The dissociation of chicken erythrocyte deoxyribonucleoprotein and some properties of its partial nucleoproteins

Histones were completely dissociated from their native complex with DNA in 2.0m-sodium chloride. Histone fractions IIb, V and I were dissociated in 1.2m-sodium chloride, fractions V and I in 0.7m-sodium chloride and fraction I in 0.45m-sodium chloride. Repeated extraction of partial dRNP (deoxyribonucleoprotein) preparations with sodium chloride of the same concentration as that from which they were prepared resulted in release of histones that previously had remained associated with the DNA of the complex. Gradual removal of histones from dRNP was paralleled by an improvement in solubility, a decrease in wavelength of the u.v.-absorption minimum, and a fall in sedimentation coefficient of the remaining partial dRNP. X-ray diffraction patterns of partial dRNP preparations showed that removal of histone fractions I and V from dRNP did not destroy the super-coil structure of the dRNP, but further removal of histones did. Infrared spectra of partial dRNP preparations showed that in native dRNP histone fraction I was present in the form of extended, isolated polypeptide chains, and that the other histone fractions probably contain a helical component that lies roughly parallel to the polynucleotide chains in the double helix and an extended polypeptide component that is more nearly parallel to the DNA helix axis. An analysis of the sedimentation of partial dRNP preparations on sucrose gradients showed that native dRNP consists of DNA molecules each complexed with histone fractions of all types.     

Integrating genetic and epidemiological data to determine transmission pathways of foot-and-mouth disease virus

Estimating detailed transmission trees that reflect the relationships between infected individuals or populations during a disease outbreak often provides valuable insights into both the nature of disease transmission and the overall dynamics of the underlying epidemiological process. These trees may be based on epidemiological data that relate to the timing of infection and infectiousness, or genetic data that show the genetic relatedness of pathogens isolated from infected individuals. Genetic data are becoming increasingly important in the estimation of transmission trees of viral pathogens due to their inherently high mutation rate. Here, we propose a maximum-likelihood approach that allows epidemiological and genetic data to be combined within the same analysis to infer probable transmission trees. We apply this approach to data from 20 farms infected during the 2001 UK foot-and-mouth disease outbreak, using complete viral genome sequences from each infected farm and information on when farms were first estimated to have developed clinical disease and when livestock on these farms were culled. Incorporating known infection links due to animal movement prior to imposition of the national movement ban results in the reduction of the number of trees from 41472 that are consistent with the genetic data to 1728, of which just 4 represent more than 95% of the total likelihood calculated using a model that accounts for the epidemiological data. These trees differ in several ways from those constructed prior to the availability of genetic data.     

DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: Synthesis and antibacterial activity

The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.     

Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing

The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10(-4) per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.     

Glutamate-mediated synaptic transmission between neuron B4 and salivary cells ofHelisoma trivolvis

In this study, we have further characterized the morphology and physiology of the neuroglandular synapse between the identified buccal neuron, B4, and the salivary gland ofHelisoma. We demonstrate that the coupling coefficient between salivary cells within an individual acinus is approximately 1.0. We also demonstrate that synapses within the salivary gland are located near a superficial muscle layer. We examine the effects of glutamate on the salivary gland and on the B4-salivary gland EPSP.l-glutamate produces a transient, rapid onset depolarization of salivary gland cells. The response is mimicked by high concentrations ofl-homocysteic acid, but not by NMDA,l-aspartate,d-glutamate or kainate. The response is blocked by the presence ofl- ord-glutamate in the bath, but not by CNQX, DNQX, DGG,d-AP5, orl-AP3. The depolarization is primarily dependent on the presence of calcium in the bathing solution. When eitherl- ord-glutamate is present in the bathing solution, the amplitude of the B4-salivary gland EPSP is reversibly reduced. The similar pharmacological properties of the response of the salivary gland to glutamate and the B4 epsp indicate thatl-glutamate is a strong candidate for the fast excitatory neurotransmitter at theHelisoma neuroglandular synapse.     

