications for the structure determination of natural products.
- Author
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Feng X and Siegel MM
- Subjects
- Drug Industry instrumentation, Drug Industry methods, Magnetics, Molecular Structure, Biological Products chemistry, Mass Spectrometry instrumentation, Mass Spectrometry methods
- Abstract
Natural products are a source of unique chemical entities with specific biological activities of great value to the pharmaceutical industry. However, the determination of unknown structures is usually time consuming and often becomes a bottleneck in the effort to develop natural products into effective drugs. The high-performance features of high magnetic field FTMS have greatly alleviated the structural elucidation bottleneck to meet increasingly shorter discovery timelines for drug candidates based on natural products. The high-performance features of high field FTMS include unsurpassed mass measurement accuracy for elemental formula determination, ultra-high mass resolution for component separation, the ability to perform multiple levels of tandem mass spectrometry for structural elucidation, and moderate sensitivity for limited supply of isolates. A number of applications utilizing these properties of FTMS have been reported recently for the structural elucidation of novel natural product structures originating from terrestrial and marine microorganisms. In this review, FTMS methods and their applications for the structural elucidation and characterization of natural products will be reviewed.
- Published
- 2007
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7. Septocylindrins A and B: peptaibols produced by the terrestrial fungus Septocylindrium sp. LL-Z1518.
- Author
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Summers MY, Kong F, Feng X, Siegel MM, Janso JE, Graziani EI, and Carter GT
- Subjects
- Alamethicin chemistry, Amino Acid Sequence, Antifungal Agents chemistry, Antifungal Agents pharmacology, Microbial Sensitivity Tests, Molecular Sequence Data, Molecular Structure, Nuclear Magnetic Resonance, Biomolecular, Peptaibols, Peptides chemistry, Peptides pharmacology, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents isolation & purification, Anti-Bacterial Agents pharmacology, Antifungal Agents isolation & purification, Fungi chemistry, Peptides isolation & purification
- Abstract
Two new peptaibols, septocylindrin A (1) and septocylindrin B (2), related to the well-studied membrane-channel-forming peptaibol alamethicin, were obtained from a terrestrial isolate of the fungus Septocylindrium sp. Both 1 and 2 are linear 19-amino acid peptides with a modified phenylalanine C-terminus. Analysis of the HRMS data indicated that they differ only in the 18th residue, where 1 contains Glu and 2 contains Gln. The structures of these two peptaibols were determined by extensive NMR and HRMS analysis. The absolute configurations of amino acids present in 1 were determined using Marfey's methodology. Both compounds were isolated through bioassay-guided fractionation and exhibited significant antibacterial and antifungal activity.
- Published
- 2007
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8. 2-(Quinazolin-4-ylamino)-[1,4]benzoquinones as covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2.
- Author
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Wissner A, Floyd MB, Johnson BD, Fraser H, Ingalls C, Nittoli T, Dushin RG, Discafani C, Nilakantan R, Marini J, Ravi M, Cheung K, Tan X, Musto S, Annable T, Siegel MM, and Loganzo F
- Subjects
- Adenosine Triphosphate metabolism, Angiogenesis Inhibitors chemistry, Angiogenesis Inhibitors pharmacology, Animals, Benzoquinones chemistry, Benzoquinones pharmacology, Binding Sites, Cell Line, Female, Glutathione chemistry, Humans, Kinetics, Mice, Mice, Nude, Models, Molecular, Molecular Conformation, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Quantum Theory, Quinazolines chemistry, Quinazolines pharmacology, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Structure-Activity Relationship, Vascular Endothelial Growth Factor Receptor-2 chemistry, Vascular Endothelial Growth Factor Receptor-2 metabolism, Xenograft Model Antitumor Assays, Angiogenesis Inhibitors chemical synthesis, Benzoquinones chemical synthesis, Quinazolines chemical synthesis, Vascular Endothelial Growth Factor Receptor-2 antagonists & inhibitors
- Abstract
A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.
- Published
- 2005
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9. Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry.
- Author
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Tabei K, Feng X, Venkatesan AM, Abe T, Hideki U, Mansour TS, and Siegel MM
- Subjects
- Bacterial Proteins chemistry, Binding Sites, Hydrolysis, Spectrometry, Mass, Electrospray Ionization, beta-Lactamase Inhibitors, beta-Lactams chemical synthesis, beta-Lactamases chemistry, beta-Lactams chemistry
- Abstract
The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.
- Published
- 2004
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10. FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach, and HPLC ESI capillary-skimmer CID FTMS.
- Author
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McDonald LA, Barbieri LR, Carter GT, Kruppa G, Feng X, Lotvin JA, and Siegel MM
- Subjects
- Cyclotrons, Fourier Analysis, Molecular Structure, Nucleotides, Peptides, Spectrometry, Mass, Electrospray Ionization methods, Streptomyces chemistry, Urea, Peptidoglycan analogs & derivatives, Peptidoglycan chemistry
- Abstract
The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.
- Published
- 2003
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11. Structures of the muraymycins, novel peptidoglycan biosynthesis inhibitors.
- Author
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McDonald LA, Barbieri LR, Carter GT, Lenoy E, Lotvin J, Petersen PJ, Siegel MM, Singh G, and Williamson RT
- Subjects
- Anti-Bacterial Agents isolation & purification, Anti-Bacterial Agents pharmacology, Chromatography, High Pressure Liquid, Streptomyces chemistry, Structure-Activity Relationship, Uracil chemistry, Uracil isolation & purification, Uracil pharmacology, Urea chemistry, Urea isolation & purification, Urea pharmacology, Anti-Bacterial Agents chemistry, Peptidoglycan biosynthesis, Uracil analogs & derivatives, Urea analogs & derivatives
- Abstract
The muraymycins, a family of nucleoside-lipopeptide antibiotics, were purified from the extract of Streptomyces sp. LL-AA896. The antibiotics were purified by chromatographic methods and characterized by NMR spectroscopy, degradation studies, and mass spectrometry. The structures of 19 compounds were established. The muraymycins constitute a new antibiotic family whose core structure contains a glycosylated uronic acid derivative joined by an aminopropane group to a hexahydro-2-imino-4-pyrimidylglycyl residue (epicapreomycidine) containing dipeptide that is further extended by a urea-valine moiety. Members of this family show broad-spectrum in vitro antimicrobial activity against a variety of clinical isolates (MIC 2 to >64 mug/mL). The muraymycins inhibited peptidoglycan biosynthesis. The fatty acid substituent and the presence or absence of the amino sugar play important roles in biological activity. One of the most active compounds, muraymycin A1, demonstrated protection in vivo against Staphylococcus aureus infection in mice (ED50 1.1 mg/kg).
