Ubiquitous Transport Research Articles

KpnEF, a New Member of the Klebsiella pneumoniae Cell Envelope Stress Response Regulon, Is an SMR-Type Efflux Pump Involved in Broad-Spectrum Antimicrobial Resistance

Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp.

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

Vibrio cholerae VexH encodes a multiple drug efflux pump that contributes to the production of cholera toxin and the toxin co-regulated pilus.

The resistance-nodulation-division (RND) efflux systems are ubiquitous transporters that function in antimicrobial resistance. Recent studies showed that RND systems were required for virulence factor production in Vibrio cholerae. The V. cholerae genome encodes six RND efflux systems. Three of the RND systems (VexB, VexD, and VexK) were previously shown to be redundant for in vitro resistance to bile acids and detergents. A mutant lacking the VexB, VexD, and VexK RND pumps produced wild-type levels of cholera toxin (CT) and the toxin co-regulated pilus (TCP) and was moderately attenuated for intestinal colonization. In contrast, a RND negative mutant produced significantly reduced amounts of CT and TCP and displayed a severe colonization defect. This suggested that one or more of the three uncharacterized RND efflux systems (i.e. VexF, VexH, and VexM) were required for pathogenesis. In this study, a genetic approach was used to generate a panel of V. cholerae RND efflux pump mutants in order to determine the function of VexH in antimicrobial resistance, virulence factor production, and intestinal colonization. VexH contributed to in vitro antimicrobial resistance and exhibited a broad substrate specificity that was redundant with the VexB, VexD, and VexK RND efflux pumps. These four efflux pumps were responsible for in vitro antimicrobial resistance and were required for virulence factor production and intestinal colonization. Mutation of the VexF and/or VexM efflux pumps did not affect in vitro antimicrobial resistance, but did negatively affect CT and TCP production. Collectively, our results demonstrate that the V. cholerae RND efflux pumps have redundant functions in antimicrobial resistance and virulence factor production. This suggests that the RND efflux systems contribute to V. cholerae pathogenesis by providing the bacterium with protection against antimicrobial compounds that are present in the host and by contributing to the regulated expression of virulence factors.

Transport of sucrose, not hexose, in the phloem

Several lines of evidence indicate that glucose and fructose are essentially absent in mobile phloem sap. However, this paradigm has been called into question, especially but not entirely, with respect to species in the Ranunculaceae and Papaveraceae. In the experiments in question, phloem sap was obtained by detaching leaves and placing the cut ends of the petioles in an EDTA solution. More hexose than sucrose was detected. In the present study, these results were confirmed for four species. However, almost identical results were obtained when the leaf blades were removed and only petiole stubs were immersed. This suggests that the sugars in the EDTA solution represent compounds extracted from the petioles, rather than sugars in transit in the phloem. In further experiments, the leaf blades were exposed to 14CO2 and, following a chase period, radiolabelled sugars in the petioles and EDTA exudate were identified. Almost all the radiolabel was in the form of [14C]sucrose, with little radiolabelled hexose. The data support the long-held contention that sucrose is a ubiquitous transport sugar, but hexoses are essentially absent in the phloem stream.

Transport Phenomena of Falling Liquid Film Flow on a Plate with Rectangular Holes

Falling liquid film flow is a sort of ubiquitous transport phenomenon occurring in chemical engineering. Fundamental research on the flow and mass transfer behavior of falling liquid film flow on plates with different structures can help engineers to develop more efficient equipment. In this paper, falling liquid film flow on a plate with rectangular holes is investigated with both numerical and experimental methods. Mass transfer experiments show that opening rectangle-shaped narrow holes on a plate can enhance the mass transfer efficiency. Compared to an ordinary plate, the vapor−liquid mass transfer rate on a holed plate can be increased by 10−20%. To analyze the detailed flow and mass transfer behavior, the computational fluid dynamics model (CFD) based on the volume of fluid (VOF) method is developed. In this model, the Marangoni effect induced by mass transfer is considered. The new model was demonstrated to give more satisfactory results than the previous model.

