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A Karcher

Publications and source records attributed to A Karcher.

16 recordsLinked to original sources
PubMed2001

Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase.

To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

Amino Acid Sequence↗
PubMed2001

Measurement of CP-violating asymmetries in B0 decays to CP eigenstates.

We present measurements of time-dependent CP-violating asymmetries in neutral B decays to several CP eigenstates. The measurement uses a data sample of 23x10(6) Upsilon(4S)-->BbarB decays collected by the BABAR detector at the PEP-II asymmetric B Factory at SLAC. In this sample, we find events in which one neutral B meson is fully reconstructed in a CP eigenstate containing charmonium and the flavor of the other neutral B meson is determined from its decay products. The amplitude of the CP-violating asymmetry, which in the standard model is proportional to sin2beta, is derived from the decay time distributions in such events. The result is sin2beta = 0.34+/-0.20 (stat)+/-0.05 (syst).

Journal Article↗
PubMed2001

Directly observed treatment for multidrug-resistant tuberculosis: an economic evaluation in the United States of America and South Africa.

OBJECTIVE: To estimate the cost-effectiveness of directly observed treatment compared to conventional therapy in reducing the spread of multidrug-resistant tuberculosis, for an industrialised country (represented by the United States of America) and a developing country (South Africa). METHODS: Monte Carlo analysis using published data on probability, cost and health impact. RESULTS: In both countries, directly observed treatment is the dominant strategy, yielding cost savings and improved health outcomes. Cost savings for directly observed treatment relative to conventional therapy become more significant as more expensive second-line drugs are used in treatments. CONCLUSIONS: The cost-effectiveness of directly observed treatment relative to conventional therapy is demonstrated for both the USA and South Africa. Cost savings are more pronounced (especially for South Africa) as the likelihood of multidrug-resistant tuberculosis increases and more expensive second-line therapies are used. Given that health care resources are more severely constrained in developing countries, the data contained in this study are useful in guiding the design of policies for the effective management of multidrug-resistant tuberculosis in settings with limited resources.

Antitubercular Agents↗
PubMed2000

Crystals of the urokinase type plasminogen activator variant beta(c)-uPAin complex with small molecule inhibitors open the way towards structure-based drug design.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Amiloride↗
PubMed2000

Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

Adenosine Triphosphatases↗
PubMed2000

Electrically driven microseparation methods for pesticides and metabolites: IV. Effects of the nature of fluorescent labels on the enantioseparation of pesticides and their degradation products by capillary zone electrophoresis with UV and laser-induced fluorescence detection.

Three different fluorescent tags, namely 5-aminonaphthalene-1-sulfonic acid (ANSA), 7-aminonaphthalene-1,3-disulfonic acid (ANDSA), and 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) were evaluated in the precolumn derivatization of some chiral phenoxy acid herbicides, some chiral transformation products of pyrethroid insecticides, and in their subsequent enantiomeric separation by capillary electrophoresis (CE). The electrolyte systems consisted of sodium phosphate buffers containing chiral surfactants such as octylglucoside (OG) or octylmaltoside (OM) at concentrations above the critical micellar concentration (CMC). Among the three different tags investigated, the ANDSA derivatives of the various solutes were more readily enantioseparated than the ANSA and ANTS derivatives. While the tagging with ANSA allowed the enantioseparation of a limited number of the chiral solute-ANSA derivatives investigated, the ANTS derivatization yielded derivatives that could not be enantioseparated. The polarity of the three different tags increases by increasing the number of sulfonic acid groups in the molecule in the following order: ANSA (one sulfonic acid) < ANDSA (two sulfonic acid groups) < ANTS (three sulfonic acid groups). This seems to indicate that the intermediate polarity of the ANDSA tag allowed more equitable nonpolar/polar interactions of the ANDSA-derivatized solutes with the OG or OM micelles, and consequently the enantioseparation of the solute-ANDSA derivatives. Thus, there is a solute polarity window for enantioresolution with alkylglycoside micelle by CE. Solutes of intermediate polarity that undergo more equitable nonpolar/polar interactions with the micelles exhibited chiral separations.

