Selection of resistant acute myeloid leukemia SKM-1 and MOLM-13 cells by vincristine-, mitoxantrone- and lenalidomide-induced upregulation of P-glycoprotein activity and downregulation of CD33 cell surface exposure.

Bone marrow cells and peripheral blood mononuclear cells obtained from both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients contain upregulated levels of cell surface antigen CD33 compared with healthy controls. This difference enables the use of humanized anti-CD33 antibody conjugated to cytotoxic agents for CD33 targeted immunotherapy. However, the expression of the membrane-bound drug transporter P-glycoprotein (P-gp) has been shown to be critical for resistance against the cytotoxicity of a humanized anti-CD33 antibody conjugated to maytansine-derivative DM4. The aim of the present study was to examine whether the expression of P-gp in AML cell lines is associated with changes in CD33 expression. For this purpose, we established drug resistant variants of SKM-1 and MOLM-13 AML cell lines via the selection of parental cells for resistance to vincristine, mitoxantrone and lenalidomide. All three substances induced a multidrug resistance (MDR) phenotype in SKM-1 cells associated with strong upregulation of P-gp and downregulation of CD33. However, in MOLM-13 cells, the upregulation of P-gp and downregulation of CD33 were present only in cells selected for resistance to vincristine and mitoxantrone but not lenalidomide. Inverse expression of P-gp and CD33 were observed in all resistant variants of SKM-1 and MOLM-13 cells. The MDR phenotype of resistant variants of SKM-1 and MOLM-13 cells was associated with alterations in apoptotic regulatory proteins and downregulation of the multidrug resistance associated protein 1 and breast cancer resistance protein.

Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity.

L1210/VCR cells, which express an abundant amount of P-glycoprotein (P-gp), were found to be resistant to thapsigargin--an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In the current paper, we have studied the possible differences among L1210 and L1210/VCR cells in expression of endoplasmic reticulum proteins involved in the regulation of calcium homeostasis and calcium-dependent processes. Amounts of mRNA encoding both calcium release channels (ryanodine receptor channels--RyR and IP3-receptor channels--IP3R) were found to be at similar levels in sensitive and resistant cells. However, mRNAs encoding IP3R1 or 2 were decreased in resistant cells cultivated in the presence of VCR (1.08 micromol/l), while mRNA encoding RyR remained unchanged. The amount of mRNA for SERCA2 was decreased in resistant cells when compared with sensitive cells. This decrease was more pronounced when resistant cells were cultivated in the presence of vincristine (VCR). Calnexin was found to be less expressed at the protein level in resistant as in sensitive cells. The level of mRNA encoding calnexin was decreased only when resistant cells were cultivated in the presence of VCR. Calnexin was found to be associated with immature P-gp in resistant cells. Thus, differences exist between sensitive and resistant cells in the expression of endoplasmic reticulum proteins involved in the control of intracellular calcium homeostasis or calcium-dependent processes. These changes may be at least partially responsible for the lack of sensitivity of resistant cells to thapsigargin.

Modulation of expression of the sigma receptors in the heart of rat and mouse in normal and pathological conditions.

The aim of the present work was to study the effect of various stressors (hypoxia, cold, immobilization) on the gene expression of sigma receptors in the left ventricles of rat heart. We have clearly shown that gene expression of sigma receptors is upregulated by strong stress stimuli, such as immobilization and/or hypoxia. Nevertheless, cold as a milder stressor has no effect on sigma receptor's mRNA levels. Signalling cascade of sigma receptors is dependent on IP(3) receptors, since silencing of both, type 1 and 2 IP(3) receptors resulted in decreased mRNA levels of sigma receptors. Physiological relevance of sigma receptors in the heart is not clear yet. Nevertheless, based on the already published data we can assume that sigma receptors might participate in contractile responses in cardiomyocytes.

Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress.

AIM: The Na(+)/Ca(2+) exchanger (NCX) is a major Ca(2+) extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. METHODS: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemia-reperfusion to compare response of control and stressed hearts. RESULTS: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. CONCLUSIONS: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.

Structural differences between sensitive and resistant L1210 cells.

