Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

The secY protein can act post-translationally to promote bacterial protein export

Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Opposing Roles of pka and epac in the cAMP-Dependent Regulation of Schwann Cell Proliferation and Differentiation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.     

Real-time three-dimensional imaging of lipid signal transduction: apical membrane insertion of epithelial Na(+) channels

In the distal tubule, Na⁺ resorption is mediated by epithelial Na⁺ channels (ENaC). Hormones such as aldosterone, vasopressin, and insulin modulate ENaC membrane targeting, assembly, and/or kinetic activity, thereby regulating salt and water homeostasis. Insulin binds to a receptor on the basal membrane to initiate a signal transduction cascade that rapidly results in an increase in apical membrane ENaC. Current models of this signaling pathway envision diffusion of signaling intermediates from the basal to the apical membrane. This necessitates diffusion of several high-molecular-weight signaling elements across a three-dimensional space. Transduction of the insulin signal involves the phosphoinositide pathway, but how and where this lipid-based signaling pathway controls ENaC activity is not known. We used tagged channels, biosensor lipid probes, and intravital imaging to investigate the role of lipids in insulin-stimulated Na⁺ flux. Insulin-stimulated delivery of intracellular ENaC to apical membranes was concurrent with plasma membrane-limited changes in lipid composition. Notably, in response to insulin, phosphatidylinositol 3,4,5-trisphosphate (PIP₃) formed in the basolateral membrane, rapidly diffused within the bilayer, and crossed the tight junction to enter the apical membrane. This novel signaling pathway takes advantage of the fact that the lipids of the plasma membrane's inner leaflet are not constrained by the tight junction. Therefore, diffusion of PIP₃ as a signal transduction intermediate occurs within a planar surface, thus facilitating swift responses and confining and controlling the signaling pathway. phosphatidylinositol 3,4,5-trisphosphate; insulin-stimulated Na⁺ transport; metabolic syndrome; real-time confocal imaging     

Functional studies of the kidney of living animals using multicolor two-photon microscopy

Optical microscopy, when applied to livinganimals, provides a powerful means of studying cell biology in the mostphysiologically relevant setting. The ability of two-photon microscopyto collect optical sections deep into biological tissues has opened upthe field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way inwhich two-photon microscopy can be applied to intravital studies ofkidney physiology. Because the kidney is easily externalized withoutcompromising its function, microscopy can be used to evaluate variousaspects of renal function in vivo. These include cell vitality andapoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes andsimultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of imagescontaining uncharacterized autofluorescence. The studies described heredemonstrate the way in which two-photon microscopy can provide a levelof resolution previously unattainable in intravital microscopy,enabling kinetic analyses and physiological studies of the organs ofliving animals with subcellular resolution.

     

Inversin forms a complex with catenins and N-cadherin in polarized epithelial cells

Nephrogenesis starts with the reciprocal induction of two embryonically distinct analages, metanephric mesenchyme and ureteric bud. This complex process requires the refined and coordinated expression of numerous developmental genes, such as inv. Mice that are homozygous for a mutation in the inv gene (inv/inv) develop renal cysts resembling autosomal-recessive polycystic kidney disease. The gene locus containing inv has been proposed to serve as a common modifier for some human and rodent polycystic kidney disease phenotypes. We generated polyclonal antibodies to inversin to study its subcellular distribution, potential binding partners, and functional aspects in cultured murine proximal tubule cells. A 125-kDa inversin protein isoform was found at cell-cell junctions. Two inversin isoforms, 140- and 90-kDa, were identified in the nuclear and perinuclear compartments. Plasma membrane allocation of inversin is dependent upon cell-cell contacts and was redistributed when cell adhesion was disrupted after incubation of the cell monolayer with low-calcium/EGTA medium. We further show that the membrane-associated 125-kDa inversin forms a complex with N-cadherin and the catenins. The 90-kDa nuclear inversin complexes with beta-catenin. These findings indicate that the inv gene product functions in several cellular compartments, including the nucleus and cell-cell adhesion sites.     

Lysophosphatidic acid is a modulator of cyst growth in autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of multiple fluid-filled cysts predominately in the kidney tubules and liver bile ducts. Elucidation of mechanisms that control cyst growth will provide the basis for rational therapeutic intervention. We used electrophysiological methods to identify lysophosphatidic acid (LPA) as a component of cyst fluid and serum that stimulates secretory Cl- transport in the epithelial cell type that lines renal cysts. LPA effects are manifested through receptors located on the basolateral membrane of the epithelial cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in human ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage and/or leakage of vascular LPA into the interstitial space are capable of stimulating epithelial cell secretion resulting in cyst enlargement. These observations are particularly relevant to the rapid decline in renal function in late-stage disease and to the "third hit" hypothesis that renal injury exacerbates cyst growth.