Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.     

Dynamic signal processing by ribozyme-mediated RNA circuits to control gene expression

Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the de novo design of RNA modules enabling dynamic signal processing in live cells. This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.     

Synthesis and α-Glucosidase II inhibitory activity of valienamine pseudodisaccharides relevant to N-glycan biosynthesis

Valienol-derived allylic C-1 bromides have been used as carbaglycosyl donors for α-xylo configured valienamine pseudodisaccharide synthesis. We synthesised valienamine analogues of the Glc(α1→3)Glc and Glc(α1→3)Man disaccharides representing the linkages cleaved by α-Glucosidase II in N-glycan biosynthesis. These (N1→3)-linked pseudodisaccharides were found to have some α-Glucosidase II inhibitory activity, while two other (N1→6)-linked valienamine pseudodisaccharides failed to inhibit the enzyme.     

Diabetic changes in the redox status of the microsomal protein folding machinery

Changes in assisted protein folding are largely unexplored in diabetes. In the present studies, we have identified a reductive shift in the redox status of rat liver microsomes after 4 weeks of streptozotocin-induced diabetes. This change was reflected by a significant increase in the total- and protein-sulfhydryl content, as well as in the free sulfhydryl groups of the major protein disulfide isomerases (PDIs), the 58 kDa PDI and the 57 kDa ERp57 but not other chaperones. A parallel decrease of the protein-disulfide oxidoreductase activity was detected in the microsomal fraction of diabetic livers. The oxidant of PDI, Ero1-Lalpha showed a more oxidized status in diabetic rats. Our results reveal major changes in the redox status of the endoplasmic reticulum and its redox chaperones in diabetic rats, which may contribute to the defective protein secretion of the diabetic liver.     

Lognormal distribution of firing time and rate from a single neuron?

Even a single neuron may be able to produce significant lognormal features in its firing statistics due to noise in the charging ion current. A mathematical scheme introduced in advanced nanotechnology is relevant for the analysis of this mechanism in the simplest case, the integrate-and-fire model with white noise in the charging ion current.     

Beta 2-agonist treatment enhances uterine oxytocin receptor mRNA expression in pregnant rats

The objective of this study was to disclose an interaction between Beta(2)-adrenergic (Beta(2)-ARs) and oxytocin (OT) receptors (OTRs) in the late-pregnant rat uterus. We investigated the level of uterine OTR mRNA expression after the administration of Beta(2)-AR agonists fenoterol and hexoprenaline to rats from day 18 to 22 of pregnancy, and also tested the effect of fenoterol on uterine explants. Hexoprenaline induced a maximum 24% increase of OTR mRNA. Fenoterol in vivo elicited a maximum 125% increase of OTR mRNA, in vitro produced a maximum fourfold increase in OTR mRNA. In fenoterol-treated rats the maximal contractility increasing effect of OT on isolated uterine rings was significantly higher than in intact term pregnant rats, but the EC50 values were not statistically different. It was concluded that the enhanced expression of OTR mRNA induced by Beta(2)-agonists in the late-pregnant rat uterus may be a possible drawback to effective therapy of preterm uterine contractions with Beta(2)-agonists.