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

Fluid Fluid Interactions in the Earth's Lithosphere

Fluids rich in water, carbon and sulfur species and a variety of dissolved salts are a ubiquitous transport medium for heat and matter in the Earth’s interior. Fluid transport through the upper mantle and crust controls the origin of magmatism above subduction zones and results in natural risks of explosive volcanism. Fluids passing through rocks affect the chemical and heat budget of the global oceans, and can be utilized as a source of geothermal energy on land. Fluid transport is a key to the formation and the practical utilization of natural resources, from the origin of hydrothermal mineral deposits, through the exploitation of gaseous and liquid hydrocarbons as sources of energy and essential raw materials, to the subsurface storage of waste materials such as CO2. Different sources of fluids and variable paths of recycling volatile components from the hydrosphere and atmosphere through the solid interior of the Earth lead to a broad range of fluid compositions, from aqueous liquids and gases through water-rich silicate or salt melts to carbon-rich endmember compositions. Different rock regimes in the crust and mantle generate characteristic ranges of fluid composition, which depending on pressure, temperature and composition are miscible to greatly variable degrees. For example, aqueous liquids and vapors are increasingly miscible at elevated pressure and temperature. The degree of this miscibility is, however, greatly influenced by the presence of additional carbonic or salt components. A wide range of fluid–fluid interactions results from this partial miscibility of crustal fluids. Vastly different chemical and physical properties of variably miscible fluids, combined with fluid flow from one pressure – temperature regime to another, therefore have major consequences for the chemical and physical evolution of the crust and mantle. Several recent textbooks (e.g., Walther and Wood 1986; Carrol and Holloway 1994; Shmulovich et al. 1995 …

The Mobile–Stationary Divide in Ubiquitous Computing Environments: Lessons from the Transport Industry

The emergence of ubiquitous computing offers new possibilities and opportunities for organizations attempting to improve their productivity and effectiveness. in particular, the promises of ubiquitous computing are attractive to organizations such as transport firms, in which coordination of diverse sets of mobile units is central to organizational performance. This article analyzes the use of ubiquitous transport systems in Swedish road haulage firms and discusses the opportunities and challenges for the early adopters. It pays specific attention to the mobile–stationary divide; that is, the set of challenges associated with integration of mobile and stationary people and systems into a seamless computing environment.

A novel assay of cell rubidium uptake using graphite furnace atomic absorption: Application to rats on a magnesium-deficient diet

The [Na,K]ATPase or sodium pump (SP) is a ubiquitous membrane cation transport system. Because of its potential participation in the pathophysiology of essential hypertension and cataract formation, the SP is under active investigation to detail its function and control. In this paper, we describe a novel, nonradioactive method of measuring SP ion transport activity in intact red blood cells (RBCs) using graphite furnace atomic absorption measurement of rubidium ion (Rb) uptake. This method provided sensitivity comparable to radioactive techniques, as assessed by experiments with human red blood cells (RBC) and ouabain, a known SP inhibitor, but this analytical approach eliminates the use of radioisotopes common to other Rb uptake assay methods. As a demonstration of its broader utility, the assay was used to assess the effects of dietary magnesium intake on SP-mediated ion transport in the RBCs of diet-controlled rats. Rats on 7 weeks of a magnesium-deficient (MgD) diet showed significant reductions in serum magnesium concentration, although levels remained in the lower region of the reference interval for healthy, magnesium replete animals. Red cell Rb uptake was significantly reduced in cells from the magnesium-restricted animals, demonstrating the sensitivity of Rb uptake to reduced magnesium intake, despite serum levels that fell within the reported normal range, and the utility of this Rb uptake assay in measuring physiological changes in SP function.

Expression of Na+/H+ exchanger isoforms in normal human nasal epithelial cells and functional activity of Na+/H+ exchanger 1 in intracellular pH regulation

Conclusions. Both the mRNA and protein of NHE1, −2 and −3 were expressed in NHNE cells. NHE1 is a major NHE isoform which is expressed in the basolateral membranes of NHNE cells. Objective. Na+/H+ exchangers are ubiquitous plasma membrane transport proteins implicated in the maintenance of intracellular pH. The aim of this study was to examine the expression of Na+/H+ exchangers as a function of the differentiation of normal human nasal epithelial (NHNE) cells. In addition, we investigated the functional activity of the Na+/H+ exchanger 1 (NHE1) in basolateral membranes. Material and methods. Cultured passage-2 NHNE cells were used. RNA and histological samples were collected on the day of confluence and on the 7th, 14th and 28th days after confluence in order to determine the effects of time. Cell lysates were collected on the day of confluence to investigate the presence of the proteins. Reverse transcriptase polymerase chain reaction and Western blotting were performed to investigate the presence of mRNA and protein, respectively. The functional activity of NHE1 was examined using 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate (HOE694), an NHE1-specific inhibitor, on the day of confluence. Results. The NHE1 mRNA expression level did not change as a function of differentiation. However, the NHE2 mRNA expression levels increased on the 7th, 14th and 28th days after confluence. The NHE3 mRNA expression levels increased on the 14th and 28th days after confluence. Western blot analysis confirmed the expression of NHE1 (91 kDa), NHE2 (90 kDa) and NHE3 (93 kDa). In addition, HOE694 inhibited basolateral NHE activity by 68% at 1 μM and by 85% at 5 μM in the NHNE cells.

Extracellular Na+ inhibits Na+/H+ exchange: cell shrinkage reduces the inhibition.

Na+/H+ exchangers (NHE) are ubiquitous transporters participating in regulation of cell volume and pH. Cell shrinkage, acidification, and growth factors activate NHE by increasing its sensitivity to intracellular H+ concentration. In this study, the kinetics were studied in dog red blood cells of Na+ influx through NHE as a function of external Na+ concentration ([Na+](o)). In cells in isotonic media, [Na+](o) inhibited Na+ influx >40 mM. Osmotic shrinkage activated NHE by reducing this inhibition. In cells in isotonic media + 120 mM sucrose, there was no inhibition, and influx was a hyperbolic function of [Na+](o). The kinetics of Na+-inhibited Na+ influx were analyzed at various extents of osmotic shrinkage. The curves for inhibited Na+ fluxes were sigmoid, indicating more than one Na+ inhibitory site associated with each transporter. Shrinkage significantly increased the Na+ concentration at half-maximal velocity of Na+-inhibited Na+ influx, the mechanism by which shrinkage activates NHE.

OPC-SMS: a wireless gateway to OPC-based data sources

This paper describes OPC-SMS gateway, a platform that integrates IP networks with the Short Message Service (SMS), in order to deliver an integrated service for access to data sources conforming to Object Linking and Embedding for Process Control (OPC) standard specifications, through SMS-enabled mobile devices. The gateway supports pull and push services in order to support both request-based and alarm/scheduled-based notifications, respectively. The proposed architecture is based entirely on the ubiquitous HyperText Transport Protocol (HTTP), Simple Object Access Protocol (SOAP), Extensible Markup Language (XML) protocols, and the Global System for Mobile communication (GSM) network and thus exploits the network infrastructure already in place. The capability of accessing different types of OPC data sources (real-time and historical) by any SMS-enabled device consists of a highly flexible service, supporting mobility and event-based notification. A pilot implementation of our approach has been tested in a large-scale installation for accessing OPC data sources of several automation subsystems in a hospital.

Role of two recently cloned rat liver GSH transporters in the ubiquitous transport of GSH in mammalian cells.

Recently our laboratory has cloned both the rat canalicular and sinusoidal GSH transporters (RcGshT and RsGshT, respectively; Yi, J., S. Lu, J. Fernandez-Checa, and N. Kaplowitz. 1994. J. Clin. Invest. 93:1841-1845; and 1995. Proc. Natl. Acad. Sci. USA. 92:1495-1499). The current work characterized GSH transport and the expression of these two GSH transporters in various mammalian cell lines. The average cell GSH levels (nmol/10(6) cells) were 25, 22, 32, 13, and 13 in HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. GSH efflux was temperature dependent and averaged 0.018, 0.018, 0.012, 0.007, and 0.019 nmol/10(6) cells/min from HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. Dithiothreitol (DTT), which stimulates rat sinusoidal GSH efflux, stimulated GSH efflux only in HepG2 and HeLa cells which was partially reversed by subsequent cystine treatment. GSH uptake (1 mM plus 35S-GSH) was temperature dependent, linear up to 45 min, and Na+-independent with average rates of 1.12, 0.91, 0.45, and 0.45 nmol/10(6) cells/30 min for HepG2, HeLa, CaCo-2, MDCK, and Cos-1 cells, respectively. BSP-GSH (2mM), which cis-inhibits sinusoidal GSH uptake in rat liver and HepG2 cells, inhibited GSH uptake only in HeLa cells. mRNA and polypeptide of RcGshT are expressed in all cells whereas those of RsGshT are expressed only in HepG2 and HeLa cells. In conclusion, bidirectional GSH transport, mediated by the "canalicular" GSH transporter, is ubiquitous in mammalian cells. Sinusoidal GSH transporter expression is more restricted, being present in HepG2 and HeLa cells. DTT and BSP-GSH affect GSH transport only in cells expressing the sinusoidal transporter confirming their selective action on this transporter.