Electrophoresis, Capillary↗
PubMed2000

Mre11 and Rad50 from Pyrococcus furiosus: cloning and biochemical characterization reveal an evolutionarily conserved multiprotein machine.

The processing of DNA double-strand breaks is a critical event in nucleic acid metabolism. This is evidenced by the severity of phenotypes associated with deficiencies in this process in multiple organisms. The core component involved in double-strand break repair in eukaryotic cells is the Mre11-Rad50 protein complex, which includes a third protein, p95, in humans and Xrs2 in yeasts. Homologues of Mre11 and Rad50 have been identified in all kingdoms of life, while the Nbs1 protein family is found only in eukaryotes. In eukaryotes the Mre11-Rad50 complex has nuclease activity that is modulated by the addition of ATP. We have isolated the Mre11 and Rad50 homologues from the thermophilic archaeon Pyrococcus furiosus and demonstrate that the two proteins exist in a large, heat-stable complex that possesses single-strand endonuclease activity and ATP-dependent double-strand-specific exonuclease activity. These findings verify the identification of the P. furiosus Rad50 and Mre11 homologues and demonstrate that functional homologues with similar biochemical properties exist in all kingdoms of life.

Amino Acid Sequence↗
PubMed1999

Coagulation factor IXa: the relaxed conformation of Tyr99 blocks substrate binding.

BACKGROUND: Among the S1 family of serine proteinases, the blood coagulation factor IXa (fIXa) is uniquely inefficient against synthetic peptide substrates. Mutagenesis studies show that a loop of residues at the S2-S4 substrate-binding cleft (the 99-loop) contributes to the low efficiency. The crystal structure of porcine fIXa in complex with the inhibitor D-Phe-Pro-Arg-chloromethylketone (PPACK) was unable to directly clarify the role of the 99-loop, as the doubly covalent inhibitor induced an active conformation of fIXa. RESULTS: The crystal structure of a recombinant two-domain construct of human fIXa in complex with p-aminobenzamidine shows that the Tyr99 sidechain adopts an atypical conformation in the absence of substrate interactions. In this conformation, the hydroxyl group occupies the volume corresponding to the mainchain of a canonically bound substrate P2 residue. To accommodate substrate binding, Tyr99 must adopt a higher energy conformation that creates the S2 pocket and restricts the S4 pocket, as in fIXa-PPACK. The energy cost may contribute significantly to the poor K(M) values of fIXa for chromogenic substrates. In homologs, such as factor Xa and tissue plasminogen activator, the different conformation of the 99-loop leaves Tyr99 in low-energy conformations in both bound and unbound states. CONCLUSIONS: Molecular recognition of substrates by fIXa seems to be determined by the action of the 99-loop on Tyr99. This is in contrast to other coagulation enzymes where, in general, the chemical nature of residue 99 determines molecular recognition in S2 and S3-S4. This dominant role on substrate interaction suggests that the 99-loop may be rearranged in the physiological fX activation complex of fIXa, fVIIIa, and fX.

Amino Acid Sequence↗
PubMed1999

High-performance liquid-phase separation of glycosides. III. Determination of total glucosinolates in cabbage and rapeseed by capillary electrophoresis via the enzymatically released glucose.

A selective and sensitive method for the determination of total glucosinolates (GSs) in plant extracts by capillary electrophoresis-laser-induced fluorescence (LIF) detection was developed. It was based on the enzymatically released glucose from glucosinolates in the presence of the hydrolyzing enzyme myrosinase. The released glucose was converted to gluconic acid (GA) by the action of glucose oxidase. The resulting GA was then labeled selectively with the fluorescent tag 7-aminonaphthalene-1, 3-disulfonic acid (ANDSA). The peak area resulting from the GA-ANDSA derived from free and bound glucose was subtracted from the peak area of the GA-ANDSA resulting from the free glucose in the sample. This gave the total glucosinolates in the sample. The peak areas were normalized to the internal standard, N-acetylneuraminic acid derivatized with ANDSA. The method was validated using four different plant extracts, white cabbage leaves, rapeseed leaves, rapeseed roots, and rapeseed seeds. Furthermore, a capillary electrophoresis-UV detection method for profiling GS in plant extracts was developed. In addition to providing a fingerprint of the glucosinolates in plant extracts, the method allowed the experimenter to rapidly check the various steps involved in the extraction and sample cleanup.

