Isolation of multiple sequences from the Plasmodium falciparum genome that encode conserved domains homologous to those in erythrocyte-binding proteins.

Open reading frames in the Plasmodium falciparum genome encode domains homologous to the adhesive domains of the P. falciparum EBA-175 erythrocyte-binding protein (eba-175 gene product) and those of the Plasmodium vivax and Plasmodium knowlesi Duffy antigen-binding proteins. These domains are referred to as Duffy binding-like (DBL), after the receptor that determines P. vivax invasion of Duffy blood group-positive human erythrocytes. Using oligonucleotide primers derived from short regions of conserved sequence, we have developed a reverse transcription-PCR method that amplifies sequences encoding the DBL domains of expressed genes. Products of these reverse transcription-PCR amplifications include sequences of single-copy genes (including eba-175) and variably transcribed genes that cross-hybridize to multiple regions of the genome. Restriction patterns of the multicopy genes show a high degree of polymorphism among different parasite lines, whereas single-copy genes are generally conserved. Characterization of the single-copy genes has identified a gene (ebl-1) that is related to eba-175 and is likely to be involved in erythrocyte invasion.

Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of SAR gene expression and pathogen resistance, followed by assays for resistance to the fungal pathogen Peronospora parasitica. Mutants from this screen were subsequently examined to assess their defense responses. We describe here a recessive mutation that causes a phenotype of insensitivity to chemical and biological inducers of SAR genes and resistance. These data indicate the existence of a common signaling pathway that couples these diverse stimuli to induction of SAR genes and resistance. Because of its non-inducible immunity phenotype, we call this mutant nim1. Although nim1 plants fail to respond to SA, they retain the ability to accumulate wild-type levels of SA, a probable endogenous signal for SAR. Further, the ability of nim1 plants to support growth of normally incompatible races of a fungal pathogen indicates a role for this pathway in expression of genetically determined resistance, consistent with earlier findings for transgenic plants engineered to break down SA. These results suggest that the wild-type NIM1 gene product functions in a pathway regulating acquired resistance, at a position downstream of SA accumulation and upstream of SAR gene induction and expression of resistance.

The association of feeding behavior with the resistance and tolerance to parasites in recently diverged sticklebacks

Divergent natural selection regimes can contribute to adaptive population divergence, but can be sensitive to human-mediated environmental change. Nutrient loading of aquatic ecosystems, for example, might modify selection pressures by altering the abundance and distribution of resources and the prevalence and infectivity of parasites. Here, we used a mesocosm experiment to test for interactive effects of nutrient loading and parasitism on host condition and feeding ecology. Specifically, we investigated whether the common fish parasite Gyrodactylus sp. differentially affected recently diverged lake and stream ecotypes of three-spined stickleback (Gasterosteus aculeatus). We found that the stream ecotype had a higher resistance to Gyrodactylus sp. infections than the lake ecotype, and that both ecotypes experienced a cost of parasitism, indicated by negative relationships between parasite load and both stomach fullness and body condition. Overall, our results suggest that in the early stages of adaptive population divergence of hosts, parasites can affect host resistance, body condition, and diet.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class (Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue (Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NACTF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor-and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Sensitivity of staphylococcus epidermidis to chlorhexidine and associated resistance properties

Staphylococcus epidermidis are common Gram-positive bacteria and are responsible for a number of life-threatening nosocomial infections. Treatment of S. epidermidis infection is problematic because the organism is usually resistant to many antibiotics. The high degree of resistance of this organism to a range of antibiotics and disinfectants is widely known. The aims of this thesis were to investigate and evaluate the susceptibility of isolates of S. epidermidis from various infections to chlorhexidine (CHX) and to other disinfectants such as benzalkonium chloride (BKC), triclosan (TLN) and povidone-iodine (PI). In addition, the mechanisms of resistance of S. epidermidis to chlorhexidine (the original isolates and strains adapted to chlorhexidine by serial passage) were examined and co-resistance to clinically relevant antibiotics investigated. In 3 of the 11 S. epidermidis strains passaged in increasing concentrations of chlorhexidine, resistance to the disinfectant arose (16-fold). These strains were examined further, each showing stable chlorhexidine resistance. Co-resistance to other disinfectants such as BKC, TLN and PI and changes in cell surface hydrophobicity were observed. Increases in resistance were accompanied by an increase in the proportion of neutral lipids and phospholipids in the cell membrane. This increase was most marked in diphosphatidylglycerol. These observations suggest that some strains of S. epidermidis can become resistant to chlorhexidine and related disinfectants/antiseptics by continual exposure. The mechanisms of resistance appear to be related to changes in membrane lipid compositions.

Ordered mixing of drugs with particulate excipients

A novel direct compression tableting excipient has been made by recrystallisation of lactose. The particles produced had high porosity, high specific surface area and high surface roughness. The resistance to segregation of ordered mixes formed between a model drug; potassium chloride and the excipients recrystallised lactose, spray crystallised maltose-dextrose (Emdexl and a direct compacting sugar (Dipac) was studied using a vibrational segregation model. The highly porous excipients, Emdex and recrystallised lactose formed ordered mixes which did not segregate even at high accelerations and low frequencies whereas the relatively smooth excipient, Dipac, displayed marked segregation in most vibration conditions. The vibrations were related to practical conditions measured in pharmaceutical process machinery. The time required to form an ordered mix was inversely related to the stability of the mix when subjected to vibration. An ultracentrifuge technique was developed to determine the interparticle adhesion forces holding drug and excipient particles together as ordered units. Excipient powders such as Emdex and recrystallised lactose, which formed non-segregating ordered mixes, had high interparticle adhesion forces. Other ordered mixes that segregated when subjected to different vibration conditions were found to have large quantities of weekly-bound drug particles; such mixes included those with Dipac as the carrier excipient as well as those containing a high concentration of drug. The electrostatic properties of different drug and excipient powders were studied using a Faraday well and an electrometer. Excipient powders such as Emdex and recrystallised lactose which formed stable ordered mixes also had a widely different surface charge in comparison with drug particles, whereas Dipac had a similar surface charge to the drug particles and formed unstable ordered mixes. A specially constructed triboelectric charging apparatus based on an air cyclone was developed to increase the affinity of drug particles for different excipient particles. Using triboelectrification to increase the interparticle adhesion forces, the segregation tendencies of unstable ordered mixes were greatly reduced. The stability of ordered mixes is shown to be related to both the surface physical characteristics and the surface electrical properties of the constituent carrier (excipientl particles.

