Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells

The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy.     

Detection and identification of Escherichia coli,Shigella, and Salmonella by microarrays using the gyrB gene

Commonly, 16S ribosome RNA (16S rRNA) sequence analysis has been used for identifying enteric bacteria. However, it may not always be applicable for distinguishing closely related bacteria. Therefore, we selected gyrB genes that encode the subunit B protein of DNA gyrase (a topoisomerase type II protein) as target genes. The molecular evolution rate of gyrB genes is higher than that of 16S rRNA, and gyrB genes are distributed universally among bacterial species. Microarray technology includes the methods of arraying cDNA or oligonucleotides on substrates such as glass slides while acquiring a lot of information simultaneously. Thus, it is possible to identify the enteric bacteria easily using microarray technology. We devised a simple method of rapidly identifying bacterial species through the combined use of gyrB genes and microarrays. Closely related bacteria were not identified at the species level using 16S rRNA sequence analysis, whereas they were identified at the species level based on the reaction patterns of oligonucleotides on our microarrays using gyrB genes.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

Local caspase activation interacts with Slit-Robo signaling to restrict axonal arborization

In addition to being critical for apoptosis, components of the apoptotic pathway, such as caspases, are involved in other physiological processes in many types of cells, including neurons. However, very little is known about their role in dynamic, nonphysically destructive processes, such as axonal arborization and synaptogenesis. We show that caspases were locally active in vivo at the branch points of young, dynamic retinal ganglion cell axonal arbors but not in the cell body or in stable mature arbors. Caspase activation, dependent on Caspase-3, Caspase-9, and p38 mitogen-activated protein kinase (MAPK), rapidly increased at branch points corresponding with branch tip addition. Time-lapse imaging revealed that knockdown of Caspase-3 and Caspase-9 led to more stable arbors and presynaptic sites. Genetic analysis showed that Caspase-3, Caspase-9, and p38 MAPK interacted with Slit1a-Robo2 signaling, suggesting that localized activation of caspases lie downstream of a ligand receptor system, acting as key promoters of axonal branch tip and synaptic dynamics to restrict arbor growth in vivo in the central nervous system.     

Motor Nerve Terminals As the Site of Initial Functional Changes After Denervation

For the cat soleus nerve-muscle system, motor nerve section 48 hr prior to in situ experiment causes certain characteristic transmission losses. Responses to repetitive stimulation are sharply altered: The capacity to transmit iterative stimulation is severely reduced; post-tetanic potentiation and the post-tetanic repetition of soleus nerve terminals responsible for it are also greatly impaired; a phenomenon of post-tetanic depression was frequently observed. However, function of the extramuscular axons appears normal and single impulse transmission is usually not seriously affected. The loss of reactivity to repetitive stimulation has been traced to soleus motor nerve terminals. In view of these data and the known absence of denervation hypersensitivity at this time, the earliest functional failure may be said to occur in the unmyelinated terminals. This subacutely denervated preparation therefore offers a simple means of evaluating motor nerve terminal responsiveness.     

High-dose zolpidem abuse in a patient with insomnia comorbid with major depressive disorder

Sleep and Biological Rhythms - A 39-year-old married woman was referred to a psychiatry outpatient clinic for treatment of major depressive disorder (MDD). The dose of zolpidem she had been taking...