Hexavalent capsomers of herpes simplex virus type 2: symmetry, shape, dimensions, and oligomeric status

The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface.     

The dependence of airway smooth muscle on extracellular Ca2+ for contraction is influenced by the presence of cartilage

Isolated guinea-pig and rabbit airway smooth muscle preparations lacking cartilage are less able to contract, in response to methacholine, histamine and K+, in the absence of extracellular Ca2+ than cartilage-containing preparations removed from the same animal. Cartilage apparently provides utilizable Ca2+ for contraction of airway smooth muscle. The presence of cartilage, therefore, affects the apparent dependence of the isolated smooth muscle on extracellular Ca2+ for contraction.     

DNA-binding proteins present in varicella-zoster virus-infected cells.

DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA.     

The role of electrogenic xylem pumps in K+ absorption from the zylem of Vigna unguiculata: the effects of auxin and fusicoccin

Abstract We tested the hypothesis that electrogenic ion pumps, working at the parenchyma symplast/xylem interface of pea hypocotyls, provide the driving force for K⁺ uptake from the xylem. Solutions of known composition were perfused through a hypocotyl segment. The K⁺ activity of the solution flowing out of the xylem (K⁺_out) increased (i.e. K⁺ uptake decreased) when aerobic respiration was inhibited by lack of O₂, and this was preceded by a decrease in V_px (electrical potential difference between parenchyma symplast and xylem). Perfusion with auxin (1AA) and fusicoccin (FC) stimulated the electrogenic activity of the ‘xylem pumps’ (111 and 205% respectively) and stimulated uptake of K ⁺ from the xylem (with 71% and 29% respectively). The close correlation between xylem pump activity and K⁺ uptake corroborated the aforementioned hypothesis. Interestingly, inhibition of pump activity by anoxia was incomplete in the presence of FC. It is thought that FC increases the affinity of the ATP-requiring xylem pump for ATP, thus ensuring that ATP production during fermentation is sufficient to fuel the pump in the absence of O₂.     

Isolation of Naegleria australiensis from an Oklahoma Lake1

Eight isolates of Naegleria australiensis were obtained from a small lake in Tulsa, Oklahoma. The eight strains were isolated during the hot summer months of July through September, when water temperatures ranged from 27 to 33°C. All eight isolates were pathogenic for mice. The mean time to death for mice was 10 days (range 6–13 days). This pathogenic free-living ameba has not before been reported from the United States or the Western Hemisphere.     

Voltage-dependent calcium and calcium-activated potassium currents of a molluscan photoreceptor

Two-microelectrode voltage clamp studies were performed on the somata of Hermissenda Type B photoreceptors that had been isolated by axotomy from all synaptic interaction as well as any impulse-generating (i.e., active) membrane. In the presence of 2-10 mM 4-aminopyridine (4-AP) and 100 mM tetraethylammonium ion (TEA), which eliminated two previously described voltage-dependent potassium currents (IA and the delayed rectifier), a voltage-dependent outward current was apparent in the steady state responses to command voltage steps more positive than -40 mV (absolute). This current increased with increasing external Ca++. The magnitude of the outward current decreased and an inward current became apparent following EGTA injection. Substitution of external Ba++ for Ca++ also made the inward current more apparent. This inward current, which was almost eliminated after being exposed for approximately 5 min to a solution in which external Ca++ was replaced with Cd++, was maximally activated at approximately 0 mV. Elevation of external potassium allowed the calcium (ICa++) and calcium-dependent K+ (IC) currents to be substantially separated. Command pulses to 0 mV elicited maximal ICa++ but no IC because no K+ currents flowed at their new reversal potential (0 mV) in 300 mM K+. At a holding potential of -60 mV, which was now more negative than the potassium equilibrium potential, EK+, in 300 mM K+, IC appeared as an inward tail current after positive command steps. The voltage dependence of ICa++ was demonstrated with positive steps in 100 mM Ba++, 4-AP, and TEA. Other data indicated that in 10 mM Ca++, IC underwent pronounced and prolonged inactivation whereas ICa++ did not. When the photoreceptor was stimulated with a light step (with the membrane potential held at -60 mV), there was also a prolonged inactivation of IC. In elevated external Ca++, ICa++ also showed similar inactivation. These data suggest that IC may undergo prolonged inactivation due to a direct effect of elevated intracellular Ca++, as was previously shown for a voltage-dependent potassium current, IA. These results are discussed in relation to the production of training-induced changes of membrane currents on retention days of associative learning.     

Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.     

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.