Diffusion of Finite-Size Particles in Confined Geometries

The diffusion of finite-size hard-core interacting particles in two- or three-dimensional confined domains is considered in the limit that the confinement dimensions become comparable to the particle’s dimensions. The result is a nonlinear diffusion equation for the one-particle probability density function, with an overall collective diffusion that depends on both the excluded-volume and the narrow confinement. By including both these effects, the equation is able to interpolate between severe confinement (for example, single-file diffusion) and unconfined diffusion. Numerical solutions of both the effective nonlinear diffusion equation and the stochastic particle system are presented and compared. As an application, the case of diffusion under a ratchet potential is considered, and the change in transport properties due to excluded-volume and confinement effects is examined.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

PAI-1 has been shown to be both profibrotic and antifibrotic in animal models of hepatic fibrosis. Although these models have similarities to human fibrotic liver disease, no rodent model completely recapitulates the clinical situation; indeed, transaminase values in most models of hepatic fibrosis are much higher than in chronic liver diseases in humans. Here, wild-type and PAI-1^−/− mice were administered AngII (500 ng/kg/min) for 4 weeks. ECM accumulation was evaluated by Sirius red staining, hydroxyproline content, and fibrin and collagen immunostaining. Induction of pro-fibrotic genes was assessed by real-time RT-PCR. Despite the absence of any significant liver damage, AngII infusion increased the deposition of hepatic collagen and fibrin ECM, with a perisinusoidal pattern. PAI-1^−/− mice were protected from these ECM changes, indicating a causal role of PAI-1 in this fibrosis model. Protection in the knockout strain correlated with a blunted increase in αSMA, and elevated activities of matrix metalloproteinases (MMP2, MMP9). These data suggest that PAI-1 plays a critical role in mediating fibrosis caused by AngII and lends weight-of-evidence to a pro-fibrotic role of this protein in liver. Furthermore, the current study proposes a new model of ‘pure’ hepatic fibrosis in mice with little inflammation or hepatocyte death.     

Syntaxin requirement for Ca2+-triggered exocytosis in neurons and endocrine cells demonstrated with an engineered neurotoxin

Botulinum neurotoxins cleave synaptic SNAREs and block exocytosis, demonstrating that these proteins function in neurosecretion. However, the function of the SNARE syntaxin remains less clear because no neurotoxin cleaves it selectively. Starting with a botulinum neurotoxin that cleaves both syntaxin and SNAP-25, we engineered a version that retains activity against syntaxin but spares SNAP-25. These mutants block synaptic release in neurons and norepinephrine release in neuroendocrine cells, thus establishing an essential role for syntaxin in Ca2+-triggered exocytosis. These mutants can generate syntaxin-free cells as a useful experimental system for research and may lead to pharmaceuticals that target syntaxin selectively.     

Fibrillation of the major curli subunit CsgA under a wide range of conditions implies a robust design of aggregation

The amyloid fold is usually considered a result of protein misfolding. However, a number of studies have recently shown that the amyloid structure is also used in nature for functional purposes. CsgA is the major subunit of Escherichia coli curli, one of the most well-characterized functional amyloids. Here we show, using a highly efficient approach to prepare monomeric CsgA, that in vitro fibrillation of CsgA occurs under a wide variety of environmental conditions and that the resulting fibrils exhibit similar structural features. This highlights how fibrillation is "hardwired" into amyloid that has evolved for structural purposes in a fluctuating extracellular environment and represents a clear contrast to disease-related amyloid formation. Furthermore, we show that CsgA polymerization in vitro is preceded by the formation of thin needlelike protofibrils followed by aggregation of the amyloid fibrils.     

