Isolation and characterization of cholangiocyte primary cilia

Primary cilia are distinct organelles expressed by many vertebrate cells, including cholangiocytes; however, their functions remain obscure. To begin to explore the physiological role of these organelles in the liver, we described the morphology and structure of cholangiocyte cilia and developed new approaches for their isolation. Primary cilia were present only in bile ducts and were not observed in hepatocytes or in hepatic arterial or portal venous endothelial cells. Each cholangiocyte possesses a single cilium that extends from the apical membrane into the bile duct lumen. In addition, the length of the cilia was proportional to the bile duct diameter. We reproducibly isolated enriched fractions of cilia from normal rat and mouse cholangiocytes by two different approaches as assessed by scanning electron, transmission electron, and confocal microscopy. The purity of isolated ciliary fractions was further analyzed by Western blot analysis using acetylated tubulin as a ciliary marker and P2Y(2) as a nonciliary cell membrane marker. These novel techniques produced enriched ciliary fractions of sufficient purity and quantity for light and electron microscopy and for biochemical analyses. They will permit further assessment of the role of primary cilia in normal and pathological conditions.     

Provision of nestboxes raises the breeding density of Great Tits Parus major equally in coniferous and deciduous woodland

Nestbox provision is a technique used to increase nest-site availability for secondary cavity-nesting birds. However, little is known about the demographic consequences of nestbox provision in different habitat types. To assess how nestbox provision affects the density of hole-nesting birds simultaneously in two contrasting habitats, we compared the breeding density of Great Tits along transects without nestboxes with that in transects where nestboxes were provided. Although the initial density of breeders was considerably higher in the deciduous habitat than in the coniferous habitat, provision of nestboxes increased density by a similar number of additional pairs in each habitat type. Thus, the provision of nestboxes in managed coniferous forests may be as effective in increasing the breeding opportunities of cavity nesters as in deciduous stands. Moreover, previous research showed that pairs in deciduous habitat with nestboxes have consistently lower breeding success than those in coniferous habitat with nestboxes. It is possible that the addition of nestboxes in the preferred habitat increased density to such an extent that density-dependent effects became apparent.     

Type II Opsins: Evolutionary Origin by Internal Domain Duplication?

Opsins are a large group of proteins with seven transmembrane segments (TMSs) that are found in all domains of life. There are two types of opsins that are sometimes considered nonhomologous: type I is known from prokaryotes and some eukaryotes, while type II is known only from Eumetazoan animals. Type II opsins are members of the family of G-protein coupled receptors (GPCRs), which facilitate signal transduction across cell membranes. While previous studies have concluded that multiple transmembrane-containing protein families-including type I opsins-originated by internal domain duplication, the origin of type II opsins has been speculated on but never tested. Here we show that type II opsins do not appear to have originated through a similar internal domain duplication event. This provides further evidence that the two types of opsins are nonhomologous, indicating a convergent evolutionary origin, in which both groups of opsins evolved a seven-TM structure and light sensitivity independently. This convergence may indicate an important role for seven-TM protein structure for retinal-based light sensitivity.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Proteolytic cleavage and nuclear translocation of fibrocystin is regulated by intracellular Ca2+ and activation of protein kinase C

Fibrocystin, a type I membrane protein of unknown function, is the protein affected in the autosomal recessive form of polycystic kidney disease. Here we show that fibrocystin undergoes regulated proteolysis. Several proteolytic cleavages occur within the predicted ectodomain, whereas at least one cleavage occurs within the cytoplasmic portion. The latter generates a C-terminal intracellular fragment that harbors the nuclear localization signal KRKVSRLAVTGERTATPAPKIPRIT and translocates to the nucleus. Proteolytic cleavage of fibrocystin occurs constitutively in long term cultures of polarized inner medullary collecting duct cells (mIMCD-3). Activation of protein kinase C and release of intracellular Ca2+ are required for proteolysis under these conditions. In short term cultures of human embryonic kidney 293 cells (HEK-293), proteolytic cleavage of fibrocystin can be elicited by stimulation of intracellular Ca2+ release or activation of protein kinase C. These results identify a novel Ca2+-dependent pathway that signals from fibrocystin located in the cell membrane to the nucleus.