- Published
- 2002
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12. Multiple ion isolation applications in FT-ICR MS: exact-mass MSn internal calibration and purification/interrogation of protein-drug complexes.
- Author
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Kruppa G, Schnier PD, Tabei K, Van Orden S, and Siegel MM
- Subjects
- Angiotensin I analysis, Angiotensin I pharmacokinetics, Animals, Binding Sites, Bradykinin analysis, Bradykinin metabolism, Calibration, Cyclotrons, ErbB Receptors analysis, ErbB Receptors metabolism, Fourier Analysis, Humans, Ions, Mass Spectrometry, Melitten analysis, Melitten metabolism, Molecular Weight, Pharmacokinetics, Proteins analysis, Sirolimus analysis, Sirolimus pharmacokinetics, Pharmaceutical Preparations analysis, Proteins metabolism
- Abstract
Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a MSn experiment, under high-resolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies < 1.0 ppm are demonstrated and routinely achieved. Purification of a mixture is illustrated by isolating multiple charge states of a protein-drug complex from residual protein for further MSn studies to elucidate the site of covalent drug bonding using IRMPD for a mixture of epidermal growth factor receptor (EGFr) protein and EGFr-drug complex. The procedure developed for multiple ion isolations is referred to as multi-CHEF, multiple correlated harmonic excitation fields, in which tailored waveforms are used to notch out multiple mass regions of a spectrum with minimal off-resonance excitation.
- Published
- 2002
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13. Further evidence that a cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc] serves as an acceptor in a sorting reaction.
- Author
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Ruzin A, Severin A, Ritacco F, Tabei K, Singh G, Bradford PA, Siegel MM, Projan SJ, and Shlaes DM
- Subjects
- Bacterial Proteins, Cysteine Endopeptidases, Membrane Proteins metabolism, Aminoacyltransferases physiology, Cell Wall metabolism, Peptidoglycan biosynthesis, Streptomyces metabolism, Uridine Diphosphate N-Acetylmuramic Acid analogs & derivatives, Uridine Diphosphate N-Acetylmuramic Acid metabolism
- Abstract
Previous studies suggested that a Gly-containing branch of cell wall precursor [C(55)-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.
- Published
- 2002
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14. Early discovery drug screening using mass spectrometry.
- Author
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Siegel MM
- Subjects
- Drug Design, Drug Evaluation, Preclinical methods, Spectrometry, Mass, Electrospray Ionization instrumentation, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization instrumentation, Structure-Activity Relationship, Drug Evaluation, Preclinical instrumentation, Spectrometry, Mass, Electrospray Ionization methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometric methods useful for early discovery drug screening are reviewed. All methods described involve studies of non-covalent complexes between biopolymer receptors and small molecule ligands formed in the condensed phase. The complexes can be sprayed intact directly into the gas phase by ESI-MS using gentle experimental conditions. Gas phase screening applications are illustrated for drug ligand candidates non-covalently interacting with peptides, proteins, RNA, and DNA. In the condensed phase, the complexes can be also isolated, denatured and analyzed by ESI-MS to identify the small molecule ligands. Condensed phase drug screening examples are illustrated for the ESI-MS ancillary techniques of affinity chromatography, ultrafiltration, ultracentrifugation, gel permeation chromatography (GPC), reverse phase-high performance liquid chromatography (RP-HPLC) and capillary electrophoretic methods. Solid phase drug screening using MALDI-MS is illustrated for small molecule ligands bound to MALDI affinity probe tips and to beads. Since ESI and MALDI principally produce molecular ions, high throughput screening is achieved by analyzing mass indexed mixtures.
- Published
- 2002
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15. Biopolymer sequencing using a triple quadrupole mass spectrometer in the ESI nozzle-skimmer/precursor ion MS/MS mode.
- Author
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Chen H, Tabei K, and Siegel MM
- Abstract
A variety of model biopolymers, including oligonucleotides, oligosaccharides and a synthetic pharmaceutical agent, were sequenced using a triple quadrupole mass spectrometer equipped with an electrospray source and operated in a scan mode referred to as pseudo-MS3. This scan mode consists of three steps: (1) in-source collision-induced dissociation (CID) in the nozzle-skimmer (NS) region, (2) scanning of the fragment ions into the collision cell for further CID, and (3) passing of the secondary fragment ions through the final mass filter at a preselected mass, generally corresponding to the mass of a terminal sequence ion for the biopolymer. The mass spectra are recorded in the precursor ion MS/MS mode where ion selection and detection occur at the third stage of the triple quadrupole but the scan function is determined by the first stage. The advantages and limitations in using this pseudo-MS3 NS/precursor ion MS/MS scan mode for biopolymer sequencing are discussed.
- Published
- 2001
- Full Text
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16. MS/NMR: a structure-based approach for discovering protein ligands and for drug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy.
- Author
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Moy FJ, Haraki K, Mobilio D, Walker G, Powers R, Tabei K, Tong H, and Siegel MM
- Subjects
- Humans, Ligands, Protein Conformation, Recombinant Proteins metabolism, Chromatography, Gel methods, Drug Design, Magnetic Resonance Spectroscopy methods, Mass Spectrometry methods, Matrix Metalloproteinase 1 metabolism
- Abstract
A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.
- Published
- 2001
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17. Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry.
- Author
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Yang Y, Janota K, Tabei K, Huang N, Siegel MM, Lin YI, Rasmussen BA, and Shlaes DM
- Subjects
- Chromatography, High Pressure Liquid, Mass Spectrometry methods, Penicillanic Acid pharmacology, Recombinant Proteins antagonists & inhibitors, Serine Endopeptidases metabolism, Tazobactam, Trypsin metabolism, beta-Lactamases, Enzyme Inhibitors pharmacology, Penicillanic Acid analogs & derivatives, beta-Lactamase Inhibitors
- Abstract
The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde.
- Published
- 2000
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18. Isolation and structural elucidation of AC326-alpha, a new member of the moenomycin group.
