Rapid turnover rate of phosphoinositides at the front of migrating MDCK cells

Phosphoinositides (PtdInss) play key roles in cell polarization and motility. With a series of biosensors based on Förster resonance energy transfer, we examined the distribution and metabolism of PtdInss and diacylglycerol (DAG) in stochastically migrating Madin-Darby canine kidney (MDCK) cells. The concentrations of phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), phosphatidylinositol (3,4)-bisphosphate, and DAG were higher at the plasma membrane in the front of the cell than at the plasma membrane of the rear of the cell. The difference in the concentrations of PtdInss was estimated to be less than twofold between the front and rear of the migrating MDCK cells. To decode the spatial activities of PtdIns metabolic enzymes from the obtained concentration maps of PtdInss, we developed a one-dimensional reaction diffusion model of PtdIns metabolism. In this model, the activities of phosphatidylinositol monophosphate 5-kinase, phosphatidylinositol 3-kinase, phospholipase C, and PIP₃ 5-phosphatases were higher at the plasma membrane of the front than at the plasma membrane of the rear of the cell. This result suggests that, although the difference in the steady-state level of PtdInss is less than twofold, PtdInss were more rapidly turned over at the front than the rear of the migrating MDCK cells.     

Associations between Diurnal 24-Hour Rhythm in Ambulatory Heart Rate Variability and the Timing and Amount of Meals during the Day Shift in Rotating Shift Workers

It has not hitherto been clarified whether there is an association between dietary behavior and circadian variation in autonomic nervous system activity among shift workers. This study examines diurnal 24-h rhythm in heart rate variability (HRV) and dietary behavior among rotating shift workers, while taking into account the sleep-wake cycle and physical activity. The subjects were 11 female and 2 male nurses or caregivers working in a rotating 2-shift system at a health care facility. All the subjects were asked to undergo 24-h electrocardiograph and step count recordings, and to record the time of each meal and the amounts of each food and beverage consumed. Coarse graining spectral analysis was used for approximately 10-min segments of HRV to derive the total power (TOT: >0.04 Hz) of the periodic components and the integrated power of periodic components in the low-frequency (LF: 0.04–0.15 Hz) and high-frequency (HF: >0.15 Hz) ranges. Then the ratio of HF power to TOT (HF nu) and the ratio of LF power to HF power (LF/HF) were calculated to assess cardiac vagal tone and cardiac sympathovagal balance, respectively. Single cosinor analysis was used to obtain 24-h period variations in both variables of HRV. Acrophases of HF nu and LF/HF expressed in time since awakening were significantly (p<0.05) delayed for subjects having breakfast at a later time after awakening. Multivariable regression analysis indicated that the timing of breakfast, the ratio of energy intake at dinner to total energy intake, and total energy intake were correlated to the acrophases of HF nu and/or LF/HF. These results suggest that the phase angle between circadian variation in cardiac autonomic nervous system activity and the sleep-wake cycle may be associated with dietary behavior in shift workers.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

Bombesin-like peptides in human small cell carcinoma of the lung

The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma.     

Activity of Rho-family GTPases during cell division as visualized with FRET-based probes

Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.