Liposome formulation of poorly water soluble drugs:optimisation of drug loading and ESEM analysis of stability

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C 24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 μmol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 μmol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 μmol:4 μmol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 μmol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome. © 2004 Elsevier B.V. All rights reserved.

Paths of the least resistance:understanding how motives form in international retail joint venturing

Developing the premise that strategies are forged through an ongoing mutual process of developing motives and responses to multiple degrees of resistance, this paper examines the motives underpinning the adoption of joint venture strategies using empirical details from four British retail firms. The findings point to multiple motives forming from multiple paths of resistance in the foreign market, but also among individuals within the firm as well as across the whole international programme. Moreover, this study reveals a paradoxical tension between management's operational impatience to immediately ground the retail format and an overall wariness or gloomy perceptions associated with adopting an international retail joint venture. The paper therefore concludes that the motives and barriers are manifestations of the struggles involved in internationalising retail operations.

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E-coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Lys9 for Glu9 substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His7 and Ala8, can greatly improve resistance to this enzyme. Little research has assessed what effect Glu9-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu9 of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue (Lys9)GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys9)GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor.

Multiple predator effects and native prey responses to two non-native Everglades cichlids

Non-native predators may have negative impacts on native communities, and these effects may be dependent on interactions among multiple non-native predators. Sequential invasions by predators can enhance risk for native prey. Prey have a limited ability to respond to multiple threats since appropriate responses may conflict, and interactions with recent invaders may be novel. We examined predator–prey interactions among two non-native predators, a recent invader, the African jewelfish, and the longer-established Mayan cichlid, and a native Florida Everglades prey assemblage. Using field enclosures and laboratory aquaria, we compared predatory effects and antipredator responses across five prey taxa. Total predation rates were higher for Mayan cichlids, which also targeted more prey types. The cichlid invaders had similar microhabitat use, but varied in foraging styles, with African jewelfish being more active. The three prey species that experienced predation were those that overlapped in habitat use with predators. Flagfish were consumed by both predators, while riverine grass shrimp and bluefin killifish were eaten only by Mayan cichlids. In mixed predator treatments, we saw no evidence of emergent effects, since interactions between the two cichlid predators were low. Prey responded to predator threats by altering activity but not vertical distribution. Results suggest that prey vulnerability is affected by activity and habitat domain overlap with predators and may be lower to newly invading predators, perhaps due to novelty in the interaction.

Drying Times: Integrating Citizen Science to Examine Survival of Florida Largemouth Bass in a Coastal Refuge Habitat

In aquatic systems refuge habitats increase resistance to drying events and are necessary for maintaining populations in disturbed environments. However, reduced water availability and altered flow regimes threaten the existence and function of these habitats. To test refuge function I conducted a capture-mark-recapture (CMR) study, integrating citizen science angler sampling into fisheries-independent methods. The objectives of this study were twofold: 1) To determine the contribution of citizen science anglers to improving CMR research, and 2.) to quantify apparent survival of Florida Largemouth Bass, Micropterus salmoides floridanus, in a coastal refuge habitat across multiple years of drying severity. The inclusion of angler sampling was determined to be an effective and feasible method for increasing capture probability. Apparent survival of Florida Bass varied among hydrologic periods with lowest survival when marshes functionally dried (< 10 cm). Overall mortality from drying events increased with the duration of marsh drying upstream.

Antarctic crustacean grazer assemblages exhibit resistance following exposure to decreased pH

Anthropogenic atmospheric CO2 concentrations are increasing rapidly, resulting in declining seawater pH (ocean acidification). The majority of ocean acidification research to date has focused on the effects of decreased pH in single-species experiments. To assess how decreased pH may influence natural macroalgal-grazer assemblages, we conducted a mesocosm experiment with the common, chemically defended Antarctic brown macroalga Desmarestia menziesii and natural densities of its associated grazer assemblage, predominantly amphipods. Grazer assemblages were collected from the immediate vicinity of Palmer Station (64°46'S, 64°03'W) in March 2013. Assemblages were exposed for 30 days to three levels of pH representing present-day mean summer ambient conditions (pH 8.0), predicted near-future conditions (2100, pH 7.7), and distant-future conditions (pH 7.3). A significant difference was observed in the composition of mesograzer assemblages in the lowest pH treatment (pH 7.3). The differences between assemblages exposed to pH 7.3 and those maintained in the other two treatments were driven primarily by decreases in the abundance of the amphipod Metaleptamphopus pectinatus with decreasing pH, reduced copepod abundance at pH 7.7, and elevated ostracod abundance at pH 7.7. Generally, the assemblages maintained at pH 7.7 were not significantly different from those at ambient pH, demonstrating resistance to short-term decreased pH. The relatively high prevalence of generalist amphipods may have contributed to a net stabilizing effect on the assemblages exposed to decreased pH. Overall, our results suggest that crustacean grazer assemblages associated with D. menziesii, the dominant brown macroalgal species of the western Antarctic Peninsula, may be resistant to short-term near-future decreases in seawater pH.