- Author
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He H, Shen B, Korshalla J, Siegel MM, and Carter GT
- Subjects
- Actinomycetales metabolism, Anti-Bacterial Agents isolation & purification, Bambermycins chemistry, Candida albicans drug effects, Fermentation, Gram-Positive Bacteria drug effects, Magnetic Resonance Spectroscopy, Microbial Sensitivity Tests, Molecular Structure, Spectrometry, Mass, Fast Atom Bombardment, Actinomycetales chemistry, Aminoglycosides, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology
- Published
- 2000
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19. Comparative mass spectrometric analyses of Photofrin oligomers by fast atom bombardment mass spectrometry, UV and IR matrix-assisted laser desorption/ionization mass spectrometry, electrospray ionization mass spectrometry and laser desorption/jet-cooling photoionization mass spectrometry.
- Author
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Siegel MM, Tabei K, Tsao R, Pastel MJ, Pandey RK, Berkenkamp S, Hillenkamp F, and de Vries MS
- Subjects
- Dihematoporphyrin Ether chemistry, Polymers, Spectrometry, Mass, Fast Atom Bombardment, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Spectrophotometry, Infrared, Spectrophotometry, Ultraviolet, Dihematoporphyrin Ether analysis
- Abstract
Photofrin (porfimer sodium) is a porphyrin derivative used in the treatment of a variety of cancers by photodynamic therapy. This oligomer complex and a variety of porphyrin monomers, dimers and trimers were analyzed with five different mass spectral ionization techniques: fast atom bombardment, UV and IR matrix-assisted laser desorption/ionization, electrospray ionization, and laser desorption/jet-cooling photoionization. All five approaches resulted in very similar oligomer distributions with an average oligomer length of 2.7 +/- 0.1 porphyrin units. In addition to the Photofrin analysis, this study provides a side-by-side comparison of the spectra for the five different mass spectrometric techniques.
- Published
- 1999
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20. Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry.
- Author
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Hileman RE, Siegel MM, Tabei K, Balagurunathan K, and Linhardt RJ
- Subjects
- Carbohydrate Sequence, Molecular Sequence Data, Cyclodextrins analysis, Electrophoresis, Capillary methods, Electrophoresis, Polyacrylamide Gel methods, Mass Spectrometry methods, beta-Cyclodextrins
- Abstract
A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.
- Published
- 1998
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21. Rapid methods for screening low molecular mass compounds non-covalently bound to proteins using size exclusion and mass spectrometry applied to inhibitors of human cytomegalovirus protease.
- Author
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Siegel MM, Tabei K, Bebernitz GA, and Baum EZ
- Subjects
- Binding, Competitive, Chromatography, Gel, Humans, Mass Spectrometry, Molecular Weight, Protein Binding, Ultrafiltration, Cytomegalovirus enzymology, Protease Inhibitors chemistry
- Abstract
General and rapid methods were developed for determining the extent of non-covalent binding between small molecules and proteins, using the model system of human cytomegalovirus protease and several drug candidates which inhibit the protease by non-covalently binding to it. The assay was performed by off-line coupling of size-exclusion methods with mass spectrometry in the following manner. The protease and inhibitor were incubated together under native conditions and then subjected to separation based on size, by use of a spin column (gel permeation chromatography) and/or a microconcentrator (ultrafiltration). The spin column selectively passed the high molecular mass (M(r)) protease and trapped low M(r) molecules. Alternatively, the microconcentrator passed low M(r) molecules and retained the protease. If the inhibitor bound non-covalently to the protease, both the inhibitor and protease passed through the spin column (or were retained by the microconcentrator). Electrospray ionization mass spectrometry was used to assay the spin column eluate (or the microconcentrator retentate) and to characterize the amounts of protease and inhibitor based on known standards. An advantage of these techniques is that a mixture containing inhibitors can be analyzed in the presence of the protease, and inhibitors with the greatest binding affinity can be identified. Non-covalent binding specificity was demonstrated using spin columns by comparing the binding affinity of inhibitors using several mutants of cytomegalovirus protease. The techniques described are applicable to the rapid screening of compound libraries for selecting substances which bind non-covalently to a known protein.
- Published
- 1998
- Full Text
- View/download PDF
22. Characterization of a recombinant fragment that contains a carbohydrate recognition domain of the filamentous hemagglutinin.
- Author
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Liu DF, Phillips E, Wizemann TM, Siegel MM, Tabei K, Cowell JL, and Tuomanen E
- Subjects
- Animals, Bacterial Adhesion, Binding Sites, Carbohydrate Metabolism, Escherichia coli genetics, Mice, Pertussis Vaccine immunology, Rabbits, Recombinant Proteins immunology, Adhesins, Bacterial immunology, Bordetella pertussis immunology, Hemagglutinins immunology, Peptide Fragments immunology, Virulence Factors, Bordetella
- Abstract
The filamentous hemagglutinin (FHA) of Bordetella pertussis plays an important role in establishing infection by attaching the bacteria to the ciliated respiratory epithelial cells. Expression of DNA encoding residues 1141 to 1279 of FHA in Escherichia coli yields a protein of 18,000 Da that exhibits some of the carbohydrate recognition properties of FHA (S. M. Prasad, Y. Yin, E. Rodzinski, E. I. Tuomanen, and H. R. Masure, Infect. Immun. 61:2780-2785, 1993). We have constructed an E. coli strain that expresses this protein, designated fragment A, in a soluble form at markedly elevated levels. Fragment A could be purified with high purity and yields and was immunogenic in mice. Both fragment A and anti-fragment A sera inhibited the binding of B. pertussis to asialo-GM2 and to rabbit ciliated cells. These observations demonstrate that this fragment of FHA contains a cellular binding domain capable of eliciting functional antibodies.
- Published
- 1997
- Full Text
- View/download PDF
23. Calicheamicin derivatives conjugated to monoclonal antibodies: determination of loading values and distributions by infrared and UV matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization mass spectrometry.
- Author
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Siegel MM, Tabei K, Kunz A, Hollander IJ, Hamann RR, Bell DH, Berkenkamp S, and Hillenkamp F
- Subjects
- Anti-Bacterial Agents analysis, Antibiotics, Antineoplastic analysis, Antibodies, Monoclonal chemistry, Carbohydrate Sequence, Drug Delivery Systems, Infrared Rays, Mass Spectrometry, Molecular Sequence Data, Molecular Structure, Ultraviolet Rays, Aminoglycosides, Anti-Bacterial Agents chemistry, Antibiotics, Antineoplastic chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
Calicheamicin derivatives (MW approximately 1500) and monoclonal antibodies (MoAbs) conjugated to calicheamicin derivatives (MW approximately 150,000) were analyzed by UV-MALDI/MS, IR-MALDI/MS, and ESI/MS. These materials are potent anticancer agents. Calicheamicin derivatives and conjugates rapidly degrade upon UV irradiation but are relatively stable during IR irradiation and under ESI conditions. A unique feature of IR-MALDI/MS is a 2 times enhancement in resolution relative to UV-MALDI/MS for masses above approximately 50,000 Da resulting in a molecular ion envelope containing a series of partially resolved peaks of the calicheamicin-MoAb conjugates. The mass shift difference between the peak maxima corresponded to the mass change due to the covalent addition of calicheamicin derivatives to the monoclonal antibody. The distribution of the calicheamicin derivatives in the monoclonal antibodies was computed by deconvoluting the partially resolved peak envelope. A unique feature of the ESI mass spectra, under unit resolution conditions, is that the distribution of the carbohydrates can be well resolved for pure MoAbs and can be only partially resolved for conjugated MoAbs. Average loading values for calicheamicia derivatives when conjugated to MoAbs were computed from UV-MALDI/MS, IR-MALDI/MS, and ESI/MS data and the results compared with the average loading values obtained by UV absorption spectrometry. Very low average loading values were computed from UV-MALDI/MS data due to the degradation of the conjugated calicheamicin derivatives during the UV irradiation process. The IR-MALDI/MS average loading values, obtained with glycerol as the matrix, were consistent with the UV absorption spectrometry values for conjugates having hydrolytically stable linkers, but not when the linker contained a hydrolytically labile hydrazone. ESI/MS average loading values were generally lower than the corresponding values obtained by IR-MALDI/MS. The average loading values and distributions obtained using IR-MALDI/MS were more reliable than the corresponding ESI/MS values because the partially resolved, singly and doubly charged peaks in the IR-MALDI spectra can be mathematically deconvoluted, while the overlapping, highly multiply charged peaks of the electrospray spectra can only be partially deconvoluted.
- Published
- 1997
- Full Text
- View/download PDF
24. Polysulfated carbohydrates analyzed as ion-paired complexes with basic peptides and proteins using electrospray negative ionization mass spectrometry.
- Author
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Siegel MM, Tabei K, Kagan MZ, Vlahov IR, Hileman RE, and Linhardt RJ
- Subjects
- Cyclodextrins analysis, Mass Spectrometry, Molecular Weight, Sucrose analogs & derivatives, Sucrose analysis, Carbohydrates analysis, Oligosaccharides analysis, Peptides analysis, Proteins analysis, Sulfates analysis
- Abstract
Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.
- Published
- 1997
- Full Text
- View/download PDF
25. Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.
- Author
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Baum EZ, Ding WD, Siegel MM, Hulmes J, Bebernitz GA, Sridharan L, Tabei K, Krishnamurthy G, Carofiglio T, Groves JT, Bloom JD, DiGrandi M, Bradley M, Ellestad G, Seddon AP, and Gluzman Y
- Subjects
- Base Sequence, Binding Sites, Chromatography, High Pressure Liquid, Cysteine chemistry, Cytomegalovirus genetics, DNA Primers genetics, DNA, Viral genetics, Dacarbazine pharmacology, Disulfides chemistry, Endopeptidases genetics, Humans, Molecular Sequence Data, Molecular Structure, Mutagenesis, Site-Directed, Oxidation-Reduction, Point Mutation, Viral Proteins genetics, Cytomegalovirus enzymology, Dacarbazine analogs & derivatives, Endopeptidases chemistry, Endopeptidases metabolism, Flavins pharmacology, Protease Inhibitors pharmacology, Viral Proteins chemistry, Viral Proteins metabolism
- Abstract
Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.
- Published
- 1996
- Full Text
- View/download PDF
26. Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation.
- Author
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Baum EZ, Siegel MM, Bebernitz GA, Hulmes JD, Sridharan L, Sun L, Tabei K, Johnston SH, Wildey MJ, Nygaard J, Jones TR, and Gluzman Y
- Subjects
- Amino Acid Sequence, Binding Sites genetics, Cysteine chemistry, Cytomegalovirus genetics, Disulfides chemistry, Endopeptidases genetics, Escherichia coli genetics, Humans, Mass Spectrometry, Molecular Sequence Data, Molecular Structure, Mutagenesis, Site-Directed, Point Mutation, Viral Proteins genetics, Biguanides pharmacology, Cytomegalovirus enzymology, Endopeptidases chemistry, Endopeptidases metabolism, Protease Inhibitors pharmacology, Viral Proteins chemistry, Viral Proteins metabolism
- Abstract
A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.
- Published
- 1996
- Full Text
- View/download PDF
27. Determination of loading values and distributions for drugs conjugated to proteins and antibodies by MALDI-MS and ESI-MS.
- Author
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Siegel MM
- Subjects
- Sensitivity and Specificity, Antibodies chemistry, Mass Spectrometry methods, Pharmaceutical Preparations chemistry, Proteins chemistry
- Published
- 1996
- Full Text
- View/download PDF
28. Sulfinemycin, a new anthelmintic antibiotic: fermentation, isolation and structure determination.
- Author
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Lee TM, Siegel MM, Morton GO, Goodman JJ, Testa RT, and Borders DB
- Subjects
- Animals, Anthelmintics chemistry, Anti-Bacterial Agents chemistry, Fermentation, Gerbillinae, Magnetic Resonance Spectroscopy, Thioamides chemistry, Thioamides pharmacology, Anthelmintics isolation & purification, Anti-Bacterial Agents isolation & purification, Streptomyces chemistry, Thioamides isolation & purification
- Published
- 1995
- Full Text
- View/download PDF
29. Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus sp. LL-42D005. I. Isolation and structure determination.
- Author
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Ding W, Williams DR, Northcote P, Siegel MM, Tsao R, Ashcroft J, Morton GO, Alluri M, Abbanat D, and Maiese WM
- Subjects
- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents isolation & purification, Macrolides, Streptomyces metabolism
- Abstract
Pyrroindomycins A and B were isolated from fermentations of culture LL-42D005, a strain of Streptomyces rugosporus. Pyrroindomycins possess potent antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. Their structures have been determined by using 1- and 2-D NMR, mass spectroscopy and chemical degradations. Pyrroindomycins are the first natural products that contain the highly unsaturated pyrroloindole moiety.
- Published
- 1994
- Full Text
- View/download PDF
30. Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry.
- Author
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Siegel MM, Huang J, Lin B, Tsao R, and Edmonds CG
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Mass Spectrometry methods, Molecular Sequence Data, Peptides, Cyclic chemistry, Bacitracin chemistry
- Abstract
The bacitracin antibiotic complex consists principally of bacitracin A, a peptide antibiotic containing seven amino acid residues in a ring and five amino acid residues in a blocked side chain, together with a mixture of minor components presumably related but of unknown structures. A preparative high-performance liquid chromatographic method was developed for isolating the minor components A2, B1 and B2 which were then characterized by amino acid analysis, exact mass fast atom bombardment (FAB) mass spectrometry, FAB tandem mass spectrometry (MS/MS) and electrospray ionization (ESI) mass spectrometry. For bacitracins A (MW 1421), A2 (MW 1421), B1a (MW 1407), B1b (MW 1407), B2 (MW 1407) and F (MW 1419), the side chain sequences were determined by ESI MS/MS and ESI nozzle-skimmer collision-induced dissociation (CID) mass spectrometry and the ring sequences elucidated by ESI nozzle-skimmer CID MS/MS. Relative to bacitracin A, bacitracin A2a has the modified isoleucine residue at position 1 replaced by a modified allo-isoleucine residue, bacitracin B1a has the isoleucine residue at position 8 replaced by a valine residue, bacitracin B1b has the isoleucine residue at position 5 replaced by a valine residue and bacitracin B2 has the modified isoleucine residue at position 1 replaced by a modified valine residue. FAB tandem mass spectra were shown to be consistent with the above structural assignments for the isolated bacitracin components. Structures were also proposed for the trace bacitracin components C1 (MW 1393) and D1 (MW 1379) using ESI MS/MS data obtained from the analysis of the bacitracin complex without isolation.
- Published
- 1994
- Full Text
- View/download PDF
31. Multisample probe for fast-atom bombardment / mass spectrometry.
- Author
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Siegel MM, Tsao R, Bouley W, Kornas T, and Brubaker G
- Abstract
An inexpensive multisample fast-atom bombardment (FAB) probe assembly was designed for high-throughput analysis of samples on a VG ZAB-SE mass spectrometer. The system consists of a vacuum lock system and a FAB probe whose tip contains five or more sample wells. The probe enters the mass spectrometer source region perpendicular to the secondary ion beam axis, The probe is maintained at high voltage on contact with a spring clip attached to the screen plate of the source block. Sample throughput with the multisample probe is twice that of a coaxial probe, with about twice the sensitivity and no sample cross contamination.
- Published
- 1993
- Full Text
- View/download PDF
32. Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content.
- Author
-
Siegel MM, Hollander IJ, Hamann PR, James JP, Hinman L, Smith BJ, Farnsworth AP, Phipps A, King DJ, and Karas M
- Subjects
- Lasers, Mass Spectrometry, Spectrophotometry, Ultraviolet, Antibodies, Monoclonal analysis, Carbohydrates analysis, Chelating Agents analysis, Pharmaceutical Preparations analysis
- Abstract
The chemically averaged molecular weights of a variety of native and conjugated monoclonal antibodies, approximately 150,000, were measured by matrix-assisted UV-laser desorption/ionization mass spectrometry. The average mass of the carbohydrate present in a monoclonal antibody was estimated from the difference between the measured mass of the monoclonal antibody and the mass of the protein present in the monoclonal antibody computed from the amino acid translation of the DNA sequence. The loading of chelators and anticancer drugs conjugated to a monoclonal antibody was quantitated from the difference in the measured masses for the conjugated and untreated monoclonal antibody relative to the expected mass change upon conjugation of 1 mol of chelator or drug. The loading results obtained by mass spectrometry were consistent in most cases with measurements obtained by radioactivity trace assay or UV spectrometry. Similar matrix-assisted UV-laser desorption/ionization mass spectrometric studies were also made after reducing untreated and conjugated monoclonal antibodies with dithiothreitol to determine the distribution of carbohydrate and chelator between the light and heavy chains of the molecules. Matrix-assisted UV-laser desorption/ionization mass spectra were used to compute loading values for covalently bound drugs and proteins, while the loading values obtained by use of gel-filtration HPLC and UV spectrometry cannot distinguish between covalently and noncovalently bound drugs and proteins.
- Published
- 1991
- Full Text
- View/download PDF
33. LL-AF283 antibiotics, cyclic biphenyl peptides.
- Author
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Chang CC, Morton GO, James JC, Siegel MM, Kuck NA, Testa RT, and Borders DB
- Subjects
- Animals, Antimicrobial Cationic Peptides, Bacteria drug effects, Chromatography, Gel, Chromatography, Ion Exchange, Dipeptides chemistry, Dipeptides isolation & purification, Dipeptides pharmacology, Dipeptides therapeutic use, Fermentation, Magnetic Resonance Spectroscopy, Mass Spectrometry, Mice, Molecular Structure, Peptides, Cyclic chemistry, Peptides, Cyclic isolation & purification, Peptides, Cyclic pharmacology, Peptides, Cyclic therapeutic use, Soil Microbiology, Spectrophotometry, Ultraviolet, Streptomyces metabolism, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents isolation & purification, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents therapeutic use, Peptides, Staphylococcal Infections drug therapy
- Published
- 1991
- Full Text
- View/download PDF
34. 252Cf-plasma desorption and cesium-ion liquid secondary-ion mass spectrometric analysis of recombinant proteins.
- Author
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Tsarbopoulos A, Pramanik BN, Reichert P, Siegel MM, Nagabhushan TL, and Trotta PP
- Subjects
- Animals, Granulocyte-Macrophage Colony-Stimulating Factor chemistry, Humans, Interferon-alpha chemistry, Interleukin-4 chemistry, Mass Spectrometry methods, Molecular Weight, Swine, Recombinant Proteins chemistry
- Abstract
The 35 keV Cs+ liquid secondary-ion mass spectrometry (LSIMS) and 252Cf-plasma desorption (PD) mass spectra of recombinant proteins in the 10-25 kDa mass range are compared. Both techniques showed comparable mass accuracy and sensitivity, and in the case of LSIMS, remarkably short analysis time. Analysis by the PD/nitrocellulose method demonstrated slightly higher sensitivity and relatively lower dependence on the salt and buffer content of the protein sample.
