Membrane-associated Transport Research Articles

EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone

BackgroundProstate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells.ResultsThe studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells.ConclusionThis study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by blast searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified solute-binding protein (designated ArtJ) was demonstrated to bind L-arginine with high affinity (Kd=0.39+/-0.06 microM). Competition experiments revealed only partial inhibition by excess L-lysine (38 %) and L-ornithine (46 %), while no inhibition was observed with L-histidine or other amino acids tested. The membrane-associated transport complex, composed of a permease (designated ArtM) and an ATPase component (designated ArtP), was solubilized from E. coli membranes by decanoylsucrose and purified by metal-affinity chromatography. The ArtMP complex, when incorporated into liposomes formed from a crude extract of G. stearothermophilus lipids, displayed ATPase activity in the presence of ArtJ only. Addition of L-arginine further stimulated the activity twofold. ATP hydrolysis was optimal at 60 degrees C and sensitive to the specific inhibitor vanadate. Analysis of kinetic parameters revealed a maximal velocity of ATP hydrolysis of 0.71 micromol Pi min(-1) (mg protein)(-1) and a Km(ATP) of 1.59 mM. Together, these results identify the ArtJMP complex as a high-affinity arginine ABC transporter.

Identification and characterisation of a multidrug resistance-related protein mRNA in the blue mussel Mytilus edulis

Membrane-associated transport proteins were discovered in the 1970s in mammals and were considered to be expressed in response to chemotherapy during cancer treatment. Prominent members of this class of proteins are multidrug resistance-related or -associated proteins (MRPs). Besides their expression in cancer cells, MRPs are ubiquitously expressed in normal tissues and are active transporters of reduced glutathione, glucuronate and organic anions of toxicological re- levance, either conjugated or unconjugated with sulphate. MRPs may also provide aquatic organisms with resistance to chemicals in a polluted environment by binding xenobiotics and excreting them from cells in an energy-dependent manner. The present study investigated expression of MRPs as part of the multixenobiotic resistance (MXR) system in the blue mussel Mytilus edulis. We isolated and characterised 2 putative mrp cDNA fragments, mrp1 and mrp2, which showed 50 to 70% homo- logy on the protein level with MRPs of other species. The mrp1 fragment could not be linked with any mRNA in Northern blots of M. edulis tissues, whereas the mrp2 fragment hybridised with an mRNA of approximately 4.6 kb. Mrp2 showed tissue-specific expression patterns. Highest expression was found in digestive gland and gill tissue. Its expression could be induced 2-fold by the model carcino- gen 2-acetylaminofluorene (AAF), whereas mrp1 expression was unaffected. The cDNA fragment of the inducible form was then integrated into a multiplex PCR system for analysis of multixenobiotic resistance in the blue mussel, in concert with other detoxification and biotransformation genes.

A null mutation in H-FABP only partially inhibits skeletal muscle fatty acid metabolism.

The low-molecular-mass, cytosolic heart-type fatty acid-binding protein (H-FABP) is thought to be required for shuttling FA through the cytosol. Therefore, we examined the effects of an H-FABP-null mutation on FA and carbohydrate metabolism in isolated soleus muscle at rest and during a period of increased metabolic demand (30-min contraction). There were lower concentrations of creatine phosphate (-41%), ATP (-22%), glycogen (-34%), and lactate (-31%) (P < 0.05) in H-FABP-null soleus muscles, but no differences in citrate synthase and beta-3-hydroxyacyl-CoA dehydrogenase activities or in the intramuscular triacylglycerol (TAG) depots. There was a 43% increase in subsarcolemmal mitochondria in H-FABP-null solei. FA transport was reduced by 30% despite normal content of sarcolemmal long-chain fatty acid transporters fatty acid translocase/CD36 and plasma membrane-associated FABP transport proteins. Compared with wild-type soleus muscles, the H-FABP-null muscles at rest hydrolyzed less TAG (-22%), esterified less TAG (-49%), and oxidized less palmitate (-71%). The H-FABP-null soleus muscles retained a substantial capacity to increase FA metabolism during contraction (TAG esterification by +72%, CO2 production by +120%), although these rates remained lower (TAG esterification -26% and CO2 production -64%) than in contracting wild-type soleus muscles. Glycogen utilization during 30 min of contraction did not differ, whereas glucose oxidation was lower at rest (-24%) and during contraction (-32%) in H-FABP-null solei. Although these studies demonstrate that the absence of H-FABP alters rates of FA metabolism, it is also apparent that glucose oxidation is downregulated. The substantial increase in FA metabolism in contracting H-FABP-null muscle may indicate that other FABPs are also present, a possibility that we were not able to completely eliminate.

