Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.     

Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos

Genomic imprinting regulates parental-specific expression of particular genes and is required for normal mammalian development. How imprinting is established during development is, however, largely unknown. To address this question, we studied the mouse Kcnq1 imprinted cluster at which paternal-specific silencing depends on expression of the noncoding RNA Kcnq1ot1. We show that Kcnq1ot1 is expressed from the zygote stage onward and rapidly associates with chromatin marked by Polycomb group (PcG) proteins and repressive histone modifications, forming a discrete repressive nuclear compartment devoid of RNA polymerase II, a configuration also observed at the Igf2r imprinted cluster. In this compartment, the paternal Kcnq1 cluster exists in a three-dimensionally contracted state. In vivo the PcG proteins Ezh2 and Rnf2 are independently required for genomic contraction and imprinted silencing. We propose that the formation of a parental-specific higher-order chromatin organization renders imprint clusters competent for monoallelic silencing and assign a central role to PcG proteins in this process.     

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Background

Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.

Methodology/Principal Findings

Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca²⁺ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.

Conclusions/Significance

These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.     

Origin of cells and network information

All cells are derived from one cell, and the origin of different cell types is a subject of curiosity. Cells construct life through appropriately timed networks at each stage of development. Communication among cells and intracellular signaling are essential for cell differentiation and for life processes. Cellular molecular networks establish cell diversity and life. The investigation of the regulation of each gene in the genome within the cellular network is therefore of interest. Stem cells produce various cells that are suitable for specific purposes. The dynamics of the information in the cellular network changes as the status of cells is altered. The components of each cell are subject to investigation.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli

Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 °C. Soluble-GLuc remained fully folded until 40 °C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 °C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.     

Cohesins determine the attachment manner of kinetochores to spindle microtubules at meiosis I in fission yeast

During mitosis, sister kinetochores attach to microtubules that extend to opposite spindle poles (bipolar attachment) and pull the chromatids apart at anaphase (equational segregation). A multisubunit complex called cohesin, including Rad21/Scc1, plays a crucial role in sister chromatid cohesion and equational segregation at mitosis. Meiosis I differs from mitosis in having a reductional pattern of chromosome segregation, in which sister kinetochores are attached to the same spindle (monopolar attachment). During meiosis, Rad21/Scc1 is largely replaced by its meiotic counterpart, Rec8. If Rec8 is inactivated in fission yeast, meiosis I is shifted from reductional to equational division. However, the reason rec8Delta cells undergo equational rather than random division has not been clarified; therefore, it has been unclear whether equational segregation is due to a loss of cohesin in general or to a loss of a specific requirement for Rec8. We report here that the equational segregation at meiosis I depends on substitutive Rad21, which relocates to the centromeres if Rec8 is absent. Moreover, we demonstrate that even if sufficient amounts of Rad21 are transferred to the centromeres at meiosis I, thereby establishing cohesion at the centromeres, rec8Delta cells never recover monopolar attachment but instead secure bipolar attachment. Thus, Rec8 and Rad21 define monopolar and bipolar attachment, respectively, at meiosis I. We conclude that cohesin is a crucial determinant of the attachment manner of kinetochores to the spindle microtubules at meiosis I in fission yeast.