Renal tubule Na,K-ATPase polarity in different animal models of polycystic kidney disease.

Apical mislocation of the ubiquitous transport enzyme Na,K-ATPase has been implicated as a feature of cyst development in in vitro studies of human polycystic kidney disease (PKD) epithelia. We undertook an immunohistochemical study of murine glucocorticoid-induced PKD, the pcy mouse, the cpk mouse, and the diphenylthiazole (DPT)-induced rat models of PKD to determine if this feature was common to these models of cyst development. Distribution of Na,K-ATPase was determined with a polyclonal anti-Na,K-ATPase antibody and a nickel-silver-enhanced peroxidase color development system. Results were documented objectively with densitometric techniques. Control animals appropriate to the age, strain, and species of the experimental groups demonstrated the expected polar distribution of Na,K-ATPase to the basolateral surface. This distribution was more marked in mature animals. Tubular dilatation and cystic change, however, were associated with increased apical Na,K-ATPase in all models. The murine models demonstrated decreased basolateral staining for Na,K-ATPase compared with controls, although this was not a feature of the DPT rat model. Abnormal location of Na,K-ATPase is a shared feature of a variety of animal models and human PKD. This may contribute to abnormal fluid and electrolyte flux favoring cyst formation or may represent expression of a less differentiated renal tubule epithelial phenotype.

Amino acid transport systems in the human hepatoma cell line Hep G2

The human hepatoma cell line Hep G2 was used to investigate amino acid transport systems in human liver tissue. The ubiquitous transport systems responsible for the uptake of most neutral amino acids (systems A, ASC and L) were found to be present. Transport system A was predominant for proline uptake but system ASC was the major Na +-dependent transport system, particularly for glutamine. The specific hepatic system N was functional, but only partially mediated glutamine uptake. The study of Na +-independent arginine uptake demonstrated the presence of the cationic transport system Y +, reflecting the transformed nature of Hep G2 cells.

Phosphate transport by fibroblasts from patients with hypophosphataemic vitamin D-resistant rickets.

It is accepted that renal phosphate wasting is the basis of hypophosphataemia in vitamin D-resistant hypophosphataemic rickets (VDRR). Abnormal renal adaptation to phosphate deprivation has also been reported in these patients. We studied sodium-dependent phosphate transport and its modulation by phosphate deprivation in skin fibroblasts cultured from healthy subjects and patients with VDRR. Control fibroblasts exhibited high-affinity sodium-dependent phosphate transport (77 +/- 12 mumol/l) which resembled the ubiquitous transport of renal and non-renal cells. Phosphate deprivation (incubation in low phosphate medium) increased the maximal velocity (Vmax) of the transport by 2.7-fold after 24 h, with no change in the affinity. The increase in Vmax was dependent on gene transcription and protein synthesis. The sodium-dependent phosphate transport exhibited in fibroblasts from VDRR patients did not significantly differ from that of control subjects, except that the Vmax of the phosphate transport was higher in cells from patients with VDRR under normal and phosphate-deprivation conditions, although the difference was significant only after 24 h of phosphate deprivation (Vmax: 22.6 +/- 2.4 pmol/mg protein per s in VDRR vs 16 +/- 3.6 pmol/mg protein per s in controls, P less than 0.05). These data demonstrate that sodium-coupled phosphate transport in human skin fibroblasts has the properties of ubiquitous sodium-phosphate co-transport and show that this transport is not deficient in patients with VDRR. Indeed paradoxically the Vmax was 40% higher in VDRR than in control subjects after 24 h of phosphate deprivation. The transport must be either different from that of kidney cells responsible for the phosphate leak, or differently modulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Na(+)-H+ exchanger and its role in essential hypertension and diabetes mellitus.