A novel membrane-bound respiratory complex from Desulfovibrio desulfuricans ATCC 27774

In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron–sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and −20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe–4S] 1+/0 center and at least four [4Fe–4S] 2+/1+ centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron–sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.

Novel strategies for overcoming multidrug resistance in cancer.

The problems of why metastatic cancers develop pleiotropic resistance to all available therapies, and how this might be countered, are the most pressing in cancer chemotherapy. It is likely that such resistance involves a combination of mechanisms including changes in drug transport/drug targets, reduction in the degree of drug-induced apoptosis/cell loss, and increased rate of tumour repopulation following therapy. Current research must consider not only which mechanisms contribute, eventually relating this to individual patients with cancer, but also what strategies might be utilised to counter each of the important resistance mechanisms. A considerable amount of work has been devoted to the development of inhibitors of membrane-associated transport proteins such as P-glycoprotein, which mediate drug efflux. This work is now being complemented by approaches that target cell death pathways such as those mediated by release of mitochondrial proteins and by activation of surface receptors such as Fas. Rapid progress has been made in developing small-molecular-weight drugs that influence the rate of apoptosis, for instance by binding to the bcl-2 family of proteins regulating mitochondrial permeability. Antisense approaches aimed at reducing bcl-2 expression, and thus increasing the rate of cell death, are also showing promise. Modification of repopulation kinetics provides a further approach but has not received as much attention as other aspects of tumour resistance. New therapeutic approaches will have to be complemented by improved diagnostic tests to evaluate the contributions of different resistance mechanisms in individual patients with cancer.

Characteristics of transport of fluoresceinated methotrexate in rat small intestine

The small intestinal damage induced by the methotrexate (MTX) treatment results in malabsorption and diarrhea. The fluoresceinated methotrexate (F-MTX) may possibly be useful to study such effects of MTX on the small intestine. The purpose of this study is to characterize the transport of F-MTX in the small intestine in order to use it as a membrane transport and cellular marker of MTX. The transport of F-MTX in the rat small intestine (jejunum) was examined in the in vitro everted segments of the intestine. The uptake was pH-dependent and showed a maximal effect at pH 6.0, which was the same as the results of MTX previously reported. Further, it was temperature-dependent and was inhibited by metabolic inhibitors, dinitrophenol and sodium azide, and by MTX. The transport kinetics at pH 6.0 in the mucosal solution and at pH 7.4 in the serosal solution was saturable with K m of 0.48 ± 0.23 μM and V max of 0.66 ± 0.24 pmol/cm/min and in addition, the passive diffusion was observed there. These results suggested that the transport of F-MTX was energy-dependent and was mediated by the same transporter as that of MTX, although, in addition to it, other transport mechanism might contribute to the F-MTX transport. Therefore F-MTX will be of great use to investigate the MTX transport system in the normal and diseased states of small intestine, using various fluorescence techniques like visualization of membrane-associated transport proteins.

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56 + lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500–1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56 + lymphocytes ( ic 50 = 1.2 nM) or CD56 + lymphocytes in whole blood ( ic 50 = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.

Expression and activity of NAD(P)H:quinone oxidoreductase (NMO1) in human osteoblastic cells

NAD(P)H:quinone oxidoreductase (NMO1; EC 1.6.99.2), also called DT-diaphorase, is involved in the reduction of coenzyme Q, an important cellular lipophilic antioxidant that can function as an intermediate electron carrier in plasma membrane-associated electron transport regulating cell growth. We examined the ability of normal human trabecular osteoblastic cells to express NAD(P)H:quinone oxidoreductase (NMO1) and studied its modulation during cell proliferation and growth arrest. We found that confluent primary human trabecular osteoblastic cells derived from healthy individuals constitutively express NMO1 activity, measured using 2-6 dichlorophenol indophenol (DCI) or menadione (vitamin K3) as substrate, and NADPH or NADH as electron donor. We also found that NMO1 activity was related to osteoblastic cell growth. NMO1 activity increased with osteoblastic cell density. Confluent growth-arrested cells expressed an eightfold higher NMO1 specific activity than proliferative human osteoblastic cells. Reverse-transcription polymerase chain reaction analysis showed that NMO1 mRNA levels did not differ in growth-arrested confluent cell and growing cells, suggesting that the increased NMO1 activity with cell density was due to posttranslational events. Harvesting and replating the cells at low density resulted in a 93.4% loss of NMO1 enzymatic activity. Removal of serum from high-density growth-arrested cells resulted in a 48.5% decrease in NMO1 activity. NMO1 activity does not appear to be related to induction of osteoblast differentiation because treatment with the differentiating agent 1,25(OH) 2 vitamin D 3 had no effect on NMO1 activity. The finding that human osteoblastic cells express NMO1 constitutively and that NMO1 activity increases with density-dependent growth inhibition suggest a role for NAD(P)H:quinone oxidoreductase in the control of growth arrest in normal human osteoblastic cells.

Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of Pseudomonas aeruginosa

The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [ 14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

The prognostic significance of membrane transport-associated multidrug resistance (MDR) proteins in leukemia.

A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents. There are several ways for cancer cells to develop resistance or defense mechanisms against cytotoxic drugs. This review paper will focus on membrane transport-associated multidrug resistance (MDR). The proteins involved, P-glycoprotein (P-gp), MRP1 and LRP/MVP, share the ability to act as drug transport proteins. Following upregulation of the mdr-1 gene, the energy-dependent transmembrane P-gp overexpression results in diminished intracellular concentrations of anthracyclins, vinca-alkaloids and epipodophyllotoxins. The other transmembrane protein, MRP1, also has intracellular epitopes which are involved in intracellular redistribution and sequestration of drugs. The last named mechanism has also been ascribed to LRP, a protein which only occurs intracellularly. In leukemia patients, cellular drug resistance profiles determined in vitro at the time of presentation show a strong correlation with outcome. In AML, mdr-1 overexpression at diagnosis is a strong independent predictor for CR and long-term survival. In ALL, mdr-1 expression is of minor importance for prediction of outcome. In AML, MRP1 expression at diagnosis is not correlated with clinical response and survival in most studies. In ALL, MRP1 expression at diagnosis is not associated with response and long-term survival in the few studies on this aspect which have been published. The studies on LRP in AML emphasize the importance of the correlation between LRP-expression and anthracycline accumulation and suggest that LRP-expression has prognostic value at diagnosis. However, there is an equal number of studies where a predictive value in the case of LRP-expression in de novo AML cannot be shown. The highest levels of LRP have been reported in multiple relapses of ALL. Furthermore, new membrane-associated drug transport proteins have been reported including the transporter associated with antigen processing (TAP), the anthracyclin resistance-associated protein (ARA), five new homologues of MRP (MRP2, or MOAT, MRP3, MRP4, MRP5, and MRP6), the sister of P-glycoprotein (sP-gp) and breast cancer resistance protein (BCRP). Studies on the (clinical) significance of these proteins have not yet been reported.

Identification of a multixenobiotic resistance mechanism in Xenopus laevis embryos

In the '90's a membrane-associated transport protein, discovered in aquatic organisms, was considered to be expressed in response to environmental xenobiotics. Like the multidrug resistance protein found in mammalian tumor cell lines, this protein confers resistance in organisms in polluted areas by binding xenobiotics and transporting them out of the cells in an energy-dependent manner. This study investigates the expression and the activity of a P-glycoprotein (Pgp) involved in a multixenobiotic resistance mechanism (MXRW) during the early developmental stages and in tissues of adult Xenopus laevis.

THE PHARMACOLOGICAL ROLE OF P-GLYCOPROTEIN IN THE INTESTINAL EPITHELIUM

P-glycoprotein, a membrane-associated transport protein, has recently been recognised as an important element of the intestinal epithelium. This paper summarises thein vivodata on the pharmacological role of intestinal P-glycoprotein. These data show that P-glycoprotein contributes to the elimination of many drugs by mediating their direct secretion from the blood into the intestinal lumen. In addition, there is also evidence that this protein can limit oral drug absorption. Hence, inhibition of intestinal P-glycoprotein, e.g. by a reversal agent like cyclosporin A, may be a promising strategy for improving the oral bioavailability of P-glycoprotein substrate drugs. Indeed, several preclinical and clinical studies have shown that coadministration of drugs with a reversal agent can substantially increase oral drug absorption.

Copper-specific Transcriptional Repression of Yeast Genes Encoding Critical Components in the Copper Transport Pathway

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Solubilization and Characterization of a Thyroid Ca2+‐Dependent and NADPH‐Dependent K3Fe(CN)6 Reductase

The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

Cloning and characterization of a gene cluster, phsBCDEF, necessary for the production of hydrogen sulfide from thiosulfate by Salmonella typhimurium

We cloned, by complementation of an H 2S − mutant, a cluster of Salmonella typhimurium genes, phsBCDEF, that appears to be essential for the anaerobic production of hydrogen sulfide from thiosulfate. Tn5 mutagenesis and ExoIII deletion analysis showed that approx. the entire region of a 3.3-kb subclone was necessary for H 2S production. Subsequent sequencing revealed the presence of five potential translationally coupled open reading frames (ORFs). Their putative protein products were confirmed by synthesis from a phage T7 expression system. Comparison of the encoded sequences with previously determined sequences suggests that these genes constitute part of a thiosulfatereducing operon coding for a membrane-associated electron transport chain which contains proteins potentially capable of ligating iron-sulfur clusters and heme. Immediately upstream from these genes, a region encoding the C-terminal portion of an ORF (OrfA) was identified that showed a high degree of similarity to some other anaerobic terminal reductases, polysulfide reductase (PsrA) of Wolinella succinogenes and dimethylsulfoxide reductase (DmsA), formate dehydrogenase (formate-hydrogene-lyase linked) (FdhF) and nitrate reductase (NarG) of Escherichia coli.

