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G Szakács

Publications and source records attributed to G Szakács.

18 recordsLinked to original sources
PubMed2006

Degradation of lignin-containing materials by xylanase in biopreparation of cotton.

Solubilization of lignin and carbohydrates from the lignin-holocellulose structure of cotton seed-coat fragments was investigated by UV/VIS spectrometry. Xylanase (Pulpzyme HC) pre-treatment partially destroyed the lignocellulosic structure of the seed-coat fragments, producing reducing sugars and soluble lignin in the supernatant. Furthermore, the pre-treatment by enzyme enhanced the delignification in the subsequent alkaline scouring process and increased the lightness of the substrate.

Biotechnology↗
PubMed2001

Characterization of the ATPase cycle of human ABCA1: implications for its function as a regulator rather than an active transporter.

ABCA1 plays a key role in cellular cholesterol and phospholipid traffic. To explore the biochemical properties of this membrane protein we applied a Baculovirus-insect cell expression system. We found that human ABCA1 in isolated membranes showed a specific, Mg(2+)-dependent ATP binding but had no measurable ATPase activity. Nevertheless, conformational changes in ABCA1 could be demonstrated by nucleotide occlusion, even without arresting the catalytic cycle by phosphate-mimicking anions. Addition of potential lipid substrates or lipid acceptors (apolipoprotein A-I) did not modify the ATPase activity or nucleotide occlusion by ABCA1. Our data indicate that ATP hydrolysis by ABCA1 occurs at a very low rate, suggesting that ABCA1 may not function as an effective active transporter as previously assumed. In the light of the observed conformational changes we propose a regulatory function for human ABCA1.

ATP Binding Cassette Transporter 1↗
PubMed2001

Enzymes and chelating agent in cotton pretreatment.

Desized cotton fabric and cotton seed-coat fragments (impurities) have been treated with commercial cellulase (Celluclast 1.5 L), hemicellulase-pectinase (Viscozyme 120 L) and xylanase (Pulpzyme HC) enzymes. Seed-coat fragments hydrolyzed much faster than the cotton fabric itself. This relative difference in hydrolysis rates makes possible a direct enzymatic removal of seed-coat fragments from desized cotton fabric. Addition of chelating agents such as ethylenediamine-tetra-acetic acid (EDTA) markedly enhanced the directed enzyme action. Pretreatments carried out in acidic solution at pH 5 increased the lightness of seed-coat fragments, contrary to the samples treated in neutral medium at pH 7. Alkaline scouring resulted in darker seed-coat fragments except for the samples pretreated with Pulpzyme HC plus EDTA. This effect is similar to that observed in the biobleaching process in pulp and paper industry.

Calcium↗
PubMed2001

Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells.

ABCG2 (also called MXR (3), BCRP (4), or ABCP (5) is a recently-identified ABC half-transporter, which causes multidrug resistance in cancer. Here we report that the expression of the ABCG2 protein in Sf9 insect cells resulted in a high-capacity, vanadate-sensitive ATPase activity in isolated membrane preparations. ABCG2 was expressed underglycosylated, and its ATPase activity was stimulated by daunorubicin, doxorubicin, mitoxantrone, prazosin and rhodamine 123, compounds known to be transported by this protein. ABCG2-ATPase was inhibited by low concentrations of Na-orthovanadate, N-ethylmaleimide and cyclosporin A. Verapamil had no effect, while Fumitremorgin C, reversing ABCG2-dependent cancer drug resistance, strongly inhibited this ATPase activity. The functional expression of ABCG2 in this heterologous system indicates that no additional partner protein is required for the activity of this multidrug transporter, probably working as a homodimer. We suggest that the Sf9 cell membrane ATPase system is an efficient tool for examining the interactions of ABCG2 with pharmacological agents.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2001

Role of glycine-534 and glycine-1179 of human multidrug resistance protein (MDR1) in drug-mediated control of ATP hydrolysis.

The human multidrug resistance protein (MDR1) (P-glycoprotein), a member of the ATP-binding cassette (ABC) family, causes multidrug resistance by an active transport mechanism, which keeps the intracellular level of hydrophobic compounds below a cell-killing threshold. Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D/G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes. These mutations caused a loss of measurable ATPase activity but still allowed ATP binding and the formation of a transition-state intermediate (nucleotide trapping). In contrast with the wild-type protein, in which substrate drugs accelerate nucleotide trapping, in the ABC signature mutants nucleotide trapping was inhibited by MDR1-substrate drugs, suggesting a miscommunication between the drug-binding site(s) and the catalytic domains. Equivalent mutations of the two catalytic sites resulted in a similar effect, indicating the functional equivalence of the two sites. On the basis of these results and recent structural information on an ABC-ABC dimer [Hopfner, Karcher, Shin, Craig, Arthur, Carney and Tainer (2000) Cell 101, 789-800], we propose a key role of these glycine residues in the interdomain communication regulating drug-induced ATP hydrolysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Enzyme assisted ensiling of alfalfa with enzymes by solid substrate fermentation.

