Genetic reconstitution of the high-affinity L-arabinose transport system.

Expression plasmids containing various portions of araFGH operon sequences were assayed for their ability to facilitate the high-affinity L-arabinose transport process in a strain lacking the chromosomal copy of this operon. Accumulation studies demonstrated that the specific induction of all three operon coding sequences was necessary to restore high-affinity L-arabinose transport. Kinetic analysis of this genetically reconstituted transport system indicated that it functions with essentially wild-type parameters. Therefore, L-arabinose-binding protein-mediated transport appears to require only two inducible membrane-associated components (araG and araH) in addition to the binding protein (araF).     

The mechanism of interaction between high-affinity probes and the uridine transport system of mammalian cells.

The carrier of uridine transport in hamster cells in culture is highly susceptible to the inhibitory effect of probes like S-benzylated derivatives of mercaptopurine nucleosides. The interaction between the probes and the carrier is competitive and reversible and it takes place at a site different from the substrate binding site. The Ki for the most potent derivative p-nitrobenzyl-6-mercaptoinosine is 0.15 n Molar at 20 degrees C. The effect of the probes is interpreted in terms of conformational change induced on the carrier upon binding of the probe. The carrier assumes distinct conformations depending on whether it is probe-free (form A) or probe bound (form B). Kinetic as well as chemical evidence supports the predictions of the allosteric carrier model. A single component of kinetics is observed either in the absence of inhibitor (Km form A) or at high concentrations of inhibitor (Km form B). A two component kinetics is observed at intermediate concentrations of inhibitor (some carriers in form B and others in form A). The two forms have distinct Km values for uridine: form A50 muMolar and form B 250 muMolar. Two forms have also different susceptibilities to the action of organomercurials: form A is insensitive whereas form B is highly inhibited by the chemical modified of SH groups. The existence of putative allosteric sites in carriers is discussed in terms of modifier sites capable of modulating transport activities as a result of specific membrane-ligand interactions.     

Characterization of the polyamine transport system in mouse neuroblastoma cells. Effects of sodium and system A amino acids

The biochemical properties of polyamine transport system have been studied in detail in NB-15 mouse neuroblastoma cells in culture by measuring the uptake of [14C]putrescine under various experimentally imposed pharmacological conditions. Putrescine uptake in the NB-15 mouse neuroblastoma cells appeared to be a sodium-dependent process. Iso-osmotic displacement of Na+ in the assay medium with either choline or Li+ resulted in a linear decrease of putrescine uptake. Gramicidin, a channel-former ionophore, inhibited putrescine uptake by more than 90% at 20 nM. N-Ethylmaleimide at 5 mM or p-chloromercuribenzene sulfonate at 50 microM completely abolished putrescine uptake. Conversely, oxidized glutathione at 10 mM or 5,5'-dithiobis-(2-nitrobenzoic acid) at 5 microM gave a 1.3-1.4-fold stimulation after a 1-h incubation. This polyamine transport system appeared to be subjected to adaptive regulation. Polyamine antimetabolites such as alpha-difluoromethyl ornithine stimulated putrescine uptake whereas preloading of cells with polyamines inhibited putrescine uptake. Preloading cells with neutral amino acids that belong to sodium-dependent transport System A stimulated putrescine uptake by more than 8-10-fold. These results suggested that the polyamine transport system in NB-15 mouse neuroblastoma cells was sodium dependent and shared some characteristics common to other known sodium-dependent transport systems. These characteristics included (a) sensitivity to ionophores, (b) sensitivity to sulfhydryl reagents, and (c) sensitivity to intracellular contents of substrate molecules. Our data also indicated that polyamine transport may be regulated by transport System A amino acids.     

Phosphate transport in Neurospora. Derepression of a high-affinity transport system during phosphorus starvation.

