Using genetic markers to estimate the pollen dispersal curve

Pollen dispersal is a critical process that shapes genetic diversity in natural populations of plants. Estimating the pollen dispersal curve can provide insight into the evolutionary dynamics of populations and is essential background for making predictions about changes induced by perturbations. Specifically, we would like to know whether the dispersal curve is exponential, thin-tailed (decreasing faster than exponential), or fat-tailed (decreasing slower than the exponential). In the latter case, rare events of long-distance dispersal will be much more likely. Here we generalize the previously developed TWOGENER method, assuming that the pollen dispersal curve belongs to particular one- or two-parameter families of dispersal curves and estimating simultaneously the parameters of the dispersal curve and the effective density of reproducing individuals in the population. We tested this method on simulated data, using an exponential power distribution, under thin-tailed, exponential and fat-tailed conditions. We find that even if our estimates show some bias and large mean squared error (MSE), we are able to estimate correctly the general trend of the curve - thin-tailed or fat-tailed - and the effective density. Moreover, the mean distance of dispersal can be correctly estimated with low bias and MSE, even if another family of dispersal curve is used for the estimation. Finally, we consider three case studies based on forest tree species. We find that dispersal is fat-tailed in all cases, and that the effective density estimated by our model is below the measured density in two of the cases. This latter result may reflect the difficulty of estimating two parameters, or it may be a biological consequence of variance in reproductive success of males in the population. Both the simulated and empirical findings demonstrate the strong potential of TWOGENER for evaluating the shape of the dispersal curve and the effective density of the population (d(e)).     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Polarized membrane traffic and cell polarity development is dependent on dihydroceramide synthase-regulated sphinganine turnover

Sphingoid bases have been implicated in various cellular processes including cell growth, apoptosis and cell differentiation. Here, we show that the regulated turnover of sphingoid bases is crucial for cell polarity development, i.e., the biogenesis of apical plasma membrane domains, in well-differentiated hepatic cells. Thus, inhibition of dihydroceramide synthase or sphinganine kinase activity with fumonisin B1 or N,N-dimethylsphingosine, respectively, dramatically perturbs cell polarity development, which is due to increased levels of sphinganine. Consistently, reduction of free sphinganine levels stimulates cell polarity development. Moreover, dihydroceramide synthase, the predominant enzyme responsible for sphinganine turnover, is a target for cell polarity stimulating cAMP/protein kinase A (PKA) signaling cascades. Indeed, electrospray ionization tandem mass spectrometry analyses revealed a significant reduction in sphinganine levels in cAMP/PKA-stimulated cells. These data suggest that sphinganine turnover is critical for and is actively regulated during HepG2 cell polarity development. Previously, we have identified an apical plasma membrane-directed trafficking pathway from the subapical compartment. This transport pathway, which is part of the basolateral-to-apical transcytotic itinerary, plays a crucial role in apical plasma membrane biogenesis. Here, we show that, as a part of the underlying mechanism, the inhibition of dihydroceramide synthase activity and ensuing increased sphinganine levels specifically perturb the activation of this particular pathway in the de novo apical membrane biogenesis.     

Parasite-mediated predation between native and invasive amphipods

Parasites can structure biological communities directly through population regulation and indirectly by processes such as apparent competition. However, the role of parasites in the process of biological invasion is less well understood and mechanisms of parasite mediation of predation among hosts are unclear. Mutual predation between native and invading species is an important factor in determining the outcome of invasions in freshwater amphipod communities. Here, we show that parasites mediate mutual intraguild predation among native and invading species and may thereby facilitate the invasion process. We find that the native amphipod Gammarus duebeni celticus is host to a microsporidian parasite, Pleistophora sp. (new species), with a frequency of infection of 0-90%. However, the parasite does not infect three invading species, G. tigrinus, G. pulex and Crangonyx pseudogracilis. In field and laboratory manipulations, we show that the parasite exhibits cryptic virulence: the parasite does not affect host fitness in single-species populations, but virulence becomes apparent when the native and invading species interact. That is, infection has no direct effect on G. d. celticus survivorship, size or fecundity; however, in mixed-species experiments, parasitized natives show a reduced capacity to prey on the smaller invading species and are more likely to be preyed upon by the largest invading species. Thus, by altering dominance relationships and hierarchies of mutual predation, parasitism strongly influences, and has the potential to change, the outcome of biological invasions.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

The sequence and crystal structure of the alpha-amino acid ester hydrolase from Xanthomonas citri define a new family of beta-lactam antibiotic acylases

alpha-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an alpha-amino group. As such, they can synthesize beta-lactam antibiotics from acyl compounds and beta-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-A resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an alpha/beta-hydrolase fold domain, a helical cap domain, and a jellyroll beta-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a beta-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an alpha-amino group on the substrate, and explains the low specificity toward the beta-lactam nucleus.     

