Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.     

Learning in the fast lane: new insights into neuroplasticity

The timescale of structural remodeling that accompanies functional neuroplasticity is largely unknown. Although structural remodeling of human brain tissue is known to occur following long-term (weeks) acquisition of a new skill, little is known as to what happens structurally when the brain needs to adopt new sequences of procedural rules or memorize?a cascade of events within minutes or hours. Using diffusion tensor imaging (DTI), an MRI-based framework, we examined subjects before and after a spatial learning and memory task. Microstructural changes (as reflected by DTI measures) of limbic system structures (hippocampus and parahippocampus) were significant after only 2?hr of training. This observation was also found in a supporting rat study. We conclude that cellular rearrangement of neural tissue can be detected by DTI, and that this modality may allow neuroplasticity to be localized over short timescales.     

Graft versus neuroblastoma reaction is efficiently elicited by allogeneic bone marrow transplantation through cytolytic activity in the absence of GVHD

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.     

The Ferrichrome Uptake Pathway in Pseudomonas aeruginosa Involves an Iron Release Mechanism with Acylation of the Siderophore and Recycling of the Modified Desferrichrome

The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. ⁵⁵Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-⁵⁵Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-⁵⁵Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli.Iron is essential for life for practically all living organisms and plays a number of key roles in biology. DNA and RNA synthesis, glycolysis, energy generation by electron transport, nitrogen fixation, and photosynthesis are examples of processes in which iron-containing enzymes play vital roles. However, under physiological conditions iron forms highly insoluble ferric hydroxide complexes, which severely limits its bioavailability. To overcome the problem of iron inaccessibility, bacteria excrete high-affinity iron chelators termed siderophores, which are able to solubilize iron and deliver it into the cells (3, 64).Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is capable of infecting a wide variety of animal, insects, and plants. As a human pathogen, P. aeruginosa is the leading source of Gram-negative nosocomial infections (59) and causes chronic lung infections in approximately 90% of individuals suffering from cystic fibrosis (40). Under iron-limited conditions, P. aeruginosa produces two major siderophores, pyoverdine (PVD) (62) and pyochelin (PCH) (15). P. aeruginosa is also capable of utilizing numerous siderophores secreted by other microorganisms: pyoverdins from other pseudomonas, enterobactin (49), cepabactin (45), mycobactin and carboxymycobactin (38), fungal siderophores (ferrichrome [39]; deferrioxamines [39, 60]; and desferrichrysin, desferricrocin, and coprogen [44]), and natural occurring chelators such as citrate (14, 23) (for a review, see reference 47).In Gram-negative bacteria, the uptake of ferrisiderophores always involves a specific transporter at the level of the outer membrane (4). The energy required for this process is provided by the proton motive force (PMF) of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (21, 51, 63). In silico analysis of the P. aeruginosa genome (http://www.pseudomonas.com) revealed 32 genes encoding putative TonB-dependent transporters (13), of which only 12 are involved in metal (mostly iron) uptake (38). FpvA and FpvB are the outer membrane transporters involved in the uptake of PVD-Fe (19, 48), and FptA transports PCH-Fe (25). Concerning the heterologous siderophores, there are two transporters, FoxA and FiuA, involved in the transport of ferrioxamine B and ferrichrome (39). The mechanism involved in the translocation of ferrisiderophores across the outer membrane by the TonB-dependent transporters has been studied mostly in E. coli (for a review, see reference 5) and in the case of P. aeruginosa has been studied only for the FpvA/PVD and the FptA/PCH systems. The structures of FpvA (8, 11, 65) and FptA (12) have been solved and their interactions with PVD and PCH investigated at the molecular level (26, 27, 45, 53). Three tonB genes, encoding the energy coupler TonB, have been found in the P. aeruginosa genome, i.e., tonB1, tonB2, and tonB3. Disruption of tonB1 abrogates PVD- and PCH-mediated iron uptake (50, 58) and heme uptake (67). Inactivation of tonB2 has no adverse effect on iron or heme acquisition, but tonB1 tonB2 double mutants are more compromised with respect to growth in iron-restricted medium than is a single tonB1 knockout mutant (67). Mutation of tonB3 appears to result in defective twitching motility (28), and the gene product is most likely not involved in iron uptake.In P. aeruginosa, many ferrisiderophore outer membrane transporters are also involved in a signaling cascade regulating the expression of genes involved in iron uptake. This is the case for FpvA (PVD uptake), FoxA (ferrioxamine), and FiuA (ferrichrome) (38, 39, 43, 61). Such a signaling cascade involves an extracytoplasmic function (ECF) sigma factor and an inner membrane anti-sigma factor. Equivalent cell surface signaling is present in Escherichia coli for ferricitrate uptake by FecA but not for ferrichrome, ferrioxamine, and enterobactin uptake by FhuA, FhuE, and FepA, respectively.Little is known about the translocation of ferrisiderophores across the inner membrane in P. aeruginosa. In E. coli, this step involves a specific ABC transporter for almost every siderophore used by this bacterium: FhuBCD for the uptake of ferrichrome and ferrioxamine (33-36), FecBCD for the uptake of ferricitrate (6, 56) and, FepBCDG for the uptake of ferrienterobactin (9). In P. aeruginosa, the only characterized inner membrane siderophore transport protein is FptX, a proton motive force-dependent permease, which functions in PCH-Fe utilization (16). The inner membrane FoxB is involved in the utilization of ferrichrome and ferrioxamine B, but it remains to be determined whether this protein functions in the transport of ferrisiderophore or in the release of iron from ferrichrome or ferrioxamine (17). The genome clearly shows a fepBCDG homologue for the transport of ferrienterobactin. For the other iron uptake pathways present in P. aeruginosa, the transporters involved at the level of the inner membrane have not been identified. An import ABC transporter is present in the pvd locus (PA2407 to PA2410; http://www.pseudomonas.com), but its mutation does not affect PVD-Fe uptake (46).In P. aeruginosa, the mechanism of ferrisiderophore dissociation has been investigated only for the PVD pathway. This step occurs in the periplasm by a mechanism involving no chemical siderophore modification but involving a reduction of iron and a recycling of the siderophore into the extracellular medium by the PvdRT-OpmQ efflux pump (20, 54, 66). In E. coli, the mechanism of ferrisiderophore dissociation has been investigated for the ferrichrome and ferrienterobactin pathways. Iron release from ferrichrome occurs in the cytoplasm and probably involves iron reduction (41) followed by acetylation of the siderophore and its recycling into the growth medium (24). For the ferrienterobactin pathway, a cytoplasmic esterase hydrolyzes the siderophore (7).In the present work, we have investigated the ferrichrome pathway in P. aeruginosa using both ferrichrome and a fluorescently labeled biomimetic ferrichrome analogue. We evaluated the siderophore properties of the fluorescent analogue and identified the different transporters involved in the uptake across the outer and inner membranes. Furthermore, we demonstrated that following ferrichrome uptake, iron is released from the siderophore by a mechanism involving an acetylation of the chelator and the modified desferrichrome is secreted back into the growth medium.     