In addition to the constitutive, low-affinity phosphate-transport system described previously, Neurospora possesses a second, high-affinity system which is derepressed during phosphorus starvation. At pH 5.8, System ii has a K1/2 of about 3muM and a Jmax of 5.2 mmol/1 cell water per min. System ii reaches maximal activity after about 2 h of growth in phosphorus-free minimal medium. Its formation is blocked by cycloheximide and, once made, it appears to turn over rapidly. Addition of cycloheximide to fully derepressed cultures results in the decay of System ii with a t1/2 of 14 min, very similar to the turnoacteriol. 95, 959-966) for tryptophan transport in Neurospora. Thus, these transport systems appear to be regulated by a balance between synthesis and breakdown, as affected by intracellular pools of substrate or related compounds.     

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.     

Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro

Summary The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk an analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed, We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis. L. M. is a recipient of a fellowship from the IWONL, Belgium. This work was supported by the Belgisch Werk Tegen Kanker and the Internationale Stichting Jacques Brel.     

Identification of a high-affinity glycine betaine transport system in Staphylococcus aureus.

Staphylococcus aureus accumulates proline and glycine betaine when cells are grown at low water activity. In the present study, we have identified a high-affinity glycine betaine transport system in this bacterium. Optimal activity for this transport system was measured in the presence of high NaCl concentrations, but transport activity was not stimulated by high concentrations of other solutes.     

Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.     

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.     

Phosphate transport in Neurospora. Derepression of a high-affinity transport system during phosphorus starvation

In addition to the constitutive, low-affinity phosphate-transport system described previously, Neurospora possesses a second, high-affinity system which is derepressed during phosphorus starvation. At pH 5.8, System II has a of about 3μM and a J_max of 5.2 mmol/l cell water per min.System II reaches maximal activity after about 2 h of growth in phosphorus-free minimal medium. Its formation is blocked by cycloheximide and, once made, it appears to turn over rapidly. Addition of cycloheximide to fully derepressed cultures results in the decay of System II with a of 14 min, very similar to the turnover rate previously reported (Wiley, W.R. and Matchett, W.H. (1968) J. Bacteriol. 95, 959–966) for tryptophan transport in Neurospora. Thus, these transport systems appear to be regulated by a balance between synthesis and breakdoown, as affected by intracellular pools of substrate or related compounds.     

DNA synthesis in detergent-treated mouse ascites sarcoma cells.

Mouse ascites sarcoma cells (SR-C3H/He cells) were made permeable to nucleoside triphosphates by treatment with nonionic detergents in a nearly isotonic condition. The permeable cells synthesized DNA in the presence of the four deoxyribonucleoside triphosphates, ATP, Mg2+, and the proper ionic environment. The optimum detergent concentration for DNA synthesis was 0.015--0.020% with Triton X-100, 0.020% with Nonidet P-40, and about 0.0025% with Brij 58. Higher concentrations of detergents were rather inhibitory to DNA synthesis. DNA synthesis in Triton-permeabilized cells was thought to be replicative, and the activity in the optimum conditions was much higher than that measured in hypotonic permeable cells or in isolated nuclei. These studies show the potential usefulness of detergent treatment for examining DNA replication in mammalian cells in vitro.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

Transport of sugars in chick-embryo fibroblasts. Evidence for a low-affinity system and a high-affinity system for glucose transport.

The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Antisense Tiam1 down-regulates the invasiveness of 95D cells in vitro

As a specific guanine nucleotide exchange factor of Rac 1, Tiam 1 (T-lymphoma invasion and metastasis inducing protein 1) is involved in a number of cellular events, such as cytoskeleton reorganization, cell adhesion, and cell migration. Since Tiaml was implicated in the invasion and metastasis of T-lymphoma cells and breast tumor cells, we compared the expression level of Tiaml in two human giant-cell lung carcinoma cell strains with high or low metastasis potential, and found that Tiaml expression level in high-metastatic 95D cells was higher than that in low-metastatic 95C cells. To further confirm the role of Tiam I in invasion and metastasis, we constructed the antisense Tiaml expression plasmid (pcDNA3-anti-Tiaml), which was transfected into 95D cells. A stable transfected clone with decreased Tiaml expression was screened and selected for further research. Transwell assay showed that down-regulation of endogenous Tiam1 by anti-Tiam1 can reduce the in vitro invasiveness of 95D cells. Our results suggested that Tiam1 signaling contributed to the invasion and metastasis of the human giant-cell lung carcinoma cells.