Regulation Of Ciliary Motility Research Articles

The role of -1984A> G adrenomedullin gene polymorphism in tubal ectopic pregnancy

Objective: Tubal ectopic pregnancy is a health issue that can cause maternal death in first trimester and our knowledge about its pathogenesis is limited. Adrenomedullin (ADM) is responsible from regulation of ciliary motility in fallopian tubes. Its expression is known to be reduced in tubal tissue in ectopic pregnancy. In this study, it was aimed to investigate the relationship between -1984A>G functional polymorphism affecting the expression level of the ADM gene and tubal ectopic pregnancies. Material and Methods: This prospective case-control study consisted of 64 women. Peripheral blood samples were obtained from 31 women diagnosed with tubal pregnancy (the study group) and 33 fertile women without a history of ectopic pregnancy (the control group). Genomic DNA was extracted from peripheral blood. The frequency of genotypes and alleles of â€"1984A>G ADM (rs3814700) gene polymorphism was examined by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) method. Results: For -1984A>G polymorphism the study group had an A allele frequency of 90% and a G allele frequency of 10%; the control group had an A allele frequency of 91.7% and a G allele frequency of 7.3% (p=0.75). AA genotype frequency was 80% and AG allele frequency was 20% in the study group while they were 83.3% and 16.7%, respectively, in the control group (p=0.73). There were no significant differences between the study and control groups with respect to allele and genotype frequencies. Conclusion: This study found no significant relationship between tubal ectopic pregnancy and -1984A>G ADM gene polymorphism. Further studies are needed to explore other factors that affect ADM expression.

Dynein axonemal intermediate chain 2 is required for formation of the left–right body axis and kidney in medaka

Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left–right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.

Morphogenesis of the node and notochord: The cellular basis for the establishment and maintenance of left–right asymmetry in the mouse

Establishment of left-right asymmetry in the mouse embryo depends on leftward laminar fluid flow in the node, which initiates a signaling cascade that is confined to the left side of the embryo. Leftward fluid flow depends on two cellular processes: motility of the cilia that generate the flow and morphogenesis of the node, the structure where the cilia reside. Here, we provide an overview of the current understanding and unresolved questions about the regulation of ciliary motility and node structure. Analysis of mouse mutants has shown that the motile cilia must have a specific structure and length, and that they must point posteriorly to generate the necessary leftward fluid flow. However, the precise structure of the motile cilia is not clear and the mechanisms that position cilia on node cells have not been defined. The mouse node is a teardrop-shaped pit at the distal tip of the early embryo, but the morphogenetic events that create the mature node from cells derived from the primitive streak are only beginning to be characterized. Recent live imaging experiments support earlier scanning electron microscopy (SEM) studies and show that node assembly is a multi-step process in which clusters of node precursors appear on the embryo surface as overlying endoderm cells are removed. We present additional SEM and confocal microscopy studies that help define the transition stages during node morphogenesis. After the initiation of left-sided signaling, the notochordal plate, which is contiguous with the node, generates a barrier at the embryonic midline that restricts the cascade of gene expression to the left side of the embryo. The field is now poised to dissect the genetic and cellular mechanisms that create and organize the specialized cells of the node and midline that are essential for left-right asymmetry.

Nasal and lower airway level of nitric oxide in children with primary ciliary dyskinesia.

Exhaled nitric oxide can be detected in exhaled air and is readily measured by chemiluminescence. It is thought to be involved in both the regulation of ciliary motility and host defence. Recently, upper airway NO has been found to be reduced in a small number of children with primary ciliary dyskinesia (PCD) and its measurement has been recommended as a diagnostic test for this condition. The aim of this study was to compare the levels of NO in the upper and lower airways in a larger number of children with proven PCD with those found in healthy children. Exhaled NO was measured in the upper airway by direct nasal sampling during a breath-hold and in the lower airway as the end-tidal plateau level, using a chemiluminescence NO analyser. Upper airway NO levels were significantly lower in PCD (n = 21) than in the healthy children (n = 60) (mean +/-SD, 97+/-193, 664+/-298 parts per billion (ppb), respectively, p<0.0001). In PCD, the lower airway NO levels were also reduced (2.17+/-1.18, 5.94+/-3.49 ppb, respectively, p<0.0001). The levels were not associated with steroid use and did not correlate with lung function. Although there was some overlap between normal children and those with primary ciliary dyskinesia with regard to lower airway NO, nasal NO discriminated between the two groups in all but one child in each group. Measurement of nasal NO therefore may be a useful screening test for primary ciliary dyskinesia.

