Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.     

NUTRIENTS DO NOT INFLUENCE SWIMMING BEHAVIOR OR SETTLEMENT RATES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) SPORES

Spores newly released from plurilocular sporangia of Ectocarpus siliculosus (Dillw.) Lyngb. sporophytes were assayed for chemotaxis to nutrients and for settlement stimulation by nutrients. To enable these measurements with relatively small volumes and numbers of released spores, we developed a computer-assisted motion-analysis assay for spore chemotaxis and verified the results with a more standard, capillary tube chemotaxis assay. The presence of a nutrient gradient did not influence the swimming behavior of E. siliculosus spores in the motion-analysis assay, and likewise no chemotactic effect was measured in the capillary tube assay. Microplate settlement assays previously utilized with bacteria and invertebrates were adapted for use with algal spores. E. siliculosus spores settled at higher rates on a hydrophobic plastic surface than on surfaces with either positively or negatively charged hydrophilic coatings. Nutrient mixtures had no effect on the rate of spore settlement on hydrophobic surfaces.     

Rapid purification of recombinant listeriolysin O (LLO) from Escherichia coli

Listeria monocytogenes is an emerging foodborne pathogen that is responsible for about 28% of the food-related deaths in the United States. It causes meningitis, septicaemia and in pregnant women, abortions and stillbirths. It secretes the toxin listeriolysin O (LLO) that allows the bacteria to enter the cytoplasm of host cells, where they can replicate and cause further infection. The rapid and sensitive detection of LLO in food samples is a key to monitoring and prevention of listeriosis. To facilitate the development of an assay for the specific detection of LLO, a source of LLO is essential. We outline a method of producing a large amount of functional LLO by expressing the hlyA gene (encoding LLO) in Escherichia coli and purifying the recombinant LLO using a one-step purification method. Purification of the protein takes only about 4 h. We compared three different expression constructs for the production of the toxin, which tends to interact strongly with a number of column surfaces. The first construct, using an intein fusion system, could not be purified from the column. The second LLO construct contained an N-terminus His tag; it gave a yield of 3.5–8 mg l⁻¹. The third contained a C-terminus His tag; it gave a yield of 2.5 mg l⁻¹ LLO. The purified LLO from the latter two constructs retained its activity at 4°C for over a year as determined by bovine red blood cell hemolysis assay. This paper provides a much-needed, high-yield, one-step purification method of recombinant LLO, and is the first to provide evidence of long-term stability of the toxin for further applications.     

In situ translocation of volicitin by beet armyworm larvae to maize and systemic immobility of the herbivore elicitor <Emphasis Type="Italic">in planta</Emphasis>

Volicitin (N-[17-hydroxylinolenoyl]-l glutamine) present in the regurgitant of beet armyworm (Spodoptera exigua) activates the emissions of volatile organic compounds (VOCs) when in contact with damaged corn (Zea mays L.) leaves. VOC emission in turn serves as a signaling defense for the plant by attracting female parasitic wasps that prey on herbivore larvae. Chemical tracking of volicitin within plants has yet to be reported. Here we present biochemical data that beet armyworm regurgitant serves as a vector for the introduction of volicitin to the site of leaf damage under natural feeding conditions. Corn seedlings were ¹⁴CO₂-labeled in situ, and beet armyworm larvae were allowed to feed on the labeled leaves. Herbivore oral secretions collected from late-third-instar larvae contained approximately 120 pmol volicitin (0.05 nCi pmol^–1) per larva. When radiochemically labeled larvae were placed on unlabeled leaves, the amount of volicitin introduced to the damaged site was approximately 5.0 nCi (calc. 100 pmol/larvae). The mobility of volicitin in leaves was examined by allowing radiolabeled beet armyworms to feed on unlabeled plants. In such tracking experiments, radioactivity was not detected in the upper leaves; however, the exogenous application of 5 nCi of [U-¹⁴C]sucrose to the lower leaf did result in subsequent radioactivity being detected in the upper portion of the plant. The detection of labeled sucrose with the same radioactivity as that of administered volicitin indicated that volicitin was not readily transported to undamaged leaves and that volicitin may not directly serve as a mobile messenger in triggering the emissions of VOCs systemically.Abbreviations BAW Beet armyworm (Spodoptera exigua) - dpm Disintegrations per minute - FAA Fatty acid amide - JA Jasmonic acid - VOC Volatile organic compound     

Effects of temperature and photoperiod upon adult eclosion of the sweetpotato whitefly, Bemisia tabaci

A series of experiments were conducted to characterize patterns of eclosion by Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) to their adult stage and to determine how these patterns are influenced by certain environmental parameters. Under a constant temperature of 29.5±0.6°C and a photoperiod of 14:10LD, 90% of the adults emerged from their pupal cases between 0600 and 0930 h (with lights on occurring at 0600 h). Few emerged during hours of darkness. The peak time of adult emergence was delayed when temperatures were fluctuated. Under a series of constant temperatures, a significant inverse correlation was found between the time of median emergence (i.e., eclosion of 50% of the total number of adults) and temperature (P<0.001). No emergence was observed at temperatures below 17±0.3°C. Emergence patterns persisted under conditions of continuous light and continuous darkness, suggesting the presence of a circadian system.