The Na(+)-H+ exchanger is a ubiquitous transport system that is involved in the regulation of intracellular pH, cell growth and proliferation, cell volume regulation, and transepithelial absorption of Na+, Cl-, and HCO3-. Altered activity of the Na(+)-H+ exchanger has been implicated as a mechanism contributing to the development of high blood pressure in subgroups of patients with essential hypertension and in various animal models of hypertension. Many of these studies measured Na(+)-Li+ exchange rather than Na(+)-H+ exchange, hypothesizing that Na(+)-Li+ exchange represents a functional mode of the Na(+)-H+ exchanger. However, this is a controversial assumption. Several studies have also shown an association between erythrocyte Na(+)-Li(+)-exchange rate and predisposition to nephropathy in patients with insulin-dependent diabetes mellitus. The recent cDNA cloning of at least one isoform of the Na(+)-H+ exchanger will help clarify the cellular mechanisms of regulation of the exchanger and its possible role in pathophysiological states such as hypertension.

Inhibition by analogues of L-tyrosine transport by B16/F10 melanoma cells

The effect of a number of L-tyrosine (L-Tyr) analogues on L-Tyr uptake by B16/F10 malignant melanocytes is reported. This amino acid can be taken up by two of the most ubiquitous transport systems found in animals cells, L and presumably ASC. L-Tyr analogues devoid of the amino group, like p-hydroxyphenyl pyruvic acid and related compounds, and L-Tyr analogues devoid of the carboxyl group, such as tyramine, do not affect L-Tyr uptake. The other aromatic amino acids, L-Phe and L-Trp, and the L-Tyr analogues DL-m-Tyr, L-diiodotyrosine and L-dopa, strongly inhibit the uptake of L-Tyr. This suggests that these chemicals are transported more efficiently than L-Tyr. The ASC system does not show stereospecificity, but the L system has greater affinity for L-Tyr than for D-Tyr. The ASC system also has greater affinity for tyrosine isomers with the hydroxyl group in the ortho and meta positions. The presence of a methyl group at the alpha-carbon of L-Tyr and L-dopa also increases the affinity of the ASC system for these agents. In contrast, alpha-methylation decreases the affinity of the L system in comparison to L-Tyr. Finally, L-Tyr esters do not inhibit, but stimulate the transport of L-Tyr, mainly by the ASC system.

PKC and Pi deprivation modulate differently the ubiquitous Na-dependent Pi uptake in MDCK cells

The role of protein kinase C (PKC) in the modulation of the ubiquitous sodium-dependent phosphate transport and in adaptation of that transport to phosphate deprivation was investigated in MDCK cells. Phorbol myristate acetate (PMA) had a biphasic effect on sodium-dependent phosphate uptake characterized by early inhibition (-25% at 1 h) followed by late stimulation (2.3-fold at 15 h). Late stimulation was related to a decreased apparent affinity (Km) with unchanged maximal velocity (Vmax). The 15-h stimulation of phosphate uptake was also induced by an initial 1-h PMA treatment followed by a 14-h washout of PMA or by R59 022. The stimulation was inhibited by PKC downregulation. PMA stimulation was dependent on protein synthesis but not on transcription, as shown by the respective effects of cycloheximide, 3'-deoxyadenosine, and actinomycin D. In phosphate-deprived cells PMA had also a biphasic effect. A potentiation of PMA stimulation of phosphate uptake with phosphate deprivation was observed. Adaptation to phosphate deprivation was not prevented by PKC downregulation. Cytosolic and membranous PKC activities were not changed by 15-h phosphate deprivation. We conclude that 1) PKC modulates sodium-dependent phosphate uptake in MDCK cells, and 2) phosphate deprivation and PKC modulation of sodium-dependent phosphate uptake involve different cellular pathways; that is, phosphate deprivation acts through gene regulation, and PKC acts through translation regulation.

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.