Natural selection on floral traits of Lobelia (Lobeliaceae): spatial and temporal variation

The strength and direction of natural selection on floral traits can vary spatially and temporally because of variation in the biotic and abiotic environment. High spatial variation in selection should lead to differentiation of floral traits among populations. In contrast, high temporal variation in selection should retard the evolution of population-specific floral phenotypes. To determine the relative importance of spatial vs. temporal variation in natural selection, we measured phenotypic selection on seven floral traits of the wildflowers Lobelia cardinalis and L. siphilitica in 1999 and 2000. Lobelia cardinalis experienced significant temporal variation in selection, whereas L. siphilitica experienced spatial variation in selection on the same traits. This variation in selection on floral traits was associated with spatial and temporal differences in the soil microenvironment. Although few studies of natural selection include spatial or temporal replicates, our results suggest that such replication is critical for understanding the distribution of phenotypes in nature.     

Rho proteins: linking signaling with membrane trafficking

Rho proteins are well known for their effects on the actin cytoskeleton, and are activated in response to a variety of extracellular stimuli. Several Rho family members are localized to vesicular compartments, and increasing evidence suggests that they play important roles in the trafficking of vesicles on both endocytic and exocytic pathways. In particular, RhoA, RhoB, RhoD, Rac and Cdc42 have been shown to affect various steps of membrane trafficking. The underlying molecular basis for these effects of Rho proteins are incompletely understood, but in the case of Cdc42 it appears that it can drive vesicle movement through Arp2/3 complex-mediated actin polymerization at the surface of the vesicle. This is similar to what is believed to happen when Rac and Cdc42 stimulate actin polymerization at the plasma membrane. Rho proteins may also affect membrane trafficking by altering phosphatidylinositide composition of membrane compartments, or through interactions with microtubules.     

Total synthesis and evaluation of lamellarin alpha 20-Sulfate analogues

In order to explore the influence of sulfate groups on the bioactivity profiles of marine alkaloids of the lamellarin class, three such alkaloids, lamellarin alpha, lamellarin alpha 13,20-disulfate and lamellarin H, were synthesized and their activities against HIV-1 integrase and cancer cell lines were compared with those of lamellarin alpha 20-sulfate, which is a selective inhibitor of HIV-1 integrase. Lamellarin alpha does not inhibit HIV-1 integrase but shows moderate cytotoxicity with good cell line selectivity. Lamellarin alpha 13,20-disulfate is a moderate inhibitor of both HIV-1 integrase and cancer cell lines. Lamellarin H is a more potent inhibitor of HIV-1 integrase but lacked the specificity required to be medicinally useful.     

Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP. The main reason for this is the presence of serines in the positions equivalent to Ala59 and Gln61 in Ras. The structure shows that there are still water molecules in similar positions to the waters thought to be involved in the hydrolysis reaction in other G proteins. The structure suggests three not necessarily exclusive explanations for the lack of hydrolysis. The lack of the conserved glutamine raises the energy of the transition state inhibiting hydrolysis. The serines may restrain the waters from moving closer to the GTP, a step that is required to attain the transition state. They also stabilize the GTP-bound conformation of switch II and could prevent conformational changes required during hydrolysis. By superposition of the RhoE structure on structures of Rho family proteins in complex with binding partners, we make predictions on RhoE interactions with these partners.     

Assessment of cognitive and motor deficits in a marmoset model of stroke

The Stroke Therapy Academic Industry Roundtable noted the need for standardized, well-accepted primate models of stroke to help develop both neuroprotective and restorative therapies. One primate model has been developed using the marmoset, a small New World species of monkey, in which long-term functional deficits can be assessed. The surgery and postoperative care of the animals is described, as well as the behavioral tests used to quantify the postoperative disability. The types of deficits seen are illustrated by reference to some of the findings with neuroprotective treatments. Nevertheless, the long-term nature and consistency of the motor deficits make this model ideal for assessing the worth of restorative therapies.     

The type and yield of lipopolysaccharide from symbiotically deficient rhizobium lipopolysaccharide mutants vary depending on the extraction method