2-Naphthylamine↗
PubMed1999

Capillary electrophoresis of glucosinolates and their degradation products.

Glucosinolates are important natural products occurring mainly in plants of the Cruciferae family. This review article is aimed at describing the recent progress made in capillary electrophoresis of glucosinolates and their degradation products. It describes the various electrophoretic systems and detection schemes introduced to date for the capillary electrophoresis (CE) of glucosinolates and their degradation products. Also included in this review are the applications of CE to the qualitative and quantitative determination of glucosinolates and their degradation products in plant extracts.

Electrophoresis, Capillary↗
PubMed1999

Capillary electrophoresis and electrochromatography of pesticides and metabolites.

Synthetic pesticides are important chemicals since they are widely used to control many types of weeds, insects and other pests in a wide variety of agricultural and nonagricultural settings. This review article is aimed at describing the recent progress made in capillary electrophoresis (CE) and capillary electrochromatography (CEC) of pesticides and their metabolites. The various electrophoretic systems and detection schemes that have been introduced so far for the CE and CEC of pesticides are discussed. Also included in this review article are the various approaches for trace enrichment that are involved in the analysis of dilute pesticide samples.

Chromatography↗
PubMed1999

High-performance liquid phase separation of glycosides. 5. Determination of individual glucosinolates in cabbage and rapeseed by laser-induced fluorescene capillary electrophoresis via the enzymatically released isothiocyanate aglycon.

A capillary electrophoresis (CE) method was developed for the profiling and determination of individual glucosinolates (GSs) via their isothiocyanate degradation products upon myrosinase digestion. The resulting isothiocyanates, the structures of which are reflective of the parent GS's, were then converted to their corresponding amines via base hydrolysis or reaction with 1, 2-benzenedithiol. Subsequently, the amines were fluorescently labeled to allow their sensitive detection by laser-induced fluorescence (LIF). The CE method involved the use of in situ charged micelles for the separation of isothiocyanates and their corresponding fluorescently labeled amines by micellar electrokinetic capillary chromatography (MECC). The term "in situ charged micelles" refers to micelles formed by complexing the polar hydroxyl groups of glycosidic surfactants with borate. The MECC method with on-column LIF detection was applied to the determination of GSs in white cabbage, rapeseed leaves, and rapeseed roots.

Brassica↗
PubMed1997

Converting blood coagulation factor IXa into factor Xa: dramatic increase in amidolytic activity identifies important active site determinants.

The coagulation factors IXa (fIXa) and Xa (fXa) share extensive structural and functional homology; both cleave natural substrates effectively only with a cofactor at a phospholipid surface. However, the amidolytic activity of fIXa is 10(4)-fold lower than that of fXa. To identify determinants of this poor reactivity, we expressed variants of truncated fIXa (rf9a) and fXa (rf10a) in Escherichia coli. The crystal structures of fIXa and fXa revealed four characteristic active site components which were subsequently exchanged between rf9a and rf10a. Exchanging Glu219 by Gly or exchanging the 148 loop did not increase activity of rf9a, whereas corresponding mutations abolished reactivity of rf10a. Exchanging Ile213 by Val only moderately increased reactivity of rf9a. Exchanging the 99 loop, however, dramatically increased reactivity. Furthermore, combining all four mutations essentially introduced fXa properties into rf9a: the amidolytic activity was increased 130-fold with fXa substrate selectivity. The results suggest a 2-fold origin of fIXa's poor reactivity. A narrowed S3/S4 subsite disfavours interaction with substrate P3/P4 residues, while a distorted S1 subsite disfavours effective cleavage of the scissile bond. Both defects could be repaired by introducing fXa residues. Such engineered coagulation enzymes will be useful in diagnostics and in the development of therapeutics.

