Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX transport.
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We have recently determined that human multidrug resistance protein (MRP) 3, which confers resistance to certain natural product agents and methotrexate (MTX), is competent in the MgATP-energized transport of MTX and the monoanionic bile constituent glycocholate as well as several glutathione and glucuronate conjugates. Of these capabilities, the facility of MRP3 in conferring resistance to and mediating the transport of MTX is of particular interest because it raises the possibility that this pump is a component of the previously described cellular efflux system for this antimetabolite. However, if this is to be the case, a critical property of cellular MTX efflux that must be addressed is its ability to mediate the export of MTX but not that of its intracellular polyglutamylated derivatives. Here we examine the role of MRP3 in these and related processes by determining the selectivity of this transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, we show that MRP3 is not only active in the transport of MTX but is also active in the transport the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin) and that polyglutamylation of MTX abolishes transport. Both FA and leucovorin are subject to high-capacity (V(max(FA)), 1.71 +/- 0.05 nmol/mg/min; V(max(leucovorin)), 3.63 +/- 1.20 nmol/mg/min), low-affinity (K(m(FA)), 1.96 +/- 0.13 mM; K(m(leucovorin)), 1.74 +/- 0.65 mM) transport by MRP3. Addition of a single glutamyl residue to MTX is sufficient to diminish transport by >95%. We also show that polyglutamylation similarly affects the capacity of MRP1 to transport MTX and that physiological folates are also subject to MgATP-stimulated transport by MRP1. On the basis of the capacity to transport MTX but not MTX-Glu(2), it is concluded that MRP3 and MRP1 represent components of the previously described cellular efflux system for MTX. The capacity of MRP3 to transport folates indicates that it may reduce intracellular levels of these compounds and thereby indirectly influence antifolate cytotoxicity, and it also implies that this pump may play a role in the response to chemotherapeutic regimens in which leucovorin is a component. (+info)
Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.
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An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase. (+info)
The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells.
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1. The effects of unfractionated heparin (UH) and a selectively O-desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated. 2. For comparison, the effects of poly-L-glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two different molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied. 3. UH (50 - 1000 u ml(-1)) significantly (P<0.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL-1beta (100 u ml(-1)), TNF-alpha (1000 u ml(-1)) or LPS (100 microg ml(-1)), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such effects on adhesion occurred with limited influence on expression of relevant endothelial adhesion molecules (ICAM-1 and VCAM-1). 4. UH (100 - 1000 u ml(-1)), when added to prestimulated HUVECs, significantly (P<0.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any effect on adhesion molecule expression. In contrast, the opposite effect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell specific. 5. The effects of UH on HPBMNC adhesion were shared by the non-anticoagulant ODSH (600 - 6000 microg ml(-1)) but not by sulphated dextrans or PGA (300 - 6000 microg ml(-1)). 6. Heparin affects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, effects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics. (+info)
Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant-parasitic nematode Meloidogyne artiellia.
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Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages. (+info)
JEAP, a novel component of tight junctions in exocrine cells.
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Tight junctions (TJs) consist of transmembrane proteins and many peripheral membrane proteins. To further characterize the molecular organization of TJs, we attempted here to screen for novel TJ proteins by the fluorescence localization-based expression cloning method. We identified a novel peripheral membrane protein at TJs and named it junction-enriched and -associated protein (JEAP). JEAP consists of 882 amino acids with a calculated molecular weight of 98,444. JEAP contained a polyglutamic acid repeat at the N-terminal region, a coiled-coil domain at the middle region, and a consensus motif for binding to PDZ domains at the C-terminal region. Exogenously expressed JEAP co-localized with ZO-1 and occludin at TJs in polarized Madin-Darby canine kidney cells, but not with claudin-1, JAM, or ZO-1 in L cells. Endogenous JEAP localized at TJs of exocrine cells including pancreas, submandibular gland, lacrimal gland, parotid gland, and sublingual gland, but not at TJs of epithelial cells of small intestine or endothelial cells of blood vessels. The present results indicate that JEAP is a novel component of TJs, which is specifically expressed in exocrine cells. (+info)
Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production.
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The genes required for gamma-polyglutamic acid (PGA) production were cloned from Bacillus subtilis IFO16449, a strain isolated from fermented soybeans. There were four open reading frames in the cloned 4.2-kb DNA fragment, and they were almost identical to those in the ywsC and ywtABC genes of B. subtlis 168. Northern blot analysis showed that the four genes constitute an operon. Three genes, ywsC, ywtA, and ywtB, were disrupted to determine which gene plays a central role in PGA biosynthesis. No PGA was produced in Delta ywsC and Delta ywtA strains, indicating that both of these genes are essential for PGA production. To clarify the function of the YwsC protein, histidine-tagged YwsC (YwsC-His) was produced in the Delta ywsC strain and purified from the lysozyme-treated lysate of the transformant by Ni-nitrilotriacetic acid affinity chromatography. Western blot analysis revealed that the YwsC-His protein consists of two subunits, the 44-kDa and 33-kDa proteins, which are encoded by in-phase overlapping in the ywsC gene. (14)C-labeled PGA was synthesized by the purified proteins from L-[(14)C]-glutamate in the presence of ATP and MnCl(2), through an acylphosphate intermediate, indicating that the ywsC gene encodes PGA synthetase (EC 6.3.2), a crucial enzyme in PGA biosynthesis. (+info)
Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.
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We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA. (+info)
Superior therapeutic profile of poly-L-glutamic acid-paclitaxel copolymer compared with taxol in xenogeneic compartmental models of human ovarian carcinoma.
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Previous preclinical studies with ectopic tumor models have demonstrated remarkable improvements in the therapeutic profile of paclitaxel, formulated as a copolymer with poly-L-glutamic acid, compared with paclitaxel in the clinical formulation, Taxol. In this study, we evaluated these formulations in two human ovarian carcinoma xenograft models, NMP-1 and HEY, in nude mice. i.p. implantation in female nude mice of either cell line gave rise to progressive disease within the peritoneum, in the parenchyma of visceral organs, and eventually at extraperitoneal sites; the resultant, increasing morbidity then required host sacrifice. i.p. administration of multiple-dose Taxol at its maximum tolerated dose 1 week after tumor implantation afforded minimal or no increased survival compared with controls in either model. Consistent with the predictions of drug copolymer behavior, paclitaxel, as the poly-L-glutamic acid-paclitaxel copolymer, displayed much less toxicity than Taxol in these hosts. When evaluated for antitumor efficacy in both the Taxol-resistant NMP-1 and HEY models, significant improvement in survival, and even some cures, were observed after a single i.p. treatment with this copolymer. The observed antitumor response correlated with histopathological analysis of peritoneal and extraperitoneal tumor burden in comparing control HEY mice sacrificed near the onset of morbidity with mice receiving paclitaxel copolymer. We conclude that both the i.p. NMP-1 and HEY models have significant value in establishing the efficacy of candidate agents, which might address Taxol-resistant human ovarian carcinoma. Furthermore, the poly-L-glutamic acid-paclitaxel copolymer has a superior therapeutic profile in these Taxol-resistant compartmental models. (+info)