Limits of Calcium Clearance by Plasma Membrane Calcium ATPase in Olfactory Cilia

BACKGROUND: In any fine sensory organelle, a small influx of Ca(2+) can quickly elevate cytoplasmic Ca(2+). Mechanisms must exist to clear the ciliary Ca(2+) before it reaches toxic levels. One such organelle has been well studied: the vertebrate olfactory cilium. Recent studies have suggested that clearance from the olfactory cilium is mediated in part by plasma membrane Ca(2+)-ATPase (PMCA). PRINCIPAL FINDINGS: In the present study, electrophysiological assays were devised to monitor cytoplasmic free Ca(2+) in single frog olfactory cilia. Ca(2+) was allowed to enter isolated cilia, either through the detached end or through membrane channels. Intraciliary Ca(2+) was monitored via the activity of ciliary Ca(2+)-gated Cl(-) channels, which are sensitive to free Ca(2+) from about 2 to 10 microM. No significant effect of MgATP on intraciliary free Ca(2+) could be found. Carboxyeosin, which has been used to inhibit PMCA, was found to substantially increase a ciliary transduction current activated by cyclic AMP. This increase was ATP-independent. CONCLUSIONS: Alternative explanations are suggested for two previous experiments taken to support a role for PMCA in ciliary Ca(2+) clearance. It is concluded that PMCA in the cilium plays a very limited role in clearing the micromolar levels of intraciliary Ca(2+) produced during the odor response

Rapid Mapping of Active Site of KSI by Paramagnetic NMR

Active site mapping has been done for Delta(5)-3-ketosteroid isomerase (KSI) by analyses of paramagnetic effect on H-1-N-15 HSQC spectra using 4-hydroxyl-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO) and an intermediate analog (equilenin). Our result revealed that residues in hydrophobic cavity of KSI, particularly active site region, mainly experienced a high line-broadening effect of NMR signal with HyTEMPO, while they experienced full recovery of a lineshape upon the addition of equilenin. The mapped region was very similar to the active site of KSI as described by the crystal structure. These observations indicate that a combined use of paramagnetic reagent and substrate (or analog) could rapidly identify the residues in potential active site of KSI, and can be applied to the analysis of both active site and function in unknown protein.110sciescopuskc

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice

Preeclampsia leads to dysregulation of various signaling pathways in placenta.

OBJECTIVES: To compare gene expression profiles of placentas from preeclamptic and normal pregnancies. STUDY DESIGN: We performed microarray experiments to analyze genome-wide expression profiling for 10 placentas from pregnant women with preeclampsia and 10 placentas from women who experienced noncomplicated pregnancies (CON), and to identify dysregulated signaling pathways as well as genes in preeclampsia. RT-PCR, real-time RT-PCR and/or immunofluorescence analyses were performed to validate the data obtained from microarray experiments. Results: Unsupervised hierarchical clustering showed heterogeneity of preeclampsia at the molecular levels, whereas expression profiles of preeclampsia are distinctly different from those of CON. A list of genes which are differentially expressed between preeclampsia and CON included well known preeclampsia markers, such as Flt-1, leptin, HTRA1 and SIGLEC6. Gene Set Enrichment Analysis, a pathway-oriented analysis method for expression profiles, provided evidence that a number of biological activities including pathways that regulate actin cytoskeleton, TGF\u3b2 signaling, oxidative phosphorylation, and proteasome activity were aberrantly either up-regulated or down-regulated in preeclampsia. RT-PCR and real-time-RT-PCR for genes contributing these biological pathways (gene sets) enriched in either CON or preeclampsia reinforced that these biological processes were systemically dysregulated in preeclampsia. CONCLUSIONS: Genome-wide expression profiles of preeclampsia showed heterogeneous characteristics of preeclampsia at the molecular levels. Dysregulation of genes and biological pathways could contribute to abnormal behavior of preeclmapsia. Our results will help further understand underlying mechanisms by which preeclampsia affects placental physiology. \ua9 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins