The core Escherichia coli signal recognition particle receptor contains only the N and G domains of FtsY

Previous studies have proposed that the N-terminal A domain (approximately 200 amino acid residues) of the Escherichia coli signal recognition particle (SRP) receptor, FtsY, is required for membrane targeting. In contrast to this suggestion, we show that A domain-truncated versions of FtsY, harboring only domains N and G, are functional. Therefore, we propose that N and G domains constitute the core SRP