Regulation of ciliary motility in Paramecium by cAMP and cGMP

Abstract Paramecium tetraurelia is a useful model system for the study of signal transduction mechanisms that couple changes in membrane potential to changes in ciliary motility, and cAMP and cGMP have been implicated as second messengers in this system. Changes in ciliary beat are correlated with changes in the membrane potential, V m , with hyperpolarizations resulting in increased beat frequency and increased forward swimming speed. The intercellular levels of cAMP and cGMP vary with V m , and increasing internal cAMP by microinjection or the use of membrane permeant analogs results in increased swimming speed. In addition, cAMP and cGMP modify the swimming behavior of permeabilized Paramecium , and the sensitivity of cilia to cAMP and cGMP depends upon the location of the cilia on the cell surface. However, increasing internal cAMP also results in hyperpolarization and increasing internal cAMP does not override the effect of V m . These results have lead to two conflicting models to describe the role of cAMP: (1) stimulus → hyperpolarization of V m → increased [cAMP] in → increased beat frequency, and (2) stimulus → increased [cAMP] in → hyperpolarization of V m → increased beat frequency. This review discusses the data that support these models and possible experimental approaches to resolve the paradox presented by these models.

Chapter 66 Isolation and Characterization of Protein Kinases from Paramecium Cilia

Publisher Summary Several protein kinases from the cilia of Paramecium tetraurelia have been isolated. Protein phosphorylation is involved in the regulation of ciliary motility in Paramecium. Second-messenger-dependent kinases include two forms of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), and two forms of a Ca2+-dependent protein kinase (CaPK). Biochemical evidence also suggests the presence of PKC in cilia. In addition, a family of casein kinases, not dependent on a second messenger for activation, has been isolated from cilia. The amount of each kinase obtained when purified from whole cells is much higher than from cilia, which contain only 2% of the total cell protein. Cilia are therefore an essential starting fraction for the isolation of certain kinases. This chapter describes the isolation and properties of several protein kinases from Paramecium cilia. In vitro phosphorylation of Paramecium proteins by Paramecium and bovine PKAs is distinctly different. Discussion includes the kinase assay buffers, chromatographic reagents, the assays, isolation of protein kinases from cilia—kinases A, G, soluble casein kinases, and axonemal casein kinase. Each ciliary kinase can be detected selectively in a fraction that contains several kinases by using unique conditions for optimal activation. Some fractionation may be required to detect individual kinases in crude cilia. Activation of histone-phosphorylating kinase activity in whole cilia by cAMP and 8-Br-cGMP is 2.7- and 1.6-fold, respectively, above background.

Identification of a family of casein kinases in Paramecium: biochemical characterization and cellular localization.

Protein phosphorylation is believed to play a role in the regulation of ciliary motility in the protozoan Paramecium tetraurelia. Five protein kinases from Paramecium, activated by cyclic nucleotides or Ca2+, have been characterized previously. We report here the identification of a family of second-messenger-independent casein kinases in Paramecium. Casein kinase activity was enriched in the soluble fraction of cilia, but there was also significant activity tightly associated with axonemes. Three ciliary casein kinase activities (soluble CKS1 and CKS2, and axonemal CKA) were separated by chromatography and characterized. The native forms of all three were monomeric, with molecular masses of 28-45 kDa as judged by in-gel kinase assays and sizing by gel filtration. CKS2 was inhibited by heparin, but CKA was unaffected and CKS1 was stimulated. All three activities preferred acidic substrates such as casein and phosvitin, but they could be distinguished by their preference for other substrates. Antibodies against mammalian casein kinase I recognized CKS1 and CKS2 in immunoblots (43 kDa), but did not stain CKA. The antibodies to casein kinase I were used to probe other cellular fractions. A 65 kDa antigen (particulate casein kinase, CKP) was enriched in particulate fractions of whole cells. This 65 kDa protein was found in isolated cell cortices, but was not present in the infraciliary lattice. This report represents the first biochemical identification of a casein kinase I family in protozoa.

Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.

Differential regulation of Paramecium ciliary motility by cAMP and cGMP.

cAMP and cGMP had distinct effects on the regulation of ciliary motility in Paramecium. Using detergent-permeabilized cells reactivated to swim with MgATP, we observed effects of cyclic nucleotides and interactions with Ca2+ on the swimming speed and direction of reactivated cells. Both cAMP and cGMP increased forward swimming speed two- to threefold with similar half-maximal concentrations near 0.5 microM. The two cyclic nucleotides, however, had different effects in antagonism with the Ca2+ response of backward swimming and on the handedness of the helical swimming paths of reactivated cells. These results suggest that cAMP and cGMP differentially regulate the direction of the ciliary power stroke.

Regulation of ciliary motility by membrane potential in Paramecium: A role for cyclic AMP

The membrane potential of Paramecium controls the frequency and direction of the ciliary beat, thus determining the cell's swimming behavior. Stimuli that hyperpolarize the membrane potential increase the ciliary beat frequency and therefore increase forward swimming speed. We have observed that 1) drugs that elevate intracellular cyclic AMP increased swimming speed 2-3-fold, 2) hyperpolarizing the membrane potential by manipulation of extracellular cations (e.g., K+) induced both a transient increase in, and a higher sustained level of cyclic AMP compared to the control, and 3) the swimming speed of detergent-permeabilized cells in MgATP was stimulated 2-fold by the addition of cyclic AMP. Our results suggest that the membrane potential can regulate intracellular cAMP in Paramecium and that control of swimming speed by membrane potential may in part be mediated by cAMP.