The main structural differences between sensitive L1210 mouse leukaemic cells and their multidrug resistant counterpart, obtained by adaptation of the parental cell line to vincristine (VCR), concern the size and shape of the cells, their surface properties and changes in organelles involved in proteosynthesis and transport of substances. The resistant cells are larger with higher density of microvilli. In light and electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in L1210 cells. This difference in cell aggregation suggests different surface properties which could be visualised by decreased staining of L1210/VCR cell surface coat (glycocalyx) with a polycationic dye ruthenium red. A decrease in surface to volume ratio as a consequence of increased cell size in resistant cells is compensated by proliferation of villi and cytoplasmic protrusions of the cell surface. L1210/VCR cells were further distinguished by higher amount of euchromatin, increase in density of rough endoplasmic reticulum, more developed Golgi apparatus and aggregation of free ribosomes into tetrameric and pentameric polyribosomes. These structural changes may be interpreted as a sign of increase in proteosynthesis and transport of substances.

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

Proteins released from liver after ischaemia induced an elevation of heart resistance against ischaemia-reperfusion injury: 2. Beneficial effect of liver ischaemia in situ.

We have shown earlier that proteins released from the heart during preconditioning may protect non-preconditioned heart during sustained ischaemia, similarly as preconditioning itself. In other our experiments we have documented that also proteins released from isolated rat liver during reperfusion after global ischaemia performed a protective effect on isolated rat heart against ischaemia-reperfusion injury. In the current study we examined the effect of liver ischaemia in situ on resistance of rat heart to ischaemia and reperfusion injury. Wistar rats (male) were subjected to liver ischaemia maintained by occlusion of portal vein and hepatic artery for 20 min, followed with 30-min reperfusion after reopening of both vessels. Then the hearts were isolated and perfused according to Langendorf. Hearts, after initial stabilisation (15 min), were subjected to 20-min ischaemia and 30-min reperfusion. During reperfusion, the haemodynamic parameters of hearts were measured. The protein pattern of high soluble fraction (HS fraction) isolated from rat blood by precipitation with ammonium sulphate was detected by SDS-PAGE. Our results showed improved parameters of pressure and contractility in the group after liver ischaemia (ischaemic group), presented by decreased diastolic pressure and increased LVDP((S-D)) in comparison with levels of these parameters in the control group. We also observed improved heart contraction-relaxation cycles parameters (dP/dt)(max) and (dP/dt)(min) in ischaemic group as compared with the control group. On the other hand, there were no significant differences in heart rate and coronary flow between both experimental groups. SDS-PAGE showed changed protein pattern in HS fraction, particularly the levels of several low molecular weight proteins increased. We conclude that liver ischaemia induced a higher resistance of heart against ischaemia-reperfusion injury. We propose that release of some cardioprotective proteins present in HS fraction can also contribute to this cardioprotection.

Hypoxia increases cell death in multidrug-resistant leukemia cells. Differences in viability and ultrastructure between sensitive and multidrug-resistant L1210 mouse leukemic cells under hypoxia.

The comparative study of sensitive and multidrug-resistant L1210 cells under 24 hours of hypoxia (2% O2 and 5% CO2 at 37 degrees C) was done to see if differences in energetic metabolism between both cell lines are paralleled by differences in cellular morphology. During the dye exclusion assay the viability of sensitive cells was about 70 to 90%, whereas only 30 to 50% of resistant cells were viable. Electron microscopic study of sensitive and resistant L1210 cells under hypoxia has shown cells of different ultrastructural appearance in both cell lines. Cells with necrotic changes (swollen mitochondria, lysed cells) prevailed in resistant cells. The highest incidence of cells with normal or slightly dense mitochondria was found among the sensitive L1210 cells. Additionally, cells with pyknotic nuclei, shrunken cytoplasm and dense mitochondria, reminiscent of apoptosis, could be found sporadically, especially in the sensitive L1210 cell line. These results are in agreement with flow cytometry measurements: in resistant cells the number of necrotic cells was on the average 2.3 times higher than in sensitive cells. Ultrastructural differences and differences in the numbers of necrotic cells as measured by flow cytometry between sensitive and resistant L1210 cells under hypoxia are consistent with differences in energetic metabolism between these cell lines, as described in earlier studies, and document an increased cell death in the resistant L1210 cell line.

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents.

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.

Ten isoenzymes of xyloglucan endotransglycosylase from plant cell walls select and cleave the donor substrate stochastically.

To map the preferred cleavage sites of xyloglucan endotransglycosylases (XETs; EC 2.4.1.207) along the donor substrate chain, we incubated the enzymes with tamarind (Tamarindus indica) xyloglucan (donor substrate; approximately 205 kDa; 21 microM) plus the nonasaccharide [(3)H]XLLGol (Gal(2).Xyl(3).Glc(3). [(3)H]glucitol; acceptor substrate; 0.6 microM). After short incubation times, to minimize multiple cleavages, the size of the (3)H-labelled transglycosylation products (determined by gel-permeation chromatography) indicated the positions of the cleavage sites relative to the non-reducing terminus of the donor. There was very little difference between the size profiles of the products formed by any of ten XETs tested [one native XET purified from cauliflower (Brassica oleracea) florets, four native XET isoenzymes purified from etiolated mung-bean (Phaseolus aureus) shoots, native XETs purified from lentil (Lens culinaris) and nasturtium (Tropaeolum majus) seeds, and three insect-cell-produced thale-cress (Arabidopsis thaliana) XETs (EXGT, TCH4 and MERI-5)]. All such product profiles showed a good fit to a model in which the enzyme chooses its donor substrate independently of size and attacks it, once only, at a randomly selected cleavage site. The results therefore do not support the hypothesis that different XET isoenzymes are adapted to produce longer or shorter products such as might favour either the efficient integration of new xyloglucan into the cell wall or the re-structuring of old xyloglucan within an expanding wall.

Ping-pong character of nasturtium-seed xyloglucan endotransglycosylase (XET) reaction.

Plant xyloglucan endotransglycosylase (XET, EC 2.4.1.207) degrades its substrate by a transglycosylation mechanism while endo-cleaving xyloglucan (XG) molecules at their beta-1,4-linked polyglucosyl main chain and transferring the newly generated reducing chain ends to hydroxyls at C-4 of non-reducing glucosyl ends of the main chains of other XG molecules or of low-Mr XG-fragments (OS). Kinetic data obtained with purified nasturtium seed (Tropaeolum majus, L.) XET while using high-Mr xyloglucan and 3H-labeled XGOS alditols (DP 7-9) as substrates could be best fitted to the model for Ping-Pong Bi Bi reaction mechanism. Such mechanism is typical for transglycosylases operating with retention of the anomeric configuration of the formed glycosidic bond and involving the formation of a covalent glycosyl-enzyme reaction intermediate.

Purification of xyloglucan endotransglycosylase based on affinity sorption of the active glycosyl-enzyme intermediate complex to cellulose.

Xyloglucan endotransglycosylase (XET) catalyzes the cleavage of xyloglucan (XG) molecules by a transglycosylation mechanism involving two steps: (a) endocleavage of the beta-(1,4)-linked polyglucosyl backbone of the xyloglucan molecule with formation of a glycosyl-enzyme intermediate; (b) transfer of the glycosyl residue from the intermediate to the C-4 position of the nonreducing end glucosyl unit of another molecule of XG or an XG-derived oligosaccharide with liberation of the enzyme (Z. Sulová et al., 1998, Biochem. J. 330, 1475-1480). The formation of a relatively stable active complex of XET with XG and the tendency of xyloglucan to bind tightly via hydrogen bonds to cellulose were exploited in the present method of purification of XET. Crude extracts from nasturtium (Tropaeolum majus) cotyledons and other plant sources containing the enzyme were mixed with XG in order to form the XET:XG complex, which was applied onto cellulose. Unadsorbed proteins were removed by washing and the XET was released from the adsorbed XET:XG complex by transglycosylation of its glycosyl moiety to added XG-derived oligosaccharides. The described procedure resulted in an over 100-fold increase in specific activity of XET in a single step. Further purification of the enzyme to homogeneity was achieved by gel-permeation chromatography on Bio-Gel P30. Similar procedure could be used for purification of XET from other plant sources, such as lentil (Lens culinaris) seeds, pea (Pisum sativum) epicotyls, and supernatant of suspension-cultured Catharanthus roseus cells.

Ca(2+)-induced inhibition of sodium pump: effects on energetic metabolism of mouse diaphragm tissue.