Permeation of styryl dyes through nanometer-scale pores in membranes

Styryl dyes are widely used to study synaptic vesicle (SV) recycling in neurons; vesicles are loaded with dye during endocytosis, and dye is subsequently released via exocytosis. During putative kiss-and-run exocytosis, efflux of dye from individual SVs has been proposed to occur via two sequential steps: dissociation from the membrane followed by permeation through a small fusion pore. To improve our understanding of the kinetics of efflux of dye from vesicles during kiss-and-run events, we examined the rates of efflux of different dyes through nanometer-scale pores formed in membranes by the toxins melittin and α-hemolysin; these pores approximate the size of fusion pores measured in neuroendocrine cells. We found that the axial diameter of each dye was a crucial determinant for permeation. Moreover, the two dyes with the largest cross-sectional areas were completely unable to pass through pores formed by a mutant α-hemolysin that has a slightly smaller pore than the wild-type toxin. The overall time constant for efflux (seconds) of each dye was orders of magnitude slower than the time constant for dissociation from membranes (milliseconds). Thus, the permeation step is rate-limiting, and this observation was further supported by atomistic molecular dynamics simulations. Together, the data reported here help provide a framework for interpreting dye destaining rates from secretory vesicles.     

Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein

The oligopeptide permease (Opp) of Escherichia coli is an ATP-binding cassette transporter that uses the substrate-binding protein (SBP) OppA to bind peptides and deliver them to the membrane components (OppBCDF) for transport. OppA binds conventional peptides 2-5 residues in length regardless of their sequence, but does not facilitate transport of the cell wall component murein tripeptide (Mtp, L-Ala-γ-D-Glu-meso-Dap), which contains a D-amino acid and a γ-peptide linkage. Instead, MppA, a homologous substrate-binding protein, forms a functional transporter with OppBCDF for uptake of this unusual tripeptide. Here we have purified MppA and demonstrated biochemically that it binds Mtp with high affinity (K(D) ～ 250 nM). The crystal structure of MppA in complex with Mtp has revealed that Mtp is bound in a relatively extended conformation with its three carboxylates projecting from one side of the molecule and its two amino groups projecting from the opposite face. Specificity for Mtp is conferred by charge-charge and dipole-charge interactions with ionic and polar residues of MppA. Comparison of the structure of MppA-Mtp with structures of conventional tripeptides bound to OppA, reveals that the peptide ligands superimpose remarkably closely given the profound differences in their structures. Strikingly, the effect of the D-stereochemistry, which projects the side chain of the D-Glu residue at position 2 in the direction of the main chain in a conventional tripeptide, is compensated by the formation of a γ-linkage to the amino group of diaminopimelic acid, mimicking the peptide bond between residues 2 and 3 of a conventional tripeptide.     

Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) β subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F(1) α subunit. While yeast mutants lacking β subunit have stable aggregated F(1) α subunit in the mitochondrial matrix, the human α subunit is completely degraded in cells deficient in F(1) β subunit. These results are discussed in light of the different properties of the proteins and environments in which yeast and human mitochondria exist.     

The role of cladocerans in tracking long-term change in shallow lake trophic status

Shallow lakes have been affected by a variety of human activities profoundly altering their ecological structure and function. Cladocerans have been used to track change resulting from a variety of drivers at a number of time scales. Aquatic macrophytes are well recognised as reflecting the ecological condition of a lake. Here, we compare the plant macrofossils with the sub-fossil cladoceran assemblages from 20 dated sediment cores. Co-correspondence analysis was used to determine the degree of commonality of change in community composition of the two biological groups through time. This analysis revealed very high levels of agreement in the nature and timing of change at all the sites examined with very high correlation coefficients between the axis 1 scores for macrofossils and cladocerans. Furthermore, at all sites a high proportion of the variance (min 20%, max 54%) in the macrofossil data was explained by the change in the cladoceran assemblage. Sub-fossil macrofossil and cladoceran assemblages, from at least from 1700 AD onwards, were examined in more detail at three sites: Ormesby Great Broad, Felbrigg Lake and Lake Søbygaard. There was very good accord in the main shifts of the cladoceran and macrofossil assemblages at all three sites. This may reflect the long-term shift in the principal focus of primary production from the benthic to the pelagic habitat. We suggest that the combination of their central position in the food-web and the presence of both pelagic and benthic taxa make cladocerans a strong candidate as the single best indicator of (palaeo) ecological condition related to changing trophic status and alteration in food-web structure in shallow lakes.