SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.     

Modeling the spatial reach of the LFP

The local field potential (LFP) reflects activity of many?neurons in the vicinity of the recording electrode and is therefore useful for studying local network dynamics. Much of the nature of the LFP is, however, still unknown. There are, for instance, contradicting reports on the spatial extent of the region generating the LFP. Here, we use a detailed biophysical modeling approach to investigate the size of the contributing region by simulating the LFP from a large number of neurons around the electrode. We find that the size of the generating region depends on the neuron morphology, the synapse distribution, and the correlation in synaptic activity. For uncorrelated activity, the LFP represents cells in a small region (within a radius of a few hundred micrometers). If the LFP contributions from different cells are correlated, the size of the generating region is determined by the spatial extent of the correlated activity.     

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor tissues, as discussed in invited review papers^1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is translucent and framed by fatty tissue, giving it a window-like appearance.The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in¹; (iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.Mast-cell-mediated angiogenesis was first demonstrated using this assay^3,4. The model demonstrates a high level of discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., angiogenesis-suppressive, neutral or angiogenesis-stimulating activities⁵); (ii) natural iron-unsaturated human lactoferrin, which stimulates VEGF-A-mediated angiogenesis⁶, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis⁷; and (iii) low-molecular-weight heparin fractions produced by various means^8,9. Moreover, the assay is highly suited to studies of the combined effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.Review papers (invited) discussing and appraising the assayNorrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).     

Progenitor cell injury after irradiation to the developing brain can be modulated by mild hypothermia or hyperthermia

Ionizing radiation induced acute cell death in the dentate gyrus subgranular zone (SGZ) and the subventricular zone (SVZ). Hypomyelination was also observed. The effects of mild hypothermia and hyperthermia for 4 h after irradiation (IR) were studied in postnatal day 9 rats. One hemisphere was irradiated with a single dose of 8 Gy and animals were randomized to normothermia (rectal temperature 36 degrees C for 4 h), hypothermia (32 degrees C for 4 h) or hyperthermia (39 degrees C for 4 h). Cellular injury, e.g. chromatin condensation and nitrotyrosine formation, appeared to proceed faster when the body temperature was higher. Caspase-3 activation was more pronounced in the hyperthermia group and nuclear translocation of p53 was less pronounced in the hypothermia group 6 h after IR. In the SVZ the loss of nestin-positive progenitors was more pronounced (48%) and the size was smaller (45%) in the hyperthermia group 7 days post-IR. Myelination was not different after hypo- or hyperthermia. This is the first report to demonstrate that hypothermia may be beneficial and that hyperthermia may aggravate the adverse side-effects after radiation therapy to the developing brain.     

Structural organization and Zn2+-dependent subdomain interactions involving autoantigenic epitopes in the Ring-B-box-coiled-coil (RBCC) region of Ro52

Ro52 is one of the major autoantigens targeted in the autoimmune disease Sj?gren syndrome. By sequence similarity, Ro52 belongs to the RING-B-box-coiled-coil (RBCC) protein family. Disease-related antibodies bind Ro52 in a conformation-dependent way both in the coiled-coil region and in the Zn2+-binding Ring-B-box region. Primarily associated with Sj?gren syndrome, Ro52 autoantibodies directed to a specific, partially structured epitope in the coiled-coil region may also induce a congenital heart block in the fetus of pregnant Ro52-positive mothers. To improve our understanding of the pathogenic effects of autoantibody binding to the Zn2+-binding region, a multianalytical mapping of its structural, biophysical, and antigenic properties is presented. Structure content and ligand binding of subregions, dissected by peptide synthesis and subcloning, were analyzed by fluorescence and circular dichroism spectroscopy. A novel matrix-assisted laser desorption ionization time-of-flight mass spectrometry strategy for time-resolved proteolysis experiments of large protein domains was developed to facilitate analysis and to help resolve the tertiary arrangement of the entire RBCC subregion. The linker region between the RING and B-box motifs is crucial for full folding, and Zn2+ affinity of the RING-B-box region is further protected in the entire RBCC region and appears to interact with the coiled-coil region. Murine monoclonal antibodies raised toward the RING-B-box region were primarily directed toward the linker, further supporting a highly functional role for the linker in a cellular environment. Taken together with our previous analysis of autoantigenic epitopes in the coiled-coil region, localization of autoantigenic epitopes in Ro52 appears closely related to molecular functionalities.     

DAP12 signaling directly augments proproliferative cytokine stimulation of NK cells during viral infections

NK cells vigorously proliferate during viral infections. During the course of murine CMV infection, this response becomes dominated by the preferential proliferation of NK cells that express the activation receptor Ly49H. The factors driving such selective NK cell proliferation have not been characterized. In this study, we demonstrate that preferential NK cell proliferation is dependent on DAP12-mediated signaling following the binding of Ly49H to its virally encoded ligand, m157. Ly49H signaling through DAP12 appears to directly augment NK cell sensitivity to low concentrations of proproliferative cytokines such as IL-15. The impact of Ly49H-mediated signaling on NK cell proliferation is masked in the presence of high concentrations of proproliferative cytokines that nonselectively drive all NK cells to proliferate.     

The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs

Wig-1 is a p53-induced zinc finger protein. Here we show that human Wig-1 binds long (>or=23 bp) dsRNAs with 5'-overhangs. The first zinc finger domain is necessary but not sufficient for this dsRNA-binding in vitro. Wig-1 also binds dsRNA in living cells via zinc fingers 1 and 2. Both zinc fingers 1 and 2 are important for Wig-1-mediated growth suppression. Moreover, Wig-1 binds 21 bp dsRNAs with 3'-protruding ends. These findings demonstrate that human Wig-1 can bind different types of dsRNAs, including dsRNAs resembling small interfering RNAs (siRNAs) and microRNAs (miRNAs), and indicate that dsRNA binding has a role in Wig-1-mediated regulation of cell growth.     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.     

A tissue model for the study of cell proliferation parameters in vivo

Summary An intact tissue model which can be used for detailed microscopic studies, quantitative cytochemical analysis and biochemical analysis has been explored, using a number of cell proliferation parameters. The preparations consist of mesenterial windows from rats dispersed on object slides. A technique for determining, in one and the same preparation, DNA content and mitotic activity of individual, selected cell types, and DNA synthesis in terms of incorporation of tritiated thymidine into DNA is described.Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty, University of LinköpingWe thank Brita Söderlund and Iréne Svensson for skilful technical assistance     

DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.