Tissues of mouse diaphragms were incubated in Liley solution containing 2, 4, 6 and 10 mmol/l calcium. When diaphragm tissue was incubated in 10 mmol/l calcium, an increase of intracellular calcium concentration from 314 +/- 28 to 637 +/- 26 nmol/l was estimated by fluorescent Ca2+ indicator Fura-2/AM. Moreover, incubation of the tissue in 10 mmol/l Ca2+ led to complete inhibition of electrogenic activity of the sodium pump, as measured by intracellular microelectrodes in a single muscle cell. This inhibition was fully reversible after 5 min washing with Liley solution containing 2 mmol/l CaCl2. The Ca(2+)-induced blocking effect on electrogenic activity of the sodium pump was accompanied by inhibition of glucose incorporation into the muscle tissue. Calcium at concentrations of 6 and 10 mmol/l in bath medium significantly inhibited both CO2 production and O2 consumption. A continual decrease of respiration (CO2/O2) quotient was observed under increasing concentrations of calcium. Moreover, an exponential decrease of ATP tissue levels was observed at increasing concentrations of calcium in the bath medium. On the other hand, massive acceleration of anaerobic glycolysis induced by incubation of the tissue in a medium containing high calcium concentration is improbable. This may be deduced from the fact that only about an 50% increase of lactate content in muscle tissue was observed when diaphragms were incubated for 30 min in medium containing calcium ions at 6 and 10 mmol/l as compared with the control tissue incubated for the same time in the medium containing 2 mmol/l CaCl2. In conclusion it could be stressed that increase of Ca2+ concentration in bath medium induced in diaphragm muscle tissue an elevation of intracellular Ca2+ concentration accompanied by a depression of sodium pump electrogenic activity and a depression of energy metabolism. These changes may be involved in pathology of muscle tissue during the Ca2+ overload.

Kinetic evidence of the existence of a stable enzyme-glycosyl intermediary complex in the reaction catalyzed by endotransglycosylase.

Xyloglucan-endotransglycosylase (XET) is an enzyme involved in the metabolism of xyloglucan (XG) in plant cell walls and seeds. This enzyme acts both as a hydrolase and as a transglycosylase by transferring the fragments of xyloglucan molecules to other XG molecules or xyloglucan-derived oligosaccharides (XGOs). In this work, we studied the kinetics of interaction between XET and XG. The equilibrium in the reaction of XG degradation by XET was found to depend on the initial enzyme concentration and the availability of suitable glycosyl acceptors. After reaching the equilibrium, the addition to the reaction mixture of XET or XGOs caused further degradation of XG, and a new equilibrium with a higher degree of XG depolymerization was established. These results indicated that in the course of XG depolymerization, the enzyme is bound in a relatively stable, temporarily inactive enzyme-glycosyl complex and this complex is decomposed by transferring its glycosyl moiety to suitable oligosaccharide acceptor. Mouse polyclonal antibody against XET linked to AffiGel 10 (Affi-Ab) adsorbed both XET and XET-XG complex but not [3H]XG alone. XET immobilized onto Affi-Ab was able to bind [3H]XG and catalyze transglycosylation in presence of XGOs.

Ca(2+)-induced inhibition of sodium pump: noncompetitive inhibition in respect of magnesium and sodium cations.

Calcium inhibits the activity of the (Na+/K+)-ATPase from dog kidney in a dose-dependent manner. Other 2A group cations of the periodic table such as Sr2+ and Ba2+ were able to inhibit the ATPase activity but to a lesser degree. Any considerable competition between Ca2+ (Ba2+, Sr2+) ions and magnesium or sodium ions could not be detected using enzyme kinetic analysis. Thus, the above three inhibitory acting ions depress the ATPase activity of sodium pump by interaction with loci distant from the sodium and potassium binding sites. This suggests that the (Na+/K+)-ATPase molecule contains an inhibitory acting binding site for calcium. This putative binding site could recognize magnesium ions as well as calcium, strontium and barium ions. The specificity of the binding site may describe herein be secured by a structure complementary to the coordination structure of Ca2+, Ba2+ and Sr2+ ions characterized by coordination number 8. Mg2+ ions can form coordination structure with a maximum coordination number 6, and do not interact specifically with this binding site.

Xyloglucan endotransglycosylase: evidence for the existence of a relatively stable glycosyl-enzyme intermediate.

Xyloglucan endotransglycosylases (XETs) catalyse the breakdown of xyloglucan molecules predominantly by transglycosylation. In this process, fragments of cleaved polysaccharide are preferentially transferred to other xyloglucan molecules or their oligosaccharide subunits, with overall retention of the anomeric configuration of the glycosidic bond. In accordance with the theory, we propose that the cleavage and re-formation of the glycosidic bond in xyloglucan involves the formation of a glycosyl-enzyme intermediate which decomposes by transfer of the glycosyl moiety to a suitable carbohydrate acceptor. XETs from nasturtium seed cotyledons, mung bean hypocotyls and cauliflower florets interacted with xyloglucan to form complexes of high Mr as judged by gel-permeation chromatography. The nasturtium enzyme also showed evidence of XET-xyloglucan complex-formation according to anion-exchange chromatography and adsorption of the complex to filter paper on the basis of affinity of its xyloglucan moiety for cellulose. The XET-xyloglucan complex was stable in water, 6 M urea and acidic and alkaline buffers (pH 2.5-9.5), but readily decomposed by transferring its glycosyl moiety to xyloglucan-derived oligosaccharides or by incubation with the strong nucleophile imidazole at pH 3.8-9.6. These results strongly support the assumption that XET forms a relatively stable covalently linked glycosyl-enzyme intermediate.