Quantitative export of FGF-2 occurs through an alternative, energy-dependent, non-ER/Golgi pathway.

Although basic fibroblast growth factor (bFGF/FGF-2) is found outside cells, it lacks a conventional signal peptide sequence; the mechanism underlying its export from cells is therefore unknown. Using a transient COS-1 cell expression system, we have identified a novel membrane-associated transport pathway that mediates export of FGF-2. This export pathway is specific for the 18-kD isoform of FGF-2, is resistant to the anti-Golgi effects of Brefeldin A, and is energy-dependent. In FGF-2-transfected COS-1 cells, this ER/Golgi-independent pathway appears to be constitutively active and functions to quantitatively export metabolically-labeled 18-kD FGF-2. Co-transfection and co-immunoprecipitation experiments, using a vector encoding the cytoplasmic protein neomycin phosphotransferase, further demonstrated the selectivity of this export pathway for FGF-2. When neomycin phosphotransferase was appended to the COOH-terminus of 18-kD FGF-2, the chimera was exported. However, the transmembrane anchor sequence of the integral membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) blocked export. The chimeric protein localized to the plasma membrane with its FGF-2 domain extracellular and remained cell-associated following alkaline carbonate extraction. Taken together, the data suggest that FGF-2 is "exported" from cells via a unique cellular pathway, which is clearly distinct from classical signal peptide-mediated secretion. This model system provides a basis for the development and testing of therapeutic agents which may block FGF-2 export. Such an intervention may be of considerable use for the treatment of angiogenesis-dependent diseases involving FGF-2.

Membranes and Transport Systems in Plants: An Overview

Membranes define the outer boundary of the living protoplast and the internal compartmentation of plant cells. From a structural point of view, membranes consist of a lipid bilayer and proteins essential for functions such as solute transport, signal transduction, and numerous metabolic reactions. While membranes can represent a significant barrier to the free movement of many solutes, those with sufficient lipid solubility may move across membranes by dissolving into the lipid bilayer. However, selective membrane transport is generally observed for hydrophilic solutes such as mineral nutrients and cell metabolites. Such selective transport requires an input of metabolic energy, and in plants this occurs via the production of proton electrochemical gradients across the membrane by substrate- (ex. ATP, PPi) driven proton pumps. Selective solute transport is then mediated by membrane-associated secondary transport systems which utilize the proton electrochemical gradient to drive the transport process. This review of membrane structure and transport system function provides a background for a further examination of herbicide interactions with plant membranes.

Cisplatin-induced changes in the gastric smooth muscle and its calcium modulation

Cisplatin (cis-Dichlorodiammineplatinum II; CDDP) a potent broad spectrum anti-cancer drug produces severe toxic side effects including kidney damage, nausea and vomiting, loss of hearing, peripheral neuropathy, hypomagnesemia and hypocalcemia. Our studies using rat as the model system show that these toxicities are related to calcium homeostasis and can be controlled through the exogenous administration of calcium before CDDP treatment.Cytochemical and ultrastructural studies in Wistar rats [Crl:(WI)BR] show that CDDP in its hydrolyzed state under low chloride ion concentrations, within the cell, causes uncoupling of oxidative phosphorylation, calcium efflux from the mitochondria, inhibits ATP synthesis, lowers membrane-associated calcium and various membrane transport enzymes. It also induces an increase in the number of lysosomes. CDDP causes a drop in the blood calcium levels with a corresponding increase in the dark cells of the parathyroid gland. In rats CDDP causes hypercontraction of the uterine smooth muscle and an undue distension of the stomach through the noncontractility of its smooth muscle thus inhibiting gastric motility, leading to the development of ulcers.

Translation elongation factor 3: a fungus‐specific translation factor?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), was first described in the yeast Saccharomyces cerevisiae and has subsequently been identified in a wide range of fungal species including Candida albicans and Schizosaccharomyces pombe. EF-3 exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation elongation. Recent studies on the structure of EF-3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP- and GTP-binding motifs. Overall, EF-3 shows striking amino acid similarity to members of the ATP-binding Cassette (ABC) family of membrane-associated transport proteins although EF-3 is not itself directly membrane-associated. Regions of the EF-3 polypeptide also show structural homology with other translation-associated factors including aminoacyl-tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF-3 in the translation elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF-3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.