Amidohydrolases↗
PubMed1997

Capillary electrophoresis of pesticides: V. Analysis of pyrethroid insecticides via their hydrolysis products labeled with a fluorescing and UV absorbing tag for laser-induced fluorescence and UV detection.

Some representative standard pyrethroid insecticides, namely permethrin, phenotrin, cypermethrin, sanmarton and fenpropathrin, were subjected to base hydrolysis with the aim of facilitating the indirect determination of these neutral species of low water solubilities by aqueous capillary electrophoresis. This first involved the base fragmentation of the pyrethroids in alcohol buffer (pH 12.0), and then the selective tagging of the carboxylated hydrolytic products with 7-aminonaphthalene-1,3-disulfonic acid (ANDSA) via a condensation reaction in the presence of organic soluble carbodiimide. The tagging of the hydrolytic products with ANDSA imparted each of the derivatives with two strong sulfonic acid groups whose permanent charges were necessary for achieving aqueous capillary electrophoresis. In addition, the labeling with ANDSA allowed the detection of the derivatives at low levels by capillary electrophoresis laser-induced fluorescence. The geometric and optical isomers of the ANDSA derivatives of the pyrethroid hydrolytic products were best separated when using electrolyte systems composed of sodium phosphate buffer, pH 6.5, containing n-octylglucoside chiral surfactant in the presence of small amounts of acetonitrile (e.g., 10% v/v).

Electrophoresis, Capillary↗
PubMed1994

Sequential high-dose therapy with peripheral-blood progenitor-cell support in low-grade non-Hodgkin's lymphoma.

PURPOSE: To evaluate the feasibility of a sequential high-dose therapy with peripheral-blood progenitor-cell (PBPC) support in patients with follicular lymphoma. PATIENTS AND METHODS: Since July 1991, we have included 30 patients (17 men and 13 women) with a median age of 41 years (range, 26 to 55) in the study. At the time of study entry, 17 patients were in first and six in second or higher remission. Another six patients had relapse of disease and one had tumor progression. PBPC were collected during filgrastim-supported leukocyte recovery following high-dose cytarabine (ara-C)/mitoxantrone (HAM). RESULTS: A median of two leukaphereses (range, one to seven) resulted in a median of 5.7 x 10(6) CD34+ cells/kg (range, 2.9 to 23.7 x 10(6). A distinct population of B-lymphoid progenitors (CD34+/CD19+) was not detectable in the autografts, and the content of CD19+ B cells was remarkably low, comprising a median of 0.07% of the mononuclear cells. Using the polymerase chain reaction (PCR) assay for the major breakpoint regions (MBR) of the bcl-2/immunoglobulin H (IgH) translocation, 22 patients had autografts positive for the t(14;18) translocation, whereas seven patients had PCR-negative transplants. The autograft of one patient could not be assessed. Following myeloablative therapy, hematologic recovery was rapid without cytokine support. The median times to reach a platelet count > or = 20 x 10(9)/L and neutrophil count > or = 0.5 x 10(9)/L were 11 and 13 days, respectively. Nonhematologic toxicity was moderate. Twenty-nine patients were alive in remission after a median follow-up duration of 6 months (range, 1 to 18). Of 22 patients autografted with t(14;18)-positive harvests, 11 had PCR-detectable cells in bone marrow and/or peripheral blood as long as 16 months posttransplantation. In contrast, six patients became PCR-negative between 3 and 16 months after reinfusion. Follow-up examinations with PCR data for the remaining five patients are not yet available. CONCLUSION: Conversion to PCR negativity in patients autografted with PCR-positive harvests suggests that the myeloablative regimen is effective and that any reinfused t(14;18)-positive cells may not be sustained. Because conventional chemotherapy provides no cure, we believe that high-dose therapy including total-body irradiation (TBI) should be explored in these particularly radiosensitive lymphomas.

Adult↗