We have isolated two cDNA clones, NaPi-2 and NaPi-3, by screening rat kidney cortex and human kidney cortex cDNA libraries, respectively, for expression of sodiumdependent phosphate transport in Xenopus laevis oocytes.

The interfacial profile between coexisting phases of a binary mixture (A,B) in a thin film of thickness D and lateral linear dimensions L depends sensitively on both linear dimensions and on the nature of boundary conditions and statistical ensembles applied. These phenomena generic for systems in confined geometry are demonstrated by Monte-Carlo simulations of the bond fluctuation model of symmetric polymer mixtures, using chains containing N A = N B = N = 32 effective monomers connected by effective bonds with an attractive interaction between monomers of the same type and a repulsive interaction between different types. We use short range potentials at the walls, the right wall favoring A monomers and the left wall B monomers. Periodic boundary conditions are applied in the directions parallel to the walls. Both the canonical and semi-grand-canonical ensemble are studied. We argue that the latter case is appropriate for experiments with a lateral resolution L much less than the actual lateral sample size, in thermal equilibrium. In the canonical ensemble, the interfacial width w increases (from small values which are of the same order as the "intrinsic profile") like w ∝ √ D, before a crossover to a saturation value w max (w 2 max ∝ ln L) sets in. In the semi-grand-canonical ensemble, however, one finds the same widths w ∝ √ D as in the canonical ensemble for not too large L, while for large L the interfacial profile is smeared out over a finite fraction of the film thickness (w ∝ D for D → ∞). We discuss the implications of these findings for the interpretation of both simulations and experiments.

In the present study, we have shown for the first time that both hCTGF mRNA and protein are expressed in human arteries in vivo and that hCTGF may represent a novel factor expressed at high levels specifically in advanced lesions and may play a role in the development and progression of atherosclerosis.

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We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodiumindependent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0'+ and carries cystine and dibasic amino acids with high affmity. The rBAT (bO'+ amino acid transporterrelated) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b°'+.

Abstract.A popular concept which describes the structure of polymer interfaces by "intrinsic profiles" centered around a two dimensional surface, the "local interface position", is tested by extensive Monte Carlo simulations of interfaces between demixed homopolymer phases in symmetric binary (AB) homopolymer blends, using the bond fluctuation model. The simulations are done in an L × L × D geometry. The interface is forced to run parallel to the L × L planes by imposing periodic boundary conditions in these directions and fixed boundary conditions in the D direction, with one side favoring A and the other side favoring B. Intrinsic profiles are calculated as a function of the "coarse graining length" B by splitting the system into columns of size B × B × D and averaging in each column over profiles relative to the local interface position. The results are compared to predictions of the self-consistent field theory. It is shown that the coarse graining length can be chosen such that the interfacial width matches that of the self-consistent field profiles, and that for this choice of B the "intrinsic" profiles compare well with the theoretical predictions. Our simulation data suggest that this "optimal" coarse graining length B 0 exhibits a dependence of the form B 0 = 3.8 w SCF (1 − 3.1/χN ), where w SCF is the interfacial width, N the chain length and χ the Flory-Huggins parameter.

