Making human nasal cilia beat in the cold: a real time assay for cell signalling

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.     

Nonparametric tests for differential gene expression and interaction effects in multi-factorial microarray experiments

Background  

Numerous nonparametric approaches have been proposed in literature to detect differential gene expression in the setting of two user-defined groups. However, there is a lack of nonparametric procedures to analyze microarray data with multiple factors attributing to the gene expression. Furthermore, incorporating interaction effects in the analysis of microarray data has long been of great interest to biological scientists, little of which has been investigated in the nonparametric framework.     

4alpha-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC

RIO1 and Rio-related proteins display little similarity of primary sequence with conventional protein kinases. Based on secondary structure alignments, we show that it contains the domain structure (subdomains I-XI) and conserved secondary structure elements found in conventional protein kinases. We show that recombinant wild-type Rio1p isolated from Escherichia coli displays kinase activity which depends on autophosphorylation and magnesium or manganese as ATP-activating ions. An initial biochemical characterization of Rio1p is presented.