Jane: a new tool for the cophylogeny reconstruction problem

Background  

This paper describes the theory and implementation of a new software tool, called Jane, for the study of historical associations. This problem arises in parasitology (associations of hosts and parasites), molecular systematics (associations of orderings and genes), and biogeography (associations of regions and orderings). The underlying problem is that of reconciling pairs of trees subject to biologically plausible events and costs associated with these events. Existing software tools for this problem have strengths and limitations, and the new Jane tool described here provides functionality that complements existing tools.     

Comparison of neurolin (ALCAM) and neurolin-like cell adhesion molecule (NLCAM) expression in zebrafish

Many immunoglobulin (Ig)-superfamily cell adhesion molecules influence skeletal muscle formation. In Drosophila, dumbfounded (duf/kirre), irreC, sticks and stones and hibris encode related Ig-family proteins expressed in subsets of neurons and muscle precursor cells. The family mediates cell migration, axon guidance and fusion of myoblasts. Despite the importance of these genes in invertebrate myogenesis, no obvious functional parallels are known in vertebrate myogenesis. Here we investigate the gene expression pattern and phylogenetic and protein-structural relationships of the duf-related molecules neurolin and neurolin-like cell adhesion molecule (NLCAM), members of the activated leukocyte cell adhesion molecule (ALCAM) sub-family of Ig-molecules. These proteins are among the closest to Duf/Kirre by sequence. During zebrafish development, neurolin is expressed in subsets of somite and muscle cells, heart and numerous sites of neuronal maturation. The new ALCAM-family member, NLCAM, appears to have arisen by duplication of neurolin/ALCAM. NLCAM is expressed widely during gastrulation, particularly in the nascent neural plate, but later becomes predominantly expressed in sites of muscle and nerve maturation and in the fin fold. The expression of each gene is often in groups of cells in similar parts of the embryo; for example, in the region of Rohon Beard neurons, trigeminal ganglion and fusing fast and migrating slow muscle fibres. However, expression can also be distinct and dynamic; for example, muscle pioneer fibres express neurolin but not NLCAM at high level. Both molecules are expressed in subsets of muscle precursors at times prior to fusion.