Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.     

Purine derivatives as potent γ-secretase modulators

The development of a novel series of purines as γ-secretase modulators for potential use in the treatment of Alzheimer’s disease is disclosed herein. Optimization of a previously disclosed pyrimidine series afforded a series of potent purine-based γ-secretase modulators with 300- to 2000-fold in vitro selectivity over inhibition of Notch cleavage and that selectively reduces Αβ42 in an APP-YAC transgenic mouse model.     

Fluorinated piperidine acetic acids as γ-secretase modulators

We report herein a novel series of difluoropiperidine acetic acids as modulators of γ-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective β-difluorination with Selectfluor®. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Aβ42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250 mg/kg per day, AUC_0–24 = 2100 μM h) did not exhibit Notch-related effects.     

Tree species effects on ecosystem water-use efficiency in a high-elevation, subalpine forest

Ecosystem water-use efficiency (eWUE; the ratio of net ecosystem productivity to evapotranspiration rate) is a complex landscape-scale parameter controlled by both physical and biological processes occurring in soil and plants. Leaf WUE (lWUE; the ratio of leaf CO₂ assimilation rate to transpiration rate) is controlled at short time scales principally by leaf stomatal dynamics and this control varies among plant species. Little is known about how leaf-scale variation in lWUE influences landscape-scale variation in eWUE. We analyzed approximately seven thousand 30-min averaged eddy covariance observations distributed across 9 years in order to assess eWUE in two neighboring forest communities. Mean eWUE was 19% lower for the community in which Engelmann spruce and subalpine fir were dominant, compared to the community in which lodgepole pine was dominant. Of that 19% difference, 8% was attributed to residual bias in the analysis that favored periods with slightly drier winds for the spruce-fir community. In an effort to explain the remaining 11% difference, we assessed patterns in lWUE using C isotope ratios. When we focused on bulk tissue from older needles we detected significant differences in lWUE among tree species and between upper and lower canopy needles. However, when these differences were scaled to reflect vertical and horizontal leaf area distributions within the two communities, they provided no power to explain differences in eWUE that we observed in the eddy covariance data. When we focused only on bulk needle tissue of current-year needles for 3 of the 9 years, we also observed differences in lWUE among species and in needles from upper and lower parts of the canopy. When these differences in lWUE were scaled to reflect leaf area distributions within the two communities, we were able to explain 6.3% of the differences in eWUE in 1 year (2006), but there was no power to explain differences in the other 2 years (2003 and 2007). When we examined sugars extracted from needles at 3 different times during the growing season of 2007, we could explain 3.8–6.0% of the differences in eWUE between the two communities, but the difference in eWUE obtained from the eddy covariance record, and averaged over the growing season for this single year, was 32%. Thus, overall, after accounting for species effects on lWUE, we could explain little of the difference in eWUE between the two forest communities observed in the eddy covariance record. It is likely that water and C fluxes from soil, understory plants, and non-needle tissues, account for most of the differences observed in the eddy covariance data. For those cases where we could explain some of the difference in eWUE on the basis of species effects, we partitioned the scaled patterns in lWUE into two components: a component that is independent of canopy leaf area distribution, and therefore only dependent on species-specific differences in needle physiology; and a component that is independent of species differences in needle physiology, and only dependent on species-specific influences on canopy leaf area distribution. Only the component that is dependent on species influences on canopy leaf area distribution, and independent of inherent species differences in needle physiology, had potential to explain differences in eWUE between the two communities. Thus, when tree species effects are important, canopy structure, rather than species-specific needle physiology, has more potential to explain patterns in eWUE.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

Amphipathic benzoic acid derivatives: synthesis and binding in the hydrophobic tunnel of the zinc deacetylase LpxC

The first committed step in lipid A biosynthesis is catalyzed by uridine diphosphate-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase, and inhibitors of LpxC may be useful in the development of antibacterial agents targeting a broad spectrum of Gram-negative bacteria. Here, we report the design of amphipathic benzoic acid derivatives that bind in the hydrophobic tunnel in the active site of LpxC. The hydrophobic tunnel accounts for the specificity of LpxC toward substrates and substrate analogues bearing a 3-O-myristoyl substituent. Simple benzoic acid derivatives bearing an aliphatic 'tail' bind in the hydrophobic tunnel with micromolar affinity despite the lack of a glucosamine ring like that of the substrate. However, although these benzoic acid derivatives each contain a negatively charged carboxylate 'warhead' intended to coordinate to the active site zinc ion, the 2.25A resolution X-ray crystal structure of LpxC complexed with 3-(heptyloxy)benzoate reveals 'backward' binding in the hydrophobic tunnel, such that the benzoate moiety does not coordinate to zinc. Instead, it binds at the outer end of the hydrophobic tunnel. Interestingly, these ligands bind with affinities comparable to those measured for more complicated substrate analogue inhibitors containing glucosamine ring analogues and hydroxamate 'warheads' that coordinate to the active site zinc ion. We conclude that the intermolecular interactions in the hydrophobic tunnel dominate enzyme affinity in this series of benzoic acid derivatives.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R(2)=0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.