Biocontrol of grey mould by Ulocladium atrum applied at different flower and fruit stages of strawberry

Grey mould is an important disease ofstrawberries resulting from flower and fruitinfection by Botrytis cinerea Pers. Thesaprophytic fungus Ulocladium atrumPreuss is a promising biological controlagent for control of B. cinerea instrawberry and other crops. The objective ofthis research was to determine the efficacy ofU. atrum to control grey mould by asingle application of a spore suspension (2 ×10⁶ conidia/ml) at different flowerand fruit development stages. Four experimentswere carried out in 1999, two under natural andtwo under enhanced inoculum levels of B.cinerea. In each experiment, flowers and youngfruits in six distinct stages of developmentwere sprayed with either water or U.atrum suspension. U. atrum suppressedB. cinerea sporulation on petals by 15 to54%. One to four days after spraying, U.atrum was present on less than 30% of stamensand did not affect the incidence of B.cinerea on these flower parts. The efficacy ofthe U. atrum sprays in controlling greymould was low to moderate, and resulted onaverage in a reduction of 21% in diseaseincidence on ripe fruits. Low control efficacywas probably due to poor coverage with orcolonisation of stamens by U. atrumspores, and a relatively low level ofsuppression of the colonisation of flower partsby B. cinerea. Significant reductions ofgrey mould in comparison to the control(p 0.10; on average 41% reduction) werefound most frequently when the antagonist wasintroduced at late flowering or early fruitstages. Therefore, these are the most suitablestages to apply U. atrum. Further studiesare needed to improve the spray coverage andpersistence of U. atrum inoculum.     

Movements of wintering surf scoters: predator responses to different prey landscapes

The distribution of predators is widely recognized to be intimately linked to the distribution of their prey. Foraging theory suggests that predators will modify their behaviors, including movements, to optimize net energy intake when faced with variation in prey attributes or abundance. While many studies have documented changes in movement patterns of animals in response to temporal changes in food, very few have contrasted movements of a single predator species naturally occurring in dramatically different prey landscapes. We documented variation in the winter movements, foraging range size, site fidelity, and distribution patterns of a molluscivorous sea duck, the surf scoter (Melanitta perspicillata), in two areas of coastal British Columbia with very different shellfish prey features. Baynes Sound has extensive tidal flats with abundant clams, which are high-quality and temporally stable prey for scoters. Malaspina Inlet is a rocky fjord-like inlet where scoters consume mussels that are superabundant and easily accessible in some patches but are heavily depleted over the course of winter. We used radio telemetry to track surf scoter movements in both areas and found that in the clam habitats of Baynes Sound, surf scoters exhibited limited movement, small winter ranges, strong foraging site fidelity, and very consistent distribution patterns. By contrast, in mussel habitats in the Malaspina Inlet, surf scoters displayed more movement, larger ranges, little fidelity to specific foraging sites, and more variable distribution patterns. We conclude that features associated with the different prey types, particularly the higher depletion rates of mussels, strongly influenced seasonal space use patterns. These findings are consistent with foraging theory and confirm that predator behavior, specifically movements, is environmentally mediated.     

Ultraviolet radiation: effects on risks of prostate cancer and other internal cancers

Governmental and research agencies worldwide have strongly advocated sun avoidance strategies in an attempt to counter marked increases in skin cancer incidence. Concurrently, there are reports describing widespread Vitamin D3 deficiency. Because 1,25-dihydroxyvitamin D3, through interaction with the Vitamin D receptor, exerts pleiotrophic effects, such deficiency might be expected to have clinical consequences. Indeed, various reports indicate that exposure to ultraviolet radiation (UVR) exerts a protective effect on development of some common diseases including internal cancers and multiple sclerosis. We describe studies indicating that modest exposure reduces risk of prostate cancer. The effect of UVR is mediated by skin type; at lower levels of exposure a relative inability to effect skin pigmentation is protective presumably because it allows more efficient Vitamin D3 synthesis. Polymorphic variants in genes associated with pigmentation including melanocyte stimulating hormone receptor and tyrosinase are also associated with prostate cancer risk. Overall, though preliminary and requiring cautious interpretation, these data indicate that moderate UVR exposure together with characteristics linked with less effective tanning confer reduced prostate cancer risk. Clearly, it is important to define safe levels of UVR that do not result in increased risk of skin cancers such as malignant melanoma.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Widespread state-dependent shifts in cerebellar activity in locomoting mice

Excitatory drive enters the cerebellum via mossy fibers, which activate granule cells, and climbing fibers, which activate Purkinje cell dendrites. Until now, the coordinated regulation of these pathways has gone unmonitored in spatially resolved neuronal ensembles, especially in awake animals. We imaged cerebellar activity using functional two-photon microscopy and extracellular recording in awake mice locomoting on an air-cushioned spherical treadmill. We recorded from putative granule cells, molecular layer interneurons, and Purkinje cell dendrites in zone A of lobule IV/V, representing sensation and movement from trunk and limbs. Locomotion was associated with widespread increased activity in granule cells and interneurons, consistent with an increase in mossy fiber drive. At the same time, dendrites of different Purkinje cells showed increased co-activation, reflecting increased synchrony of climbing fiber activity. In resting animals, aversive stimuli triggered increased activity in granule cells and interneurons, as well as increased Purkinje cell co-activation that was strongest for neighboring dendrites and decreased smoothly as a function of mediolateral distance. In contrast with anesthetized recordings, no 1-10 Hz oscillations in climbing fiber activity were evident. Once locomotion began, responses to external stimuli in all three cell types were strongly suppressed. Thus climbing and mossy fiber representations can shift together within a fraction of a second, reflecting in turn either movement-associated activity or external stimuli.     

Structural,antigenic and evolutionary analyses of immunoglobulins and T cell receptors

We have had the pleasure of collaborating with Allen Edmundson for the past 15 years on the structure, binding properties and evolution of immunoglobulins and T cell receptors. Among the most significant contributions of our joint efforts were: (1) the predictive use of structural features of immunoglobulin domains to model the three-dimensional structures of the immunoglobulin domains of human T-cell receptor alpha and beta chains as well as shark light chains and V(H) domains; (2) the finding that normal humans and other vertebrates express autoantibodies against combining site epitopes of their own T cell receptors; (3) the mapping of the peptide autoepitopes recognized in health, autoimmunity and retroviral infection; and (4) the determination that epitope recognition promiscuity is a characteristic property of the combining sites of IgM immunoglobulins ranging from those of sharks to those of humans. We briefly review the salient findings and status of these studies and indicate the future directions that we will pursue in their continuation.     

Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ～0.5 and led to siRNA/polymer complexes of ～100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.     

Luffa cylindrica and phytosterols bioconversion: from shake flask to jar bioreactor

Bioconversion of lipophilic compounds poorly soluble in water, such as sterols, required the use of chemicals and solubilizing agents. On the other hand, it was shown that immobilization of Mycobacterium species on the dried fruit of Luffa cylindrica (DFLC) allows a close interaction between immobilized cells and cholesterol particles and increases by then the product’s yield. In this work, the use of DFLC in a 5-l jar bioreactor with phytosterols mixture (1 g/l) as substrate was assessed without addition of any chemicals or solubilizing agents. DFLC increased by a factor of four the volumetric productivity of androstenones (0.08 g/l day). Products were accumulated in the aqueous medium while substrates remained on the fibers of DFLC. This observation lets envisage a green semi-continuous process of androstenone production. DFLC has no influence on cell growth, and is moreover natural, inexpensive, non-toxic, and mechanically strong.