A crude enzyme preparation, obtained by solid substrate fermentation (SSF) with a Gliocladium spp. and added at the 5% level to wilted or non-wilted alfalfa, improved the fermentation characteristics and stability of alfalfa silages as effectively as commercial preparations, Novo-Nordisk Celluclast 1.5 L and Viscozyme 120 L, applied at the 0.025% level. The effective dose of the crude enzyme costs about one-fourth of the cost of the commercial enzymes.

Carbohydrate Metabolism↗
PubMed2001

Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia.

In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug-resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non-responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non-responders (P = 0.0045 and P = 0.0454). Cut-off values were established between the MAFR + SEM and MAFNR - SEM values. On the basis of these cut-off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P-glycoprotein and multidrug resistance-associated protein activities, and identifies AML patients with unfavourable therapy responses.

Acute Disease↗
PubMed2000

Transition-state formation in ATPase-negative mutants of human MDR1 protein.

In this work we have studied the partial catalytic reactions in MDR1 variants carrying mutations in the conserved Walker A region (K433M and K1076M) of either the N-terminal or C-terminal ABC domain. Both mutations have been demonstrated to cause a loss of drug transport, drug-stimulated ATPase, and vanadate-dependent nucleotide trapping activity. Here we show that these mutants still allow transition state formation (nucleotide trapping) when fluoro-aluminate or beryllium fluoride is used as a complex-stabilizing anion. Drug stimulation of nucleotide trapping was found to be preserved in both mutants. Limited trypsin digestion revealed that whenever MDR1-nucleotide trapping occurred, both ABC domains were involved in the formation of the catalytic intermediates. Our results show that details of the MDR1-ATPase cycle can be studied even in ATPase-negative mutants. These data also demonstrate that the conformational alteration caused by a mutation in one of the ABC domains is propagated to the other, nonmutated domain, indicating a tight coupling between the functioning of the two ABC domains.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1).

The human multidrug resistance protein (MRP1) contributes to drug resistance in cancer cells. In addition to an MDR1-like core, MRP1 contains an N-terminal membrane-bound (TMD(0)) region and a cytoplasmic linker (L(0)), both characteristic of several members of the MRP family. In order to study the role of the TMD(0) and L(0) regions, we constructed various truncated and mutated MRP1, and chimeric MRP1-MDR1 molecules, which were expressed in insect (Sf9) and polarized mammalian (MDCKII) cells. The function of the various proteins was examined in isolated membrane vesicles by measuring the transport of leukotriene C(4) and other glutathione conjugates, and by vanadate-dependent nucleotide occlusion. Cellular localization, and glutathione-conjugate and drug transport, were also studied in MDCKII cells. We found that chimeric proteins consisting of N-terminal fragments of MRP1 fused to the N terminus of MDR1 preserved the transport, nucleotide occlusion and apical membrane routing of wild-type MDR1. As shown before, MRP1 without TMD(0)L(0) (Delta MRP1), was non-functional and localized intracellularly, so we investigated the coexpression of Delta MRP1 with the isolated L(0) region. Coexpression yielded a functional MRP1 molecule in Sf9 cells and routing to the lateral membrane in MDCKII cells. Interestingly, the L(0) peptide was found to be associated with membranes in Sf9 cells and could only be solubilized by urea or detergent. A 10-amino-acid deletion in a predicted amphipathic region of L(0) abolished its attachment to the membrane and eliminated MRP1 transport function, but did not affect membrane routing. Taken together, these experiments suggest that the L(0) region forms a distinct domain within MRP1, which interacts with hydrophobic membrane regions and with the core region of MRP1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. Different patterns in the two nucleotide binding domains.

The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Perspectives in agrobiotechnology.

This review surveys the most important and promising contributions of agricultural biotechnology to the development of sustainable, environment-friendly agriculture. It deals with the recent achievements of genetic technology for the development of new transgenic microbial, plant and animal products. It also deals with the newest developments and perspectives of microbial intervention in agricultural practices, such as biofertilizers, biocontrol agents, and various microbiological products used in modern agriculture. The review surveys the outlook for a waste-free, environment-friendly sustainable agricultural practice, including waste management, recycling and bioremediation technologies. The review lists the most important marketable agrobiotechnological products, and their present and projected sales volume.

