Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.     

Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111

In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.     

Time-resolved fluorescence anisotropy studies show domain-specific interactions of calmodulin with IQ target sequences of myosin V

Single cysteine mutants of calmodulin, Cam(S38C) and Cam(N111C), have been specifically labelled with Alexa488 maleimide to study the effects of calcium on the structural dynamics of calmodulin complexed with IQ3, IQ4 and IQ34 target peptide motifs of mouse unconventional myosin-V. Using phase fluorometry, the time-resolved anisotropy shows well-separated global and segmental correlation times. The calcium-sensitive global motion of either calmodulin domain can be independently monitored in domain-specific interactions of either apo- or Ca(4).calmodulin with IQ3 or IQ4 peptides. C-domain interactions predominate, and apo-N-domain interactions are unexpectedly weak. The 1:1 complex of Ca(4).calmodulin with IQ34 behaves as a compact globular species. The results demonstrate novel dynamic aspects of calmodulin-IQ interactions relating to the calcium regulation of motility of unconventional myosin.     

Design and synthesis of novel RXR-selective modulators with improved pharmacological profile

New RXR-selective modulators possessing a 6-fluoro trienoic acid moiety (6Z olefin) or a fluorinated/heterocyclic-substituted benzene core ring, were synthesized in an expedient and selective way. A subset of these compounds was evaluated for their metabolic properties (exposure in IRC male mice) and show a dramatic increase of exposure compared to our reference compound, 3 (LG101506).     

Correlations of climate and plant ecology to leaf size and shape: potential proxies for the fossil record

The sizes and shapes (physiognomy) of fossil leaves are widely applied as proxies for paleoclimatic and paleoecological variables. However, significant improvements to leaf-margin analysis, used for nearly a century to reconstruct mean annual temperature (MAT), have been elusive; also, relationships between physiognomy and many leaf ecological variables have not been quantified. Using the recently developed technique of digital leaf physiognomy, correlations of leaf physiognomy to MAT, leaf mass per area, and nitrogen content are quantified for a set of test sites from North and Central America. Many physiognomic variables correlate significantly with MAT, indicating a coordinated, convergent evolutionary response of fewer teeth, smaller tooth area, and lower degree of blade dissection in warmer environments. In addition, tooth area correlates negatively with leaf mass per area and positively with nitrogen content. Multiple linear regressions based on a subset of variables produce more accurate MAT estimates than leaf-margin analysis (standard errors of ±2 vs. ±3°C); improvements are greatest at sites with shallow water tables that are analogous to many fossil sites. The multivariate regressions remain robust even when based on one leaf per species, and the model most applicable to fossils shows no more signal degradation from leaf fragmentation than leaf-margin analysis.     

Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites

Parasite lactate dehydrogenase (pLDH) is a potential drug target for new antimalarials owing to parasite dependence on glycolysis for ATP production. The pLDH from all four species of human malarial parasites were cloned, expressed, and analyzed for structural and kinetic properties that might be exploited for drug development. pLDH from Plasmodium vivax, malariae, and ovale exhibit 90-92% identity to pLDH from Plasmodium falciparum. Catalytic residues are identical. Resides I250 and T246, conserved in most LDH, are replaced by proline in all pLDH. The pLDH contain the same five-amino acid insert (DKEWN) in the substrate specificity loops. Within the cofactor site, pLDH from P. falciparum and P. malariae are identical, while pLDH from P. vivax and P. ovale have one substitution. Homology modeling of pLDH from P. vivax, ovale, and malariae with the crystal structure of pLDH from P. falciparum gave nearly identical structures. Nevertheless, the kinetic properties and sensitivities to inhibitors targeted to the cofactor binding site differ significantly. Michaelis constants for pyruvate and lactate differ 8-9-fold; Michaelis constants for NADH, NAD(+), and the NAD(+) analogue 3-acetylpyridine adenine dinucleotide differ up to 4-fold. Dissociation constants for the inhibitors differ up to 21-fold. Molecular docking studies of the binding of the inhibitors to the cofactor sites of all four pLDH predict similar orientations, with the docked ligands positioned at the nicotinamide end of the cofactor site. pH studies indicate that inhibitor binding is independent of pH in the pH 6-8 range, suggesting that differences in dissociation constants for a specific inhibitor are not due to altered active site pK values among the four pLDH.     

Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.     

Human thymus exports naive CD8 T cells that can home to nonlymphoid tissues

Functionally naive CD8 T cells in peripheral blood from adult humans can be fully described by their CD45RA(bright)CCR7(+)CD62L(+) cell surface phenotype. Cord blood lymphocytes, from healthy newborns, are homogenously functionally naive. Accordingly, the majority of cord blood CD8 T cells express the same pattern of cell surface molecules. Unexpectedly, however, a significant fraction of cord blood CD8 T cells express neither CCR7 nor CD62L. Yet these cells remain functionally naive as they contain high levels of TCR excision circles, have long telomeres, display highly polyclonal TCRs, and do not exhibit immediate effector functions. In addition, these CD8 T cells already represent a significant fraction of the mature naive CD8 single-positive thymocyte repertoire and may selectively express the cutaneous lymphocyte Ag. We suggest that CD8 single-positive thymocytes comprise two pools of naive precursors that exhibit distinct homing properties. Once seeded in the periphery, naive CCR7(+)CD62L(+) CD8 T cells patrol secondary lymphoid organs, whereas naive CCR7(-)CD62L(-) CD8 T cells selectively migrate to peripheral tissues such as skin.     

Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent manners

Guinea pig mesenteric lymphatic vessels exhibit vasomotion through a pacemaker mechanism that involves intracellular Ca(2+) release and resultant spontaneous transient depolarizations (STDs) of the smooth muscle membrane potential. This study presents a detailed characterization of the effects of cyclopiazonic acid (CPA) on this pacemaker activity. Microelectrode recordings from smooth muscle in vessel segments revealed that application of CPA (1-10 microM) caused a hyperpolarization accompanied by a decrease in the frequency and amplitude of STDs. The CPA-induced hyperpolarization was abolished after destruction of the endothelium and in the presence of N(G)-nitro-L-arginine (100 microM) or 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one (10 microM), which suggests a contribution of endothelium-derived nitric oxide (EDNO) in this response. In the absence of EDNO-induced effects, CPA decreased the frequency and amplitude of STDs recorded before and in the presence of the thromboxane A(2) mimetic U-46619, norepinephrine, or thimerosal. CPA abolished U-46619-induced vasomotion as determined by measurement of constriction-associated intracellular Ca2+ concentration using the ratiometric Ca2+ indicator fura-2. The endothelial actions of CPA were compared with those of ACh, which is known to cause EDNO release in this preparation. Although CPA and ACh both increased endothelial intracellular Ca2+ concentration and depolarized the membrane potential, the kinetics of action for both parameters were markedly slower for CPA than ACh. These results suggest that CPA first hyperpolarizes the lymphatic smooth muscle and decreases STD frequency and amplitude through endothelial release of EDNO, and second, consistent with the action of CPA to inhibit sarcoplasmic reticulum Ca2+-ATPase and deplete Ca2+ stores, it further reduces STD activity. Inhibition of the lymphatic smooth muscle pacemaker mechanism is thought to abolish agonist-induced vasomotion.     

Design, synthesis and structure-activity relationship of novel RXR-selective modulators

The synthesis and in vitro characterization of novel RXR-selective ligands possessing various substituted 1-benzofuran or 1-benzothiophene moieties are described.