At least 18 lipopolysaccharide (LPS) extraction methods are available, and no single method is universally applicable. Here, the LPSs from four R.etli, one R.leguminosarum bv. trifolii mutant, 24AR, and the R.etli parent strain, CE3, were isolated by hot phenol/water (phi;/W), and phenol/EDTA/triethylamine (phi/EDTA/TEA) extraction. The LPS in various preparations was quantified, analyzed by deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE), and by immunoblotting. These rhizobia normally have two prominent LPS forms: LPS I, which has O-polysaccharide, and LPS II, which has none. The LPS forms obtained depend on the method of extraction and vary depending on the mutant that is extracted. Both methods extract LPS I and LPS II from CE3. The phi/EDTA/TEA, but not the phi/W, method extracts LPS I from mutants CE358 and CE359. Conversely, the phi;/W but not the phi;/EDTA/TEA method extracts CE359 LPS V, an LPS form with a truncated O-polysaccharide. phi/EDTA/TEA extraction of mutant CE406 gives good yields of LPS I and II, while phi/W extraction gives very small amounts of LPS I. The LPS yield from all the strains using phi/EDTA/TEA extraction is fairly consistent (3-fold range), while the yields from phi/W extraction are highly variable (850-fold range). The phi/EDTA/TEA method extracts LPS I and LPS II from mutant 24AR, but the phi/W method partitions LPS II exclusively into the phenol phase, making its recovery difficult. Overall, phi/EDTA/TEA extraction yields more forms of LPS from the mutants and provides a simpler, faster, and less hazardous alternative to phi/W extraction. Nevertheless, it is concluded that careful analysis of any LPS mutant requires the use of more than one extraction method.     

Design and construction of a uniaxial cell stretcher

In vitro mechanical cell stimulators are used for the study of the effect of mechanical stimulation on anchorage-dependent cells. We developed a new mechanical cell stimulator, which uses stepper motor technology and computer control to achieve a high degree of accuracy and repeatability. This device also uses high-performance plastic components that have been shown to be noncytotoxic, dimensionally stable, and resistant to chemical degradation from common culture laboratory chemicals. We show that treatment with glow discharge for 25 s at 20 mA is sufficient to modify the surface of the rubber to allow proper adhesion for polymerization of aligned collagen. We show through finite element analysis that the middle area of the membrane, away from the clamped ends, is predictable, homogeneous, and has negligible shear strain. To test the efficacy of the mechanical stretch, we examined the effect of mechanical stimulation on the production of beta(1)-integrin by neonatal rat cardiac fibroblasts. Mechanical stimulation was tested in the range of 0-12% stretch and 0-10-cycles/min stretch frequency. The fibroblasts respond with an increase in beta(1)-integrin at 3% stretch and a decrease at 6 and 12% stretch. Stretch frequency was found to not significantly effect the concentration of beta(1)-integrin. These studies yield a new and improved mechanical cell stimulator and demonstrate that mechanical stimulation has an effect on the expression of beta(1)-integrin.     

Rac Activation by the T-Cell Receptor Inhibits T Cell Migration

Background

T cell migration is essential for immune responses and inflammation. Activation of the T-cell receptor (TCR) triggers a migration stop signal to facilitate interaction with antigen-presenting cells and cell retention at inflammatory sites, but the mechanisms responsible for this effect are not known.

Methodology/Principal Findings

Migrating T cells are polarized with a lamellipodium at the front and uropod at the rear. Here we show that transient TCR activation induces prolonged inhibition of T-cell migration. TCR pre-activation leads to cells with multiple lamellipodia and lacking a uropod even after removal of the TCR signal. A similar phenotype is induced by expression of constitutively active Rac1, and TCR signaling activates Rac1. TCR signaling acts via Rac to reduce phosphorylation of ezrin/radixin/moesin proteins, which are required for uropod formation, and to increase stathmin phosphorylation, which regulates microtubule stability. T cell polarity and migration is partially restored by inhibiting Rac or by expressing constitutively active moesin.

Conclusions/Significance

We propose that transient TCR signaling induces sustained inhibition of T cell migration via Rac1, increased stathmin phosphorylation and reduced ERM phosphorylation which act together to inhibit T-cell migratory polarity.     

Bidirectional history of hybridization in California wild radish, Raphanus sativus (Brassicaceae), as revealed by chloroplast DNA

The evolutionary processes that take place in invasive plant populations are not well documented or understood. Interspecific hybridization between cultivated radish (Raphanus sativus) and R. raphanistrum is known to be responsible for the origin of the invasive California wild radish, but little is known about the nature of the hybridization events that produced the hybrid-derived lineage. We analyzed the trnL-rpl32 intergenic region of chloroplast DNA (cpDNA) obtained from 37 cultivated radish individuals from four different cultivars, 53 R. raphanistrum individuals from six European populations and 104 California wild radish individuals from 11 populations covering its entire range throughout the state. We found that cultivated radish and R. raphanistrum shared no cpDNA haplotypes but that they both shared haplotypes with California wild radish, evidence for bidirectional hybridization between the progenitor species in the creation of the California lineage. We also found evidence that multiple cultivars and multiple European source populations contributed to the diversity of cpDNA haplotypes within California. Studies like this will continue to be important for our understanding of the origin of invasive populations and the mechanisms by which they succeed.