Neutron and cobalt-60 gamma irradiation produce similar changes in DNA supercoiling

The effects of 60Co gamma-ray and d(20 MeV)Be neutron irradiation on DNA supercoiling have been studied using a nucleoid rewinding technique. Irradiation of viable CHO AA8 cells on ice with from 4 to 25 Gy of either radiation produced a similar resistance to rewinding of nuclear supercoils after treatment with ethidium bromide. The restitution from the effects of 12 Gy of either radiation was also similar, leaving no detectable residual damage. The discrepancy between these data and the reduced ability of neutrons to produce DNA breaks, as defined by the alkaline elution assay, is explained by the discontinuous deposition of dose associated with neutron irradiation. It is suggested from a microdosimetric analysis that the neutron radiation interacts with DNA at sites on average 5-10 times further apart than the interactions with gamma rays. The long DNA sequences which results after neutron irradiation are consequently eluted inefficiently during alkaline elution, giving a reported RBE of approximately 0.3. Restrictions in the rewinding of individual supercoils are not dependent on the interionization distance and thus give rise to an RBE of approximately 1. Furthermore, the complete removal of DNA damage, as measured by this technique, supports the hypothesis that neutron toxicity is associated with incorrect, not incomplete, rejoining of the DNA molecule.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Structure-function analysis of interleukin-5 utilizing mouse/human chimeric molecules.

Interleukin-5 (IL5) is a T cell derived glycoprotein that stimulates eosinophil production and activation. In the mouse, but apparently not in the human, it is active on B cells. The murine and human IL5 polypeptides exhibit 70% sequence similarity and yet display distinct species-specific activity. Whilst mouse and human IL5 are equally active in human cell assays, human IL5 is 100-fold less active than murine IL5 in mouse cell assays. Two restriction sites were utilized to divide the human and mouse sequences into three fragments. Hybrid molecules consisting of all combinations of these fragments were constructed and expressed. In the human cell assays [using bone marrow or the erythroleukaemic cell line (TF-1)] all the hybrid proteins generated activity comparable to that of the human and mouse IL5. This implies that replacing different domains does not result in detrimental effects to the tertiary structure of the molecule. In the mouse cell assays [using bone marrow or the pro-B cell line (B13)] the hybrids clearly identified the importance of residues in the C terminus for biological activity. The changing of only eight residues in this region of human IL5, to those of mouse IL5, resulted in the hybrid producing biological activity comparable to mouse IL5. In addition, competition binding assays showed that this region probably interacts with the receptor.     

Activity-dependent induction of facilitation,depression, and post-tetanic potentiation at an insect central synapse

Summary In Manduca sexta larvae, sensory neurons innervating planta hairs on the tips of the prolegs make monosynaptic excitatory connections with motoneurons innervating proleg retractor muscles. Tactile stimulation of the hairs evokes reflex retraction of the proleg. In this study we examined activity-dependent changes in the amplitude of the excitatory postsynaptic potentials (EPSPs) evoked in a proleg motoneuron by stimulation of individual planta hair sensory neurons. Deflection of a planta hair caused a phasic-tonic response in the sensory neuron, with a mean peak instantaneous firing frequency of >300 Hz, and a tonic firing rate of 10–20 Hz. Direct electrical stimulation was used to activate individual sensory neurons to fire at a range of frequencies including those observed during natural stimulation of the hair. At relatively low firing rates (e.g., 1 Hz), EPSP amplitude was stable indefinitely. At higher instantaneous firing frequencies (>10 Hz), EPSPs were initially facilitated, but continuous stimulation led rapidly to synaptic depression. High-frequency activation of a sensory neuron could also produce post-tetanic potentiation, in which EPSP amplitude remained elevated for several min following a stimulus train. Facilitation, depression, and post-tetanic potentiation all appeared to be presynaptic phenomena. These activity-dependent changes in sensory transmission may contribute to the behavioral plasticity of the proleg withdrawal reflex observed in intact insects.Abbreviations ACh acetylcholine - AChE acetylcholine esterase - CNS central nervous system - EPSP excitatory postsynaptic potential - I _h injected hyperpolarizing current - LTP long-term potentiation - PPR principal planta retractor motoneuron - PTP post-tetanic potentiation - R _in input resistance - V _h hyperpolarized potential - V _m membrane potential - VN ventral nerve - VNA anterior branch of the ventral nerve - V _r resting potential.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.