Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.     

The adrenocortical stress-response of Black-legged Kittiwake chicks in relation to dietary restrictions

In this study we examined hormonal responses of Black-legged Kittiwake (Rissatridactyla) chicks to experimental variations in energy content and nutritional quality (low or high lipid to protein ratio, LPR) of their food. Starting at the age of 10 days, chicks were fed either high or low LPR fish at 30, 50, 70 and 100% of ad libitum energy intake. After 20 days of treatment, chicks were exposed to a standardized acute handling and restraint stress protocol, where a baseline sample was taken immediately after taking a chick from the nest, and three additional blood samples were taken at intervals up to 50 min. Testosterone and corticosterone titres in plasma were measured via radioimmunoassay. We found that baseline testosterone levels were not significantly affected by the experimental treatments. Food-restricted chicks had elevated baseline and acute stress-induced levels of corticosterone compared to chicks fed ad libitum. An elevation of circulating levels of corticosterone in energetically stressed individuals was further magnified by low nutritional quality of food. Baseline and acute stress-induced corticosterone levels of chicks were negatively correlated with their fat reserves. We conclude that the physiological condition of Black-legged Kittiwake chicks can be assessed reliably by measuring circulating levels of corticosterone. We discuss short- and long-term effects of elevated corticosterone secretion in food-stressed nest-bound chicks. Accepted: 13 April 1999     

Phylogeny and taxonomy of species in the Grosmannia serpens complex

Grosmannia serpens was first described from pine in Italy in 1936 and it has been recorded subsequently from many countries in both the northern and southern hemispheres. The fungus is vectored primarily by root-infesting bark beetles and has been reported to contribute to pine-root diseases in Italy and South Africa. The objective of this study was to consider the identity of a global collection of isolates not previously available and using DNA sequence-based comparisons not previously applied to most of these isolates. Phylogenetic analyses of the ITS2-LSU, actin, beta-tubulin, calmodulin and translation elongation factor-1 alpha sequences revealed that these morphologically similar isolates represent a complex of five cryptic species. Grosmannia serpens sensu stricto thus is redefined and comprises only isolates from Italy including the ex-type isolate. The ex-type isolate of Verticicladiella alacris was shown to be distinct from G. serpens, and a new holomorphic species, G. alacris, is described. The teleomorph state of G. alacris was obtained through mating studies in the laboratory, confirming that this species is heterothallic. Most of the available isolates, including those from South Africa, USA, France, Portugal and some from Spain, represent G. alacris. The remaining three taxa, known only in their anamorph states, are described as the new species Leptographium gibbsii for isolates from the UK, L. yamaokae for isolates from Japan and L. castellanum for isolates from Spain and the Dominican Republic.     

Identification and localization of bound internal water in the solution structure of interleukin 1 beta by heteronuclear three-dimensional 1H rotating-frame Overhauser 15N-1H multiple quantum coherence NMR spectroscopy

The presence and location of bound internal water molecules in the solution structure of interleukin 1 beta have been investigated by means of three-dimensional 1H rotating-frame Overhauser 1H-15N multiple quantum coherence spectroscopy (ROESY-HMQC). In this experiment through-space rotating-frame Overhauser (ROE) interactions between NH protons and bound water separated by less than or equal to 3.5 A are clearly distinguished from chemical exchange effects, as the cross-peaks for these two processes are of opposite sign. The identification of ROEs between NH protons and water is rendered simple by spreading out the spectrum into a third dimension according to the 15N chemical shift of the directly bonded nitrogen atoms. By this means, the problems that prevent, in all but a very few limited cases, the interpretation, identification, and assignment of ROE peaks between NH protons and water in a 2D 1H-1H ROESY spectrum of a large protein such as interleukin 1 beta, namely, extensive NH chemical shift degeneracy and ROE peaks obscured by much stronger chemical exchange peaks, are completely circumvented. We demonstrate the existence of 15 NH protons that are close to bound water molecules. From an examination of the crystal structure of interleukin 1 beta [Finzel, B. C., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D., & Einspahr, H. M. (1989) J. Mol. Biol. 209, 779-791], the results can be attributed to 11 water molecules that are involved in interactions bridging hydrogen-bonding interactions with backbone amide and carbonyl groups which stabilize the 3-fold pseudosymmetric topology of interleukin 1 beta and thus constitute an integral part of the protein structure in solution.     

