Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelium, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.     

Investigation of CC and CXC chemokine quaternary state mutants

The chemokine family forms two different types of homodimer despite members sharing nearly identical folds. To study the formation of quaternary structure in this family, rational mutagenesis was employed on a representative member of each subfamily (MIP-1beta and IL-8). The variants were studied by analytical ultracentrifugation and NMR, and it was determined that formation of a folded monomer from a natural chemokine dimer is reasonably facile, while conversion between dimer types is not. Monomeric variants of MIP-1beta and IL-8 were randomly mutated and a lambda phage-based selection system was employed in a novel way to screen for dimerization. A total of 6,000,000 random mutants were screened, but no dimers were formed, suggesting again that the chemokine fold is robust and amenable to sequence variation, while the chemokine dimer is much more difficult to attain. This work represents a biophysical analysis of an array of chemokine quaternary state variants.     

The role of Haldane's rule in sex allocation

Sex allocation theory predicts that parents should bias their reproductive investments toward the offspring sex generating the greatest fitness return. When females are the heterogametic sex (e.g., ZW in butterflies, some lizards, and birds), production of daughters is associated with an increased risk of offspring inviability due to the expression of paternal, detrimental recessives on the Z chromosome. Thus, daughters should primarily be produced when mating with partners of high genetic quality. When female sand lizards (Lacerta agilis) mate with genetically superior males, exhibiting high MHC Class I polymorphism, offspring sex ratios are biased towards daughters, possibly due to recruitment of more Z-carrying oocytes when females have assessed the genetic quality of their partners. If our study has general applicability across taxa, it predicts taxon-specific sex allocation effects depending on which sex is the heterogametic one.     

Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR

The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.     

2H-NMR study and molecular dynamics simulation of the location, alignment, and mobility of pyrene in POPC bilayers

The alignment of pyrene in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer was investigated using two different approaches, namely solid-state (2)H-NMR spectroscopy and molecular dynamics (MD) simulations. Quadrupolar splittings from (2)H-NMR spectra of deuterated pyrene-d(10) in an oriented lipid bilayer give information about the orientation of C-D bonds with respect to the membrane normal. From MD simulations, geometric information is accessible via trajectories. By defining molecular and bond order parameters, the data from MD trajectories and NMR spectra can be compared straightforwardly. To ensure that the results from both methods are comparable, parameters of the experimental and the simulation setup were chosen to be as similar as possible. From simulations, we saw that pyrene prefers a position inside the lipid membrane near the headgroups and has no tendency to diffuse from one monolayer of the membrane to the other. The results from simulation and NMR show that the normal of the molecular plane is aligned nearly perpendicular to the bilayer normal. The long axis of pyrene lies preferentially parallel to the bilayer normal within a range of +/-30 degrees . The results from the two different methods are remarkably consistent. The good agreement can be explained by the fact that the different kind of motions of a pyrene molecule are already averaged within a few nanoseconds, which is the timescale covered by the MD simulation.     

Carbon limitation leads to suppression of first year oak seedlings beneath evergreen understory shrubs in Southern Appalachian hardwood forests

Inhibition of canopy tree recruitment beneath thickets of the evergreen shrubs Rhododendron maximum L. and Kalmia latifolia L. has long been observed in Southern Appalachian forests, yet the mechanisms of this process remain unresolved. We present a first-year account of suppression of oak seedlings in relation to Rhododendron and Kalmia basal area, light and resource availability, seedling performance and the rates of seedling damage (i.e., herbivory). We found no evidence of first-year seedling suppression or significant resource deficiencies beneath thickets of K. latifolia in mature mixed hardwood stands. Suppression beneath R. maximum was apparent during the first growing season. We found that seedling biomass, light availability prior to canopy closure, and seedling tissue C:N ratios were negatively correlated with R. maximum basal area. Basal area of R. maximum was positively correlated with seedling mortality rates, soil [Al], and early-growing season leaf herbivory rates. Seedling growth was positively correlated with light and tissue C:N, while negatively correlated with soil [Al]. Overall, our results support the inhibition model of shade-mediated carbon limitation beneath dense understory shrubs and indicate the potential importance of herbivory and aluminum toxicity as components of a suppression mechanism beneath R. maximum thickets. We present a causal model of first year inhibition beneath R. maximum in the context of our findings and the results of prior studies.     

Periphyton-macroinvertebrate interactions in light and fish manipulated enclosures in a clear and a turbid shallow lake

In a clear and a turbid freshwater lake the biomasses of phytoplankton, periphytic algae and periphytonassociated macrograzers were followed in enclosures with and without fish (Rutilus rutilus) and four light levels (100%, 55%, 7% and < 1% of incoming light), respectively. Fish and light affected the biomass of primary producers and the benthic grazers in both lakes. The biomass of primary producers was generally higher in the turbid than the clear lake, and in both lakes fish positively affected the biomass, while shading reduced it. Total biomass of benthic grazing invertebrates was higher in the clear than in the turbid lake and the lakes were dominated by snails and chironomids + ostracods, respectively. While light had no effect on the biomass of grazers in the clear lake, snail breeding was delayed in the most shaded enclosures and presence of fish reduced the number of snails and the total biomass of grazers. In the turbid lake ostracod abundance was not influenced by light, but was higher in fish-free enclosures. Density of chironomids correlated positively with periphyton biomass in summer, while fish had no effect. Generally, light-mediated regulation of primary producers was stronger in the turbid than in the clear lake, but the regulation did not nambiguously influence the primary consumers. However, regulation by fish of the benthic grazer community was stronger in the clear than in the turbid lake, and in both lakes strong top-down effects on periphyton were seen. The results indicate that if present-day climate in Denmark in the future is found in coastal areas at higher latitudes, the effect of lower light during winter in such areas will be highest in clear lakes, with typically lower fish biomass and higher invertebrate grazer density.     

Myosin 5a controls insulin granule recruitment during late-phase secretion

We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.     

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic

P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of approximately 10(3) PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.