Activation of the canonical beta-catenin pathway by histamine

Histamine signaling is a principal regulator in a variety of pathophysiological processes including inflammation, gastric acid secretion, neurotransmission, and tumor growth. We report that histamine stimulation causes transactivation of a T cell factor/beta-catenin-responsive construct in HeLa cells and in the SW-480 colon cell line, whereas histamine did not effect transactivation of a construct containing the mutated response construct FOP. On the protein level, histamine treatment increases phosphorylation of glycogen synthase kinase 3-beta in HeLa cells, murine macrophages, and DLD-1, HT-29, and SW-480 colon cell lines. Furthermore, histamine also decreases the phosphorylated beta-catenin content in HeLa cells and murine macrophages. Finally, pharmacological inhibitors of the histamine H1 receptor counteracted histamine-induced T cell factor/beta-catenin-responsive construct transactivation and the dephosphorylation of beta-catenin in HeLa cells and in macrophages. We conclude that the canonical beta-catenin pathway acts downstream of the histamine receptor H1 in a variety of cell types. The observation that inflammatory molecules, like histamine, activate the beta-catenin pathway may provide a molecular explanation for a possible link between inflammation and cancer.     

Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase

Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the dehalogenation pathway. Steady-state kinetic analysis of DhaA with a range of halopropanes showed that bromopropanes had higher k(cat) and lower K(M) values than the chlorinated analogues. The kinetic mechanism of dehalogenation was further studied using rapid-quench-flow analysis of 1,3-dibromopropane conversion. This provided a direct measurement of the chemical steps in the reaction mechanism, i.e., cleavage of the carbon-halogen bond and hydrolysis of the covalent alkyl-enzyme intermediate. The results lead to a minimal mechanism consisting of four main steps. The occurrence of a pre-steady-state burst, both for bromide and 3-bromo-1-propanol, suggests that product release is rate-limiting under steady-state conditions. Combining pre-steady-state burst and single-turnover experiments indicated that the rate of carbon-bromine bond cleavage was indeed more than 100-fold higher than the steady-state k(cat). Product release occurred with a rate constant of 3.9 s(-1), a value close to the experimental k(cat) of 2.7 s(-1). Comparing the kinetic mechanism of DhaA with that of the corresponding enzyme from Xanthobacter autotrophicus GJ10 (DhlA) shows that the overall mechanisms are similar. However, whereas in DhlA the rate of halide release represents the slowest step in the catalytic cycle, our results suggest that in DhaA the release of 3-bromo-1-propanol is the slowest step during 1,3-dibromopropane conversion.     

Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic halohydrins. To understand the kinetic mechanism and enantioselectivity of the enzyme, steady-state and pre-steady-state kinetic analysis was performed with p-nitro-2-bromo-1-phenylethanol (PNSHH) as a model substrate. Steady-state kinetic analyses indicated that the k(cat) of the enzyme with the (R)-enantiomer (22 s(-1)) is 3-fold higher than with the (S)-enantiomer and that the K(m) for the (R)-enantiomer (0.009 mM) is about 45-fold lower than that for the (S)-enantiomer, resulting in a high enantiopreference for the (R)-enantiomer. Product inhibition studies revealed that HheC follows an ordered Uni Bi mechanism for both enantiomers, with halide as the first product to be released. To identify the rate-limiting step in the catalytic cycle, pre-steady-state experiments were performed using stopped-flow and rapid-quench methods. The results revealed the existence of a pre-steady-state burst phase during conversion of (R)-PNSHH, whereas no such burst was observed with the (S)-enantiomer. This indicates that a product release step is rate-limiting for the (R)-enantiomer but not for the (S)-enantiomer. This was further examined by doing single-turnover experiments, which revealed that during conversion of the (R)-enantiomer the rate of bromide release is 21 s(-1). Furthermore, multiple turnover analyses showed that the binding of (R)-PNSHH is a rapid equilibrium step and that the rate of formation of product ternary complex is 380 s(-1). Taken together, these findings enabled the formulation of an ordered Uni Bi kinetic mechanism for the conversion of (R)-PNSHH by HheC in which all of the rate constants are obtained. The high enantiopreference for the (R)-enantiomer can be explained by weak substrate binding of the (S)-enantiomer and a lower rate of reaction at the active site.     

Age-related changes in the proteoglycans of human skin. Specific cleavage of decorin to yield a major catabolic fragment in adult skin

Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.