The suppression of salinity-associated oxygen radicals production, in pepper (Capsicum annuum) fruit, by manganese, zinc and calcium in relation to its sensitivity to blossom-end rot

We investigated the possibility that oxidative stress contributes to blossom-end rot (BER) initiation in bell pepper ( Capsicum annuum L.) grown under high salinity. Pepper plants (cv. Mazurka, Rijk Zwaan, the Netherlands) were grown in a greenhouse and irrigated with nutrient solution made up with either desalinated water (control — rising from E.C. 1.9 to 2.4 dS m⁻¹) or saline water (salinity – rising from E.C. 3.2 to 7.0 dS m⁻¹). Irrigation was by a circulation system. BER symptoms were observed throughout the experiment but were highly enhanced in the salinity–grown plants during the spring and summer. The fruit calcium concentration was not affected by salinity, but manganese concentrations in both leaves and fruits were significantly reduced under these conditions. Under salinity there was an enhancement of apoplast reactive oxygen species (ROS) production, which was partly a result of increase in NAD(P)H oxidase activity in the pericarp of pepper fruit at the stage that it was most sensitive to BER. Apoplast ROS production and extracted NAD(P)H oxidase activity were inhibited by manganese, zinc and to a lesser extent by calcium. These cations also negated the enhancement of ROS production caused by incubation of fruit pericarp discs in NaCl solutions. Manganese, zinc and calcium also inhibited NAD(P)H oxidase activity, extracted following their infiltration into fruit pericarp discs. The results suggest that generation and scavenging of oxygen free radicals in the apoplast may contribute to the appearance of BER symptoms in pepper fruits under saline conditions. It is suggested that manganese may serve as antioxidant in pepper fruit and that manganese addition to peppers grown under salinity may alleviate BER symptoms in the fruits.     

Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.     

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.     

Glandular-like morphogenesis of the human submandibular tumor cell line A253 on basement membrane components

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor.     

Lead identification of a potent benzopyranone selective estrogen receptor modulator

Starting from a phenol screening hit (6), three series of benzopyranone selective estrogen receptor modulators (SERMs) have been designed, synthesized, and analyzed for both estrogen receptor alpha binding affinity and in vitro activity in two cell assays. The lead compound identified, SP500263 (13), was more potent than raloxifene and tamoxifen in a cell-based assay measuring inhibition of interleukin-6 release.     

A non-invasive method for studying an index of pupil diameter and visual performance in the rhesus monkey

BACKGROUND: A non-invasive model has been developed to estimate gaze direction and relative pupil diameter, in minimally restrained rhesus monkeys, to investigate the effects of low doses of ocularly administered cholinergic compounds on visual performance. METHODS: Animals were trained to co-operate with a novel device, which enabled eye movements to be recorded using modified human eye-tracking equipment, and to perform a task which determined visual threshold contrast. Responses were made by gaze transfer under twilight conditions. 4% w/v pilocarpine nitrate was studied to demonstrate the suitability of the model. RESULTS: Pilocarpine induced marked miosis for >3 h which was accompanied by a decrement in task performance. CONCLUSIONS: The method obviates the need for invasive surgery and, as the position of point of gaze can be approximately defined, the approach may have utility in other areas of research involving non-human primates.     

Sex-biased dispersal in a salmonid fish

We tested the hypothesis that dispersal is sex biased in an unexploited population of brook trout, Salvelinus fontinalis, on Cape Race, Newfoundland, Canada. Based on the assumptions that trout are promiscuous and that reproductive success is limited primarily by either number of mates (males) or fecundity (females), we predicted that males would disperse greater distances than females. We also tested the hypothesis that trout populations comprise stationary and mobile individuals, predicting that males have greater mobility than females. Based on a mark-recapture study of 943 individually tagged fishes, 191 of which were recaptured over 5 years, we find strong support for our hypothesis of male-biased dispersal in brook trout. Averaged among all 11 resampling periods, males dispersed 2.5 times as far as females; during the spawning period only, male dispersal exceeded that by females almost fourfold. Both sexes were heterogeneous with respect to movement, with a lower incidence of mobility among females (29.6%) than males (41.1%); mobile males dispersed six times further than mobile females. We conclude that this sex bias reduces mate competition among male kin and decreases the probability that males will reproduce with related females.     

Isolation of the antibiotic pseudopyronine B and SAR evaluation of C3/C6 alkyl analogs

Natural products are an abundant source of structurally diverse compounds with antibacterial activity that can be used to develop new and potent antibiotics. One such class of natural products is the pseudopyronines. Here we present the isolation of pseudopyronine B (2) from a Pseudomonas species found in garden soil in Western North Carolina, and SAR evaluation of C3 and C6 alkyl analogs of the natural product for antibacterial activity against Gram-positive and Gram-negative bacteria. We found a direct relationship between antibacterial activity and C3/C6 alkyl chain length. For inhibition of Gram-positive bacteria, alkyl chain lengths between 6 and 7 carbons were found to be the most active (IC₅₀ = 0.04–3.8 µg/mL) whereas short alkyl chain analogs showed modest activity against Gram-negative bacteria (IC₅₀ = 223–304 µg/mL). This demonstrates the potential for this class of natural products to be optimized for selective activity against either Gram-positive or Gram-negative bacteria.     

Activity patterns and reproductive behavior of the Critically Endangered Bermuda skink (Plestiodon longirostris)

The study of rare or cryptic species in zoos can provide insights into natural history and behavior that would be difficult to obtain in the field. Such information can then be used to refine population assessment protocols and conservation management. The Bermuda skink (Plestiodon longirostris) is an endemic Critically Endangered lizard. Chester Zoo's successful conservation breeding program is working to safeguard, increase and reinforce skink populations in the wild. A key aim of this program is to develop our understanding of the behavior of this species. In this study, using 24 h video recordings, we examined the daily activity patterns, basking behavior and food preferences of four pairs of Bermuda skinks. The skinks displayed a bimodal pattern of activity and basking, which may have evolved to avoid the strength of the midday sun in exposed habitats in Bermuda. Captive Bermuda skinks appear to prefer a fruit-based diet to orthopteran prey. We also documented their reproductive behavior and compared it against two closely related species. Although there were many similarities between the courtship and mating behaviors of the three species, there was a significantly shorter period of cloacal contact in the Bermuda skink. Oophagia was also documented for the first time in this species. This knowledge has enabled the evaluation of the current ex-situ management practices of this species, filled gaps in knowledge that would be challenging to obtain in the field, and enabled the enhancement of both animal husbandry and reproductive success for the conservation breeding program.     

Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.