Pathogenicity of Bursaphelenchus xylophilus on Pines in Minnesota and Wisconsin

The pinewood nematode, Bursaphelenchus xylophilus, was inoculated into established native jack and red pines (Pinus banksiana and P. resinosa) and exotic Austrian pine (P. nigra) in Minnesota and Wisconsin forests during summer 1981. The nematode isolates did not kill established nonstressed pine trees growing in the forest. However, the same nematode isolates killed pine seedlings under greenhouse conditions. Girdling the main stem of some trees to induce stress resulted in the death of the majority of inoculated and noninoculated branches of Austrian and jack pines, but no branch death was observed on red pine. Greater numbers of nematodes were extracted from branches of inoculated, girdled trees than from nongirdled trees. The mean number of nematodes extracted from branches of inoculated, nongirdled trees was 0.3 - 14 nematodes per gram of wood.     

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.     

Cryptic speciation in Fusarium subglutinans

Fusarium isolates that form part of the Gibberella fujikuroi species complex have been classified using either a morphological, biological, or phylogenetic species concept. Problems with the taxonomy of Fusarium species in this complex are mostly experienced when the morphological and biological species concepts are applied. The most consistent identifications are obtained with the phylogenetic species concept. Results from recent studies have presented an example of discordance between the biological and phylogenetic species concepts, where a group of F. subglutinans sensu stricto isolates, i.e., isolates belonging to mating population E of the G. fujikuroi complex, could be sub-divided into more than one phylogenetic lineage. The aim of this study was to determine whether this sub-division represented species divergence or intraspecific diversity in F. subglutinans. For this purpose, we included 29 F. subglutinans isolates belonging to the E-mating population that were collected from either maize or teosinte, from a wide geographic range. DNA sequence data for six nuclear regions in each of these isolates were obtained and used in phylogenetic concordance analyses. These analyses revealed the presence of two major groups representing cryptic species in F. subglutinans. These cryptic species were further sub-divided into a number of smaller groups that appear to be reproductively isolated in nature. This suggests not only that the existing F. subglutinans populations are in the process of divergence, but also that each of the resulting lineages are undergoing separation into distinct taxa. These divergences did not appear to be linked to geographic origin, host, or phenotypic characters such as morphology.     

Endocrine responses to unpredictable environmental events: stress or anti-stress hormones?

In addition to seasonal changes in morphology, physiology andbehavior that occur in predictable annual cycles, there arefacultative responses to unpredictable events known as labile(i.e., short-lived) perturbation factors (LPFs). These rapidbehavioral and physiological changes have been termed the "emergency"life history stage (ELHS) and serve to enhance life-time fitness.Glucocorticosteroids interacting with other hormones in thehypothalamo-pituitary-adrenal (HPA) cascade, initiate and orchestratethe ELHS within minutes to hours. Components of the ELHS include:redirection of behavior from a normal life history stage toincreased foraging, irruptive-type migration during the day,enhanced restfulness at night, elevated gluconeogenesis andrecovery once the perturbation passes. These physiological andbehavioral changes allow an individual to avoid potential deleteriouseffects of stress that may result from chronically elevatedlevels of circulating glucocorticosteroids over days and weeks.In other words, acute rises in glucocorticosteroids followingperturbations of the environment may actually avoid chronicstress and serve primarily as "anti-stress" hormones. Severalfield studies in diverse habitats indicate that free-livingpopulations have elevated circulating levels of corticosteroidswhen in an ELHS. However, expression of an ELHS may not alwaysbe advantageous and there is accumulating evidence from birdsthat the adrenocortical responses to LPFs are modulated bothon seasonal and individual levels. These data suggest that glucocorticosteroidsecretions in response to LPFs not only trigger physiologicaland behavioral responses but also allow flexibility so thatthe response is integrated in relation to time of year (normalLHS) as well as individual differences owing to body condition,disease and social status.     

Morphological,cultural and pathogenic characteristics of Coniothyrium zuluense isolates from different plantation regions in South Africa

Coniothyrium canker caused by Coniothyrium zuluense, is a serious stem canker disease of Eucalyptus species in sub-tropical regions of South Africa. This disease is typified by necrotic bark lesions that coalesce to form large kino-impregnated cankers along the stems of trees. The strategy currently used to manage Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Thus, forest managers are interested in the genetic diversity of the pathogen and its potential to overcome disease resistance in planting stock. In this study, 344 isolates of C. zuluense from different plantation regions in South Africa were compared on the basis of colony colour, conidial morphology, growth characteristics on agar and pathogenicity to a susceptible E. grandis clone. Conidia of all C. zuluense isolates measured were similar in size and shape. The fungus is slow growing in culture, which is indicative of its apparent biotrophic habit, with optimum growth observed at 30 °C. Isolates of C. zuluense displayed considerable variation in colony colour and pathogenicity in inoculation trials. Variation in morphology and pathogenicity amongst isolates suggests that C. zuluense has been present in South Africa for an extended period of time, or that it is changing rapidly due to strong directional selection pressures. This revised version was published online in June 2006 with corrections to the Cover Date.