A cDNA library from rabbit kidney cortex was screened for expression of Na-dependent transport of phosphate (P.) using Xenopus laevis oocytes as an expression system. A single clone was eventually isolated (designated NaPi-1) that stimulated expression of Na/P1 cotransport -700-fold compared to total mRNA. The predicted sequence of the Na/P1 cotransporter consists of 465 amino acids (relative molecular mass, 51,797); hydropathy profile predictions suggest six (possibly eight) membrane-spanning segments. In vitro translation of NaPi-1/complementary RNA in the presence of pancreatic microsomes indicated NaPi-1 to be a glycosylated protein; four potential N-glycosylation sites are present in the amino acid sequence. Northern blot analysis demonstrated the presence of NaPi-1/mRNA in kidney cortex and liver; no hybridization signal was obtained with mRNA from other tissues (including small intestine). Kinetic analysis of Na/P1 cotransport expressed by NaPi-1/complementary RNA demonstrated characteristics (sodium interaction) similar to those observed in cortical apical membranes. The alignment of 5 amino acid residues (Gly32/Alae'-Xaa-Xaa-Xaa-Xaa-Leu38-Xaa-XaaXaa-Pro3W.Arg39l) is consistent with a motif proposed for Na-dependent transport systems. We conclude that we have cloned a cDNA for a Na/P1 cotransport system present in rabbit kidney cortex.Reabsorption of phosphate (P) in the proximal tubule of the kidney contributes essentially to maintenance of the body Pi homeostasis (1). Influx of Pi at the brush border membrane of epithelial cells is mediated by a Na/Pt cotransporter and is driven by the transmembrane electrochemical potential gradient of sodium (2). Thereafter, P1 moves to the blood across the basolateral membrane, most likely via an anion-exchange mechanism and/or another Na-dependent Pi transport system. This transepithelial transport of P1 is controlled in a complex manner by various hormonal (e.g., parathyroid hormone) and nonhormonal (e.g., dietary Pi/Pi demand) factors (2-4).By using different experimental systems such as isolated tubules, isolated brush border membranes, and established cell cultures, it has been demonstrated that regulation of proximal tubular Pi reabsorption is accomplished mainly by modulation of the apically localized Na/Pi cotransport system (2). Thus, this Na/Pa cotransport system is a central target within the complex control of P1 homeostasis. Studies with established cell lines (mainly opossum kidney cells) demonstrated that inhibition of the Na/Ps cotransport (by, for example, parathyroid hormone) is mediated by activation of protein kinase C and/or A followed by an internalization step (endocytosis) of the transport system. On the other hand, stimulation of Na/P, cotransport by, for example, reduction of the concentration of extracellular Pi has been demonstrated to be dependent on de novo protein synthesis (2, 5).Despite the detailed knowledge of kinetic and regulatory properties of the renal (proximal tubular) Na/Pa cotransport system and despite several bioc...

The two electrode voltage clamp technique was used to investigate the steady-state and presteady-state kinetic properties of the type II Na+/Pi cotransporter NaPi-5, cloned from the kidney of winter flounder (Pseudopleuronectes americanus) and expressed in Xenopus laevis oocytes. Steady-state Pi-induced currents had a voltage-independent apparent K(m) for Pi of 0.03 mM and a Hill coefficient of 1.0 at neutral pH, when superfusing with 96 mM Na+. The apparent K(m) for Na+ at 1 mM Pi was strongly voltage dependent (increasing from 32 mM at -70 mV to 77 mM at -30 mV) and the Hill coefficient was between 1 and 2, indicating cooperative binding of more than one Na+ ion. The maximum steady-state current was pH dependent, diminishing by 50% or more for a change from pH 7.8 to pH 6.3. Voltage jumps elicited presteady-state relaxations in the presence of 96 mM Na+ which were suppressed at saturating Pi (1 mM). Relaxations were absent in non-injected oocytes. Charge was balanced for equal positive and negative steps, saturated at extremes of potential and reversed at the holding potential. Fitting the charge transfer to a Boltzmann relationship typically gave a midpoint voltage (V0.5) close to zero and an apparent valency of approximately 0.6. The maximum steady-state transport rate correlated linearly with the maximum Pi-suppressed charge movement, indicating that the relaxations were NaPi-5-specific. The apparent transporter turnover was estimated as 35 sec-1. The voltage dependence of the relaxations was Pi-independent, whereas changes in Na+ shifted V0.5 to -60 mV at 25 mM Na+. Protons suppressed relaxations but contributed to no detectable charge movement in zero external Na+. The voltage dependent presteady-state behavior of NaPi-5 could be described by a 3 state model in which the partial reactions involving reorientation of the unloaded carrier and binding of Na+ contribute to transmembrane charge movement.