A colorimetric assay for xyloglucan-endotransglycosylase from germinating seeds.

One of the key enzymes involved in the breakdown of reserve xyloglucan in seeds of some dicotyledonous plants during germination is the specific endo-beta-(1,4)-glucanase. The enzyme operates predominantly by a transglycosylic mechanism, i.e., by random splitting the beta-(1,4)-linked polyglucose backbone of xyloglucan molecules and rejoining the newly created reducing ends by beta-(1,4) glycosidic bonds to nonreducing ends of other xyloglucan molecules or xyloglucan subunit oligosaccharides. For this reason, the enzyme is regarded primarily as xyloglucan-endotransglycosylase (XET). Since almost no net formation of reducing ends occurs in the course of transglycosylation, the conventional reductometric methods used for the assessment of glycanase activities are not applicable for detection and determination of XET activity. The described colorimetric assay is based on the property of xyloglucan-derived subunit oligosaccharides (DP 5-10) to stimulate selectively the breakdown of xyloglucan by endotransglycosylation while serving as additional glycosyl acceptors. The depolymerization of xyloglucan in the course of reaction is followed colorimetrically by measuring the disappearance of the blue--green-colored iodine:xyloglucan complex. The transglycosylase activity is calculated as the difference of activities measured in the presence of stimulating xyloglucan-derived oligosaccharides and in their absence. The advantages of the described colorimetric method include its low cost, simplicity, speed, and the possibility to analyze multiple samples simultaneously.

Cleavage of xyloglucan by nasturtium seed xyloglucanase and transglycosylation to xyloglucan subunit oligosaccharides.

Oligosaccharide subunits were prepared from xyloglucan (XG) by partial hydrolysis with cellulase and added back at micro- to millimolar concentrations to XG in the presence of nasturtium seed xyloglucanase (XG-ase). The oligosaccharides (0.2 mM) stimulated the capacity of this XG-ase to reduce the viscosity of XG solutions by 10- to 20-fold. Purification and fractionation of seed XG-ase activity by gel permeation fast protein liquid chromatography produced a single peak that was much more active in the presence than absence of added XG oligosaccharide. [14C]Fucose-labeled XG nonasaccharide was synthesized by pea fucosyltransferase and shown to be incorporated into polymeric XG in the presence of seed XG-ase without the net production of new reducing chain ends, even while the loss of XG viscosity and XG depolymerization were enhanced. It is concluded that in vitro seed XG-ase can transfer cleavage products of XG to XG oligosaccharides via endotransglycosylation reactions, thereby reducing XG M(r) without hydrolysis. Since this is the only XG-cleaving enzyme that develops in nasturtium seeds during germination, it may be that its transglycosylase and hydrolase capacities are both necessary to account for the rapid and complete depolymerization of XG that takes place.

Adaptation of mouse leukemia cells L1210 to vincristine. Evidence for expression of P-glycoprotein.

A vincristine resistant cell line was obtained from mouse leukemia cells L1210 by long-term adaptation in a medium with stepwise increasing concentrations of vincristine. By Western blotting using monoclonal antibody C219, positive signal on the presence of P-glycoprotein was observed in the resistant cells. Moreover, hybridization of mRNA from vincristine resistant cells with radiolabeled MDR1 cDNA probe gave evidence about the expression of MDR1 gene. The observed resistance may be depressed by application of "chemosensitizers" such as (1) calcium entry blockers (verapamil and nifedipine); (2) neuroleptics (trifluorperasine) and (3) local anesthetics (lidocaine) directly to the grow medium. Any significant effect in O2 consumption as well as incorporation of [U-14C]-glucose by the sensitive or resistant cells was not detected in the absence of vincristine. Presence of vincristine induced increasing velocity of O2 consumption by resistant cells from 2.5 +/- 0.3 to 3.3 +/- 0.2 microliters/min.10(6) cells, and, on the other hand, decreasing O2 consumption by sensitive cells from 2.3 +/- 0.2 to 1.7 +/- 0.1 ml/min.10(6) cells. The presence of vincristine induced less potent decrease in glucose incorporation by resistant cells in comparison with values which were observed in sensitive cells.