Agriculture↗
PubMed1998

Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain.

The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.

Animals↗
PubMed1998

Diagnostics of multidrug resistance in cancer.

Multidrug resistance (MDR), caused by the overexpression of two membrane proteins, MDR1-Pgp and/or MRP, is a major obstacle in the chemotherapy of cancer. The proper laboratory diagnosis of clinical multidrug resistance is still an unresolved question, and this uncertainty, in a vicious cycle, does not allow the correct evaluation of the clinical relevance of the MDR phenomenon. More-over, inefficient MDR diagnostics hinders the development of effective resistance-modulation strategies. In this review, after describing the basic features of the MDR drug pump proteins, the currently employed diagnostic methods are discussed. We suggest that a quantitative, functional method developed in our laboratory may provide a major help in the laboratory assessment of cancer MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

[Clinical application of analysis of microsatellite markers in the prenatal diagnosis of cystic fibrosis].

Cystic Fibrosis (CF) is an autosomal recessive hereditary disease, caused by the defect of a membrane transport protein. The defect is due to the mutation of the gene coding this protein. To date, these mutations have been analysed by direct mutational analyses in prenatal diagnosis. During gene sequencing, intragenic polymorphic markers (microsatellites) were identified, enabling the indirect analysis of the mutant allele. The markers characterize the given allele, so that the inheritance according to the Mendelian rules could be followed. We introduced a DNA-diagnostic method based on the amplification of three intragenic microsatellites. This new and efficient prenatal diagnostic tool would provide more reliable test results for previously screened CF families, in which direct mutation analysis was not informative.

Cystic Fibrosis↗
PubMed1994

Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B-12-2.

The endoxylanase complex from Streptomyces sp. strain B-12-2 was purified and characterized. The organism forms five distinct xylanases in the absence of significant cellulase activity when grown on oat spelt xylan. This is the largest number of endoxylanases yet reported for a streptomycete. On the basis of their physiochemical characteristics, they can be divided into two groups: the first group (xyl 1a and xyl 1b) consists of low-molecular-mass (26.4 and 23.8 kDa, respectively) neutral- to high-pI (6.5 and 8.3, respectively) endoxylanases. Group 1 endoxylanases are unable to hydrolyze aryl-beta-d-cellobioside, have low levels of activity against xylotetraose (X(4)) and limited activity against xylopentaose, produce little or no xylose, and form products having a higher degree of polymerization with complex substrates. These enzymes apparently carry out transglycosylation. The second group (xyl 2, xyl 3, and xyl 4) consists of high-molecular-mass (36.2, 36.2, and 40.5 kDa, respectively), low-pI (5.4, 5.0, and 4.8, respectively) xylanases. Group 2 endoxylanases are able to hydrolyze aryl-beta-d-cellobioside, show higher levels of activity against X(4), and hydrolyze xylopentaose completely with the formation of xylobiose and xylotriose plus limited amounts of X(4) and xylose. The enzymes display intergroup synergism when acting on kraft pulp. Despite intragroup similarities, each enzyme exhibited a unique action pattern and physiochemical characteristic. xyl 2 was highly glycosylated, and xyl 1b (but no other enzyme) was completely inhibited by p-hydroxymercuribenzoate.

Journal Article↗
PubMed1992

Xylanase Activity of Phanerochaete chrysosporium.

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30 degrees C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-beta-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. beta-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. beta-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.

Journal Article↗
PubMedDate not supplied

Parallel functional and immunological detection of human multidrug resistance proteins, P-glycoprotein and MRP1.

The proper assessment of the expression and drug extrusion activity of multidrug resistance proteins in various tumor cells is a challenging clinical laboratory problem. Recently, we have introduced a fluorescent dye (calcein) accumulation assay for the estimation of the functional expression of both P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP1). Since both MDR1 and MRP1 decrease the intracellular accumulation of the fluorescent free calcein, by applying appropriate inhibitors of MDR1 and MRP1, the transport activity of these proteins could be quantitatively and selectively estimated in fluorometry or flow-cytometry assays. In the present work single-cell fluorescence digital imaging has been applied to characterize the kinetics and inhibitor-sensitivity of calcein accumulation in a mixture of HL60 MRP1 and NIH 3T3 MDR1 cells. Subsequent immunofluorescence labeling was performed by the anti-MDR1 monoclonal antibody (mAb) UIC2 in the same cell population. We report that the double labeling approach, based on the single cell calcein accumulation assay and an immunofluorescence detection, provides good sensitivity and selectivity for the simultaneous functional and immunological detection of cellular MDR1 and MRP1.

3T3 Cells↗