The effects of homologous series of anaesthetics on a resting potassium conductance of the squid giant axon

The effects of n-alkanes (n-pentane to n-octane), n-alkanols (n-pentanol to n-undecanol) and two carboxylic esters (methyl pentanoate and methyl octanoate) on the conductance of squid giant axons in a high potassium, zero sodium bathing solution have been examined. Sodium and delayed rectifier potassium channels were as far as possible pharmacologically blocked. A substantial fraction of the measured conductance is attributed to a recently-described, voltage-independent, potassium channel. Anaesthetics block this channel but its sensitivity is markedly different from those of other squid axon ion channels.     

Hydroxamate Recognition During Iron Transport from Hydroxamate-Iron Chelates

Kinetics of radioactive iron transport from three structurally different secondary hydroxamate-iron chelates (schizokinen-iron, produced by Bacillus megaterium ATCC 19213; Desferal-iron, produced by an actinomycete; and aerobactin-iron, produced by Aerobacter aerogenes 62-1) revealed that B. megaterium SK11 (a mutant which cannot synthesize schizokinen) has a specific transport system for utilization of ferric hydroxamates with a recognition capacity based on the chemical structure of the hydroxamate. Both Desferal and schizokinen enhanced iron uptake in this organism; however, Desferal-iron delivered only one-sixth the level of iron incorporated from the schizokinen-iron chelate. Desferal-iron did not generate the rapid rates of iron transport noted with schizokinen-iron at elevated iron concentrations. Assays containing large excesses of either iron-free Desferal or iron-free schizokinen suggested that the iron-free hydroxamate may compete with the ferric hydroxamate for acceptance by the transport system although the system has greater affinity for the iron chelate. Aerobactin-iron did not stimulate iron uptake in B. megaterium SK11 and aerobactin inhibited growth of this organism, indicating that B. megaterium SK11 cannot efficiently process the aerobactin-iron chelate.     

Ion transfer across lipid membranes in the presence of gramicidin A: II. The ion selectivity

A detailed examination has been carried out of the ion selectivity of gramicidin A. Membranes of pure lipids and of various thicknesses have been used, and electrolyte concentrations have been varied over a wide range. It was first confirmed that anion permeability is low, even at very acid pH. Only in CaCl₂ was the transference number of the cation found to be less than unity (0.8). The measurement of biionic potentials was used to give the selectivity sequence for univalent cations. The results are compared with those already in the literature and with the conductivity data for the gramicidin single channel reported in a previous paper. The latter comparison suggests that a complex theoretical model is required to account for the kinetics of ion transfer.     

Formation, maintenance, and functional uncoupling of connections between identified Helisoma neurons in situ

Previous work with identified Helisoma neurons has characterized an array of neuroplastic responses to axotomy that include the generation of new neuritic outgrowth, the reinnervation of target organs, and the formation of new electrical synapses. These responses are not random, but rather occur in a precise, predictable manner under a variety of culture conditions. The present investigation demonstrates that specific identified neurons display similar neuroplastic "behavior" within the living animal. In response to in situ nerve crushes, neurons B4 and B5 generate new neuritic outgrowth, neuron B4 functionally reinnervates the salivary glands, and new electrical synapses form between the left and right neurons B5. The in situ paradigm employed in the present experiments made it possible to examine responses to axotomy over longer periods than in earlier studies with organ cultures. New B5R-B5L connections, previously found to be stable over the short term in culture, gradually decreased in strength in situ, and the cells effectively uncoupled by 8 weeks after axotomy. This uncoupling did not depend upon target reinnervation and occurred in the continued presence of neurites in the buccal commissure. It is suggested that the stability of new connections is related to whether the connection previously existed in the unperturbed nervous system. The similarities between the ability of identified neurons to grow and to form synaptic connections in situ and in culture suggests that neurons are endowed with a specific program of regenerative responses that can be expressed reliably in a wide variety of environmental conditions.