- Published
- 1991
- Full Text
- View/download PDF
35. Fast atom bombardment mass spectral analyses of Photofrin II and its synthetic analogs.
- Author
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Pandey RK, Siegel MM, Tsao R, McReynolds JH, and Dougherty TJ
- Subjects
- Antineoplastic Agents chemical synthesis, Cross-Linking Reagents analysis, Dihematoporphyrin Ether, Esters analysis, Ethers analysis, Mass Spectrometry, Hematoporphyrins, Porphyrins
- Abstract
Fast atom bombardment (FAB) mass spectroscopy was used to characterize the components of Photofrin II. The data indicate that the oligomeric components of Photofrin II contain up to nine porphyrin units. In order to elucidate the nature of covalent linkages between porphyrin units, Photofrin II methyl ester and a porphyrin derivative-2 (2A) were synthesized and characterized by FAB mass spectroscopy. Model studies were also performed by analyzing possible components of Photofrin II, namely, porphyrin dimers 3, 4, 5, 6 and timer 7 with ether or ester linkages. The higher porphyrin oligomers joined by only ether linkages were also synthesized and characterized by FAB mass spectroscopy. The model studies indicate that the porphyrin units in Photofrin II are linked with ether or ether/ester linkages.
- Published
- 1990
- Full Text
- View/download PDF
36. In vitro and in vivo preclinical chemotherapy studies of human neuroblastoma.
- Author
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Siegel MM, Chung HS, Rucker N, Siegel SE, Seeger RC, Isaacs H Jr, and Benedict WF
- Subjects
- Animals, Cell Line, Cell Survival drug effects, Drug Evaluation, Preclinical, Humans, Mice, Mice, Nude, Neoplasms, Experimental drug therapy, Neoplasms, Experimental pathology, Neuroblastoma pathology, Cyclophosphamide therapeutic use, Doxorubicin therapeutic use, Neuroblastoma drug therapy, Vincristine therapeutic use
- Abstract
Two human neuroblastoma cell lines, LA-N-1 and SK-N-MC growing in vitro and as subcutaneous tumors in athymic nude mice, were evaluated for their sensitivity to cyclophosphamide, doxorubicin (Adriamycin), and vincristine. In vitro, cyclophosphamide, following liver S-9 metabolic activation, and vincristine were significantly more cytotoxic to SK-N-MC than to LA-N-1 cells; doxorubicin was equally cytotoxic to both cell types. Treatment of nude mice bearing LA-N-1 and SK-N-MC tumors with cyclophosphamide and vincristine produced significant reduction (> 50%) in SK-N-MC tumor weights but not in LA-N-1 tumor weights. Doxorubicin failed to produce significant reduction in the weight of either the LA-N-1 or the SK-N-MC tumor. These sensitivities were generally similar to the clinical response of the tumors to these same agents. Such an in vitro and in vivo system using these and other neuroblastoma cell lines may provide a preclinical model for evaluating the activity of chemotherapeutic agents against human neuroblastoma.
- Published
- 1980
37. Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A.
- Author
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Lopez DM, Siegel MM, and Lee JC
- Subjects
- Animals, Binding Sites, Antibody, Cell Separation, Cells, Cultured, Thymidine metabolism, Tritium, Antibody Formation, Biological Evolution, Concanavalin A, Lectins, Sharks immunology, T-Lymphocytes immunology
- Published
- 1974
- Full Text
- View/download PDF
38. Thermospray mass spectrometer interface used as a flow reactor for in situ thermal degradation studies: applications to beta-lactam antibiotics.
- Author
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Siegel MM, Isensee RK, and Beck DJ
- Subjects
- Mass Spectrometry, beta-Lactams, Anti-Bacterial Agents analysis
- Published
- 1987
- Full Text
- View/download PDF
39. Isolation and identification of piperacillin amide as an impurity in piperacillin.
- Author
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Siegel MM, Mills R, Gehrlein L, Gore WE, Morton G, Chang T, Cosulich D, Medwid J, and Mirando P
- Subjects
- Chemical Phenomena, Chemistry, Drug Contamination, Magnetic Resonance Spectroscopy, Mass Spectrometry, Molecular Weight, Spectrophotometry, Infrared, Spectrophotometry, Ultraviolet, Piperacillin analogs & derivatives, Piperacillin analysis
- Abstract
Piperacillin amide (IV) was successfully identified as the predominant impurity in commercial lots of piperacillin monohydrate (III). The impurity was isolated via a preparative liquid chromatographic scheme utilizing Florisil as the adsorbent and a mobile phase of water-acetonitrile (4:96, v/v). The isolated component had nearly the same reverse-phase HPLC properties as piperacillin and was chemically and thermally unstable. This labile impurity was spectroscopically identified by field desorption (FD), fast atom bombardment (FAB) with collision activation decomposition (CAD), and desorption chemical ionization (DCI) mass spectrometries , and NMR and IR spectrometries . Identity was confirmed on comparison of the chromatographic and spectrometric data of the impurity with an independently synthesized sample of piperacillin amide.
- Published
- 1984
- Full Text
- View/download PDF
40. Primary chemotherapeutic management of unresectable and metastatic hepatoblastoma in children: report of four cases.
- Author
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Siegel MM, Siegel SE, Isaacs H, Weitzman JJ, Hanson BA, Higgins GR, and Shore NA
- Subjects
- Adolescent, Child, Child, Preschool, Doxorubicin therapeutic use, Drug Therapy, Combination, Female, Humans, Infant, Male, Neoplasm Metastasis drug therapy, Recurrence, Remission, Spontaneous, Antineoplastic Agents therapeutic use, Carcinoma, Hepatocellular drug therapy, Liver Neoplasms drug therapy
- Abstract
Four children presenting with unresectable hepatoblastomas and one with metastatic disease are reported. Following initial biopsy all were treated with chemotherapy which included Adriamycin. Three of the four children showed a significant reduction in tumor size, and in two, delayed resection of the primary lesion was possible. Chemotherapy including Adriamycin represents effective initial cytoreductive therapy for childhood hepatoblastoma, thereby reducing the morbidity and mortality associated with the extensive hepatic resection usually required for an untreated lesion.