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Zusammenfassung Um das Verständnis über den Lebenslauf von Bemisia tabaci zu ergänzen, wurde eine Serie von Experimenten durchgeführt, deren Zweck die Charakterisierung des Ausschlüpfvorgangs in das Endstadium war und die Feststellung, wie dieser Vorgang von gewissen Umweltparametern beeinflusst wird. Bei einer konstanten Temperatur von 29.5±0.6°C und einem Beleuchtungszyklus von 14: 10 LD (Licht/Dunkelheit) schlüpften 90% der Ausgewachsenen zwischen 0600 Uhr and 0930 Uhr (ab 0600 Uhr mit Licht) aus ihren Puppenhüllen aus. Wenig Ausschlüpfen geschah während der unbeleuchteten Stunden. Der Höhepunkt des Ausschlüpfens wurde bei wechselnden Temperaturen verschoben. Bei einer Serie von gleichbleibenden Temperaturen wurde eine bedeutende inverse Korrelation zwischen der medianen Ausschlüpfzeit (d.h. 50% der gesamten Ausgewachsenen schlüpften aus) und der Temperatur festgestellt (P<0.001). Kein Ausschlüpfen wurde beobachtet bei Temperaturen unter 17°C. Das Ausschlüpfschema war gleichbleibend bei dauerndem Licht oder dauernder Dunkelheit, was auf das Vorhandensein eines circadianen Systems hinweist.

     

Phosphoramidate protides of carbocyclic 2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine with potent activity against HIV and HBV

Synthesis of phosphoramidate protides of carbocyclic D- and L-2',3'-dideoxy-2',3'-didehydro-7-deazaadenosine by treatment of the nucleoside with phosphorochloridates in the presence of pyridine and t-BuMgCl is described. Several of these protides showed significantly improved antiviral potency over the parent nucleosides against both HIV and HBV.     

Patterns of MHC selection in African mole-rats, family Bathyergidae: the effects of sociality and habitat

African mole-rats are a family of rodents exhibiting an eclectic range of social behaviour and occupying a variety of habitat types. These differences are likely to impact upon the risk of parasite transmission and virulence, with increasing sociality predicted to correspond to an increased risk of transmission. We investigate these factors by analysing the major histocompatibility complex (MHC), a set of genes responsible for encoding highly variable intermediaries of the vertebrate adaptive immune response. To this end we assessed selection at exons 2 and 3 of the MHC class II DQalpha1 gene of four African mole-rat species representing a range of social behaviours. We demonstrate that: (i) the overall pattern of selection at these exons differentiates according to the predicted function of different regions, with the presence of positive selection indicating the likely influence of host-parasite coevolution; and (ii) contrary to the often observed and predicted positive correspondence between sociality and the risk of parasite transmission, two highly social African mole-rat species in fact appear to have comparatively weak positive selection, suggesting diminished host immunity and thus a low overall risk of parasite transmission.     

ASK1, a SKP1 homolog, is required for nuclear reorganization, presynaptic homolog juxtaposition and the proper distribution of cohesin during meiosis in Arabidopsis

Nuclear reorganization and juxtaposition of homologous chromosomes at late leptotene/early zygotene are essential steps before chromosome synapsis at pachytene. We report the results of detailed studies, which demonstrate that nuclear reorganization and homolog juxtapositioning processes are defective in a null mutant, ask1-1. Our results from 4, 6-diamino-2-phenylindole (DAPI)-stained spreads showed that the “synizetic knot”, which is typically found in wild type (WT) meiosis during late leptotene and zygotene, was missing in the ask1-1 mutant. Furthermore, ask1-1 meiocytes exhibited only limited homolog juxtaposition at centromere regions at early zygotene. Immunodetection of the cohesin protein SYN1 identified ask1 defects in cohesin distribution from zygotene to anaphase I. Analysis of meiotic chromosomes in ask1-1 and syn1 single mutants, as well as an ask1-1 syn1 double mutant indicate that ASK1 is required for normal SYN1 distribution during meiotic prophase I and suggest that ask1 associated defects may be primarily related to SYN1 mislocalization.