No abstract

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

In nano-ESI MS, the qualitative and quantitative characteristics of mass spectra vary considerably upon the use of different spraying conditions, i.e., aperture of the spraying needle and the voltage applied. The major parameters affected by the aperture size is the liquid flow rate which determines the initial droplet size and the current emitted upon the spray process, as described by different models of the ESI process. In the present study, the effect of flow rate on ion signals was studied systematically using mixtures of compounds with different physicochemical properties (i.e., detergent/oligosaccharide and oligosaccharide/peptide). For these model systems, the functional dependence of certain analyte-ion ratios upon the flow rate can be correlated to changes in analyte partition during droplet fission prior to ion release.

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)α1β2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTα1β2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTβR-Fc antagonized development of organized lymphoid structures. LTα1β2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTα1β2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

The epithelial–mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin α8β1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin α8β1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, α8β1-AP detects a novel ligand of 70–90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin α8β1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas α8β1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by α8β1-AP and forms a complex with α8β1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating α8β1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

To investigate the functional role of different ␣ 1 -adrenergic receptor (␣ 1 -AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the ␣ 1b -AR (␣ 1b ؊͞؊). Reverse transcription-PCR and ligand binding studies were combined to elucidate the expression of the ␣ 1 -AR subtypes in various tissues of ␣ 1b ؉͞؉ and ؊͞؊ mice. Total ␣ 1 -AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. Because of the large decrease of ␣ 1 -AR in the heart and the loss of the ␣ 1b -AR mRNA in the aorta of the ␣ 1b ؊͞؊ mice, the in vivo blood pressure and in vitro aorta contractile responses to ␣ 1 -agonists were investigated in ␣ 1b ؉͞؉ and ؊͞؊ mice. Our findings provide strong evidence that the ␣ 1b -AR is a mediator of the blood pressure and the aorta contractile responses induced by ␣ 1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in ␣ 1b ؊͞؊ as compared with ؉͞؉ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in ␣ 1b ؊͞؊ mice. The ␣ 1b -AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different ␣ 1 -AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.

The formation of neuronal networks in the central nervous system (CNS) requires precise control of axonal branch development and stabilization. Here we show that cell-specific ablation of the murine gene Ptk2 (more commonly known as fak), encoding focal adhesion kinase (FAK), increases the number of axonal terminals and synapses formed by neurons in vivo. Consistent with this, fak mutant neurons also form greater numbers of axonal branches in culture because they have increased branch formation and reduced branch retraction. Expression of wild-type FAK, but not that of several FAK variants that prevent interactions with regulators of Rho family GTPases including the p190 Rho guanine nuclear exchange factor (p190RhoGEF), rescues the axonal arborization phenotype observed in fak mutant neurons. In addition, expression of a mutant p190RhoGEF that cannot associate with FAK results in a phenotype very similar to that of neurons lacking FAK. Thus, FAK functions as a negative regulator of axonal branching and synapse formation, and it seems to exert its actions, in part, through Rho family GTPases.