- Published
- 1978
- Full Text
- View/download PDF
41. Congenital pernicious anemia: report of seven patients, with studies of the extended family.
- Author
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Heisel MA, Siegel SE, Falk RE, Siegel MM, Carmel R, Lechago J, Skaff G, Roessel T, Nielsen PG, and Cummings P
- Subjects
- Anemia, Pernicious congenital, Anemia, Pernicious diagnosis, Child, Child, Preschool, Female, Hispanic or Latino, Humans, Infant, Male, Pedigree, Anemia, Pernicious genetics
- Abstract
Seven children ages 1 1/2 to 12 years with congenital pernicious anemia were detected in an extended Mexican family. All affected children had megaloblastic anemia accompanied by low serum B12 and normal serum folate levels. Gastric fluid analysis in six patients revealed normal gastric acidity and absent intrinsic factor. Serum antibodies to intrinsic factor or parietal cells were also absent. Schilling tests performed in six of the seven patients yielded abnormal results. Of the three patients in whom gastric biopsy was done, two had normal histologic findings (including examination by electron microscopy) and one had mild atrophy. All patients responded rapidly to parenterally administered vitamin B12 therapy. In addition, 170 family members were screened for the defect with complete blood counts and serum B12 levels. Such screening detected pernicious anemia in two of the children, but no other abnormalities that could be attributed to pernicious anemia were found in other family members. Based on the family pedigree, autosomal recessive inheritance is likely. The variability of age of presentation in this family is noteworthy and suggests that expression may be modified by still undefined factors.
- Published
- 1984
- Full Text
- View/download PDF
42. Preoperative chemotherapy for unresectable primary hepatic malignancies in children.
- Author
-
Weinblatt ME, Siegel SE, Siegel MM, Stanley P, and Weitzman JJ
- Subjects
- Adolescent, Child, Child, Preschool, Cyclophosphamide therapeutic use, Dacarbazine therapeutic use, Dactinomycin therapeutic use, Doxorubicin therapeutic use, Drug Therapy, Combination, Female, Fluorouracil therapeutic use, Follow-Up Studies, Humans, Infant, Liver Neoplasms radiotherapy, Liver Neoplasms surgery, Male, Vincristine therapeutic use, Antineoplastic Agents therapeutic use, Carcinoma, Hepatocellular drug therapy, Liver Neoplasms drug therapy
- Abstract
Eight children presenting with unresectable primary hepatic malignancies were treated with chemotherapy in an attempt to decrease the size of the tumor. Adriamycin was used in all drug regimens, usually in combination with cyclophosphamide, vincristine, and 5-fluorouracil. Seven children exhibited a pronounced, clinical response with marked reduction in the size of the primary tumor as well as any pulmonary metastases present. Four children were able to have complete, uncomplicated surgical excision of residual disease, and three are alive and well off therapy. One patient with hepatocellular carcinoma had compete disappearance of all disease with chemotherapy alone. An approach utilizing preoperative chemotherapy for extensive hepatic malignancies may permit eventual resection of initially inoperable lesions, with long-term survival for these highly lethal malignancies.
- Published
- 1982
- Full Text
- View/download PDF
43. Measles pneumonia in childhood leukemia.
- Author
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Siegel MM, Walter TK, and Ablin AR
- Subjects
- Autopsy, Child, Humans, Lung pathology, Male, Measles diagnosis, Pneumonia, Viral diagnosis, Leukemia, Lymphoid complications, Measles complications, Pneumonia, Viral complications
- Abstract
Fatal measles pneumonia developed in a 7-year-old boy who was in complete remission from acute lymphoblastic leukemia. There was no detectable antibody titer in two specimens taken eight days apart. Measles virus was grown from a lung biopsy taken shortely after hospital admission. Classical measles had been diagnosed in the patient and his siblings nine months previously. Immunosuppressed children who do not develop an antibody rise after a measles infection are at risk of later development of measles giant cell pneumonia. Suggestions are offered for the prevention of this often fatal complication.
- Published
- 1977
44. Chemistry of maduramicin. II. Decarboxylation, abnormal ketalization and dehydration.
- Author
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McGahren WJ, Morton GO, Siegel MM, Borders DB, James JC, and Ellestad GA
- Subjects
- Chemical Phenomena, Chemistry, Decarboxylation, Lactones, Magnetic Resonance Spectroscopy, Coccidiostats, Pyrans
- Abstract
The behavior of the free acid and ammonium salt of maduramicin towards heat and alcohols is examined. In refluxing lower alcohols the free acid material is decarboxylated. In addition a bisketal decarboxylated compound as well as an A-ring monoketal decarboxylated derivative are formed. Heating the ammonium salt of the ionophores in suspension in water, or dissolved in inert solvents such as heptane or xylene can cause decarboxylation as well as concomitant dehydration of the F-ring. Reaction of dansyl chloride with the free acid of maduramicin can cause dehydration of the B-ring under very mild conditions.
- Published
- 1986
- Full Text
- View/download PDF
45. Variability in DNA distributions of human neuroblastomas after cyclophosphamide.
- Author
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Danon YL, Epstein MB, Siegel MM, Rucker N, Myers CP, Norman A, Benedict WF, and Seeger RC
- Subjects
- Animals, Cell Count, Cell Line, Cell Survival drug effects, Humans, Interphase drug effects, Mice, Mice, Nude, Neoplasm Transplantation, Time Factors, Cyclophosphamide pharmacology, DNA, Neoplasm metabolism, Neuroblastoma metabolism
- Abstract
Major changes in DNA distributions of two human neuroblastoma cell lines growing in vitro and in athymic nude mice occurred after treatment with cyclophosphamide. Pulse treatment of LA-N-1 cells in vitro with liver S-9-activated cyclophosphamide (10 micrograms/ml) caused approximately 50% cytotoxicity; flow microfluorometric analysis of surviving cells demonstrated an increased proportion of G2 + M cells and a decreased proportion of G1 cells, particularly at 48 hrs. Even though LA-N-1 tumors in nude mice did not regress after one dose of cyclophosphamide (250 mg/kg), the percent of G2 + M cells increased and the percent of G1 cells decreased 4-6 days after treatment; the percent of cells in S increased at 2 and again at 8 days. SK-N-MC cells were affected differently by cyclophosphamide. Pulse treatment of these cells in vitro with liver S-9-activated cyclophosphamide caused greater than 85% cytotoxicity and nearly complete elimination of cells in G2 + M at 24 and 48 hrs. Likewise, SK-N-MC tumors in nude mice regressed greater than 50% after cyclophosphamide, and the proportion of G2 + M cells decreased markedly 2-6 days after therapy. We conclude that cyclophosphamide can have different cytotoxic and cytokinetic effects on neuroblastomas. In addition, marked cytokinetic effects may occur even though changes in tumor size are minimal or not detectable.
- Published
- 1980
46. Fast atom bombardment mass spectrometry of cisplatin analogs.
- Author
-
Siegel MM, Bitha P, Child RG, Hlavka JJ, Lin YI, and Chang TT
- Subjects
- Mass Spectrometry, Molecular Conformation, Structure-Activity Relationship, Cisplatin analysis
- Abstract
Cisplatin analogs of the type PtLACl2 and PtLALB are thermally unstable, non-volatile and highly insoluble. For these platinum coordination complexes, LA is a bidentate amine ligand and LB is a bidentate carboxylate ligand. Mass spectral data for structural elucidation of these compounds are absent in the literature because they are difficult to ionize. Nevertheless, a routine fast atom bombardment mass spectroscopic method has been developed utilizing the mixed solvent system of dimethyl sulfoxide:thioglycerol in a ratio of about 1:3 v/v. Using both positive and negative ionization modes, structurally significant ions were observed from representative molecules of the two named classes of compounds. [M-H]- ions were observed in both structural classes while [M + H]+ ions were observed only in the PtLALB class of compounds. Additional ions observed are rationalized in terms of the condensed-phase solution chemistry of the cisplatin analogs and the mixed solvent system when exposed to the fast atom beam. The two mechanisms causing ionization of the cisplatin analogs in the condensed phase appear to be: displacement of the ligands with dimethyl-sulfoxide and addition of chloride and the ionized solvents [dimentyl sulfoxide + H]+ and [thioglycerol - H]- to the cisplatin analogs. It is hypothesized that the addition reactions of the ionized solvents occur because of the differences in the basicity of the solvents and their reactivity in forming platinum(II)-sulfur bonds.
- Published
- 1986
- Full Text
- View/download PDF
47. Preoperative chemotherapy for hepatoblastoma in children: report of six cases.
- Author
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Andrassy RJ, Brennan LP, Siegel MM, Weitzman JJ, Siegel SE, Stanley P, and Mahour GH
- Subjects
- Adolescent, Antineoplastic Agents administration & dosage, Carcinoma, Hepatocellular surgery, Child, Child, Preschool, Doxorubicin therapeutic use, Drug Therapy, Combination, Female, Humans, Infant, Liver Neoplasms surgery, Male, Preoperative Care, Carcinoma, Hepatocellular drug therapy, Liver Neoplasms drug therapy
- Abstract
Surgical excision has been the primary treatment for hepatoblastoma; however, at presentation, only one-third of such tumors are surgically resectable. Without operation, the disease is universally fatal. Six children with initially unresectable hepatoblastoma (two with pulmonary metastases) were treated with chemotherapy, which included Adriamycin. Four of the six children showed a significant reduction in tumor size, in three delayed resection of the primary lesion was possible, and the fourth patient died of Adriamycin cardiotoxicity. Two patients did not respond and developed pulmonary metastases after 2 and 16 mo of chemotherapy, respectively. Adriamycin alone, or in combination with other agents, has proven effective in primary and metastatic childhood hepatoblastoma. This preoperative chemotherapy regimen permits resection of previously unresectable hepatoblastoma at "second look" operation and reduces the morbidity and mortality of an otherwise extensive operation.
- Published
- 1980
- Full Text
- View/download PDF
48. An efficient algorithm for sequencing peptides using fast atom bombardment mass spectral data.
- Author
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Siegel MM and Bauman N
- Subjects
- Algorithms, Amino Acid Sequence, Angiotensin II analysis, Gonadotropin-Releasing Hormone analogs & derivatives, Gonadotropin-Releasing Hormone analysis, Mass Spectrometry, Molecular Weight, Triptorelin Pamoate, Peptides analysis
- Abstract
An efficient algorithm is described for sequencing peptides from sequence ions appearing in fast atom bombardment (FAB) and FAB tandem mass spectra. The following features are incorporated in the algorithm. The members of the set of sequence ions are represented by all possible combinations of N- and C-terminal fragment ions. From the known N- and C-terminating groups and molecular weight (MW) of the peptide, the sequence ions are mathematically re-expressed as N-terminal residue ions and arranged in ascending order. The peptide sequence is computed, in a stepwise iterative procedure, from the mass differences between the mathematically re-expressed N-terminal residue ions and the predicted peptide subsequences for the neighboring ions of lower mass. These mass differences correspond to combinations of known amino acid residues which have previously been computed and tabulated, based upon the FAB fragmentation rules for peptides. The algorithm was successfully applied to sequence the following peptides from their respective FAB or FAB tandem mass spectrum: decapeptyl (MW 1310), angiotensin II (MW 1045), and two 'unknown' peptides (MW 1227 and 1485, respectively). Two criteria used to predict the correct peptide sequence from among many possibilities are the minimum number of amino acid residues and the maximum fragmentation probability per amino acid residue.
- Published
- 1988
- Full Text
- View/download PDF
49. LL-BM726: a novel dipeptide antibiotic.
- Author
-
Ellestad GA, James JC, Morton GO, Siegel MM, and McGahren WJ
- Subjects
- Magnetic Resonance Spectroscopy, Molecular Conformation, Anti-Bacterial Agents isolation & purification, Dipeptides isolation & purification, Streptomyces metabolism
- Published
- 1985
- Full Text
- View/download PDF
50. LL-F28249 antibiotic complex: a new family of antiparasitic macrocyclic lactones. Isolation, characterization and structures of LL-F28249 alpha, beta, gamma, lambda.
- Author
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Carter GT, Nietsche JA, Hertz MR, Williams DR, Siegel MM, Morton GO, James JC, and Borders DB
- Subjects
- Chemical Phenomena, Chemistry, Parasitic Diseases drug therapy, Anti-Bacterial Agents isolation & purification, Antinematodal Agents isolation & purification, Lactones isolation & purification, Macrolides, Streptomyces metabolism
- Abstract
A new family of antiparasitic macrolides has been isolated from Streptomyces cyaneogriseus sp. noncyanogenus. The compounds, designated LL-F28249 alpha, beta, gamma and lambda, possess potent antiparasitic activity. The isolation, purification and structure determination by spectroscopic methods are presented.
- Published
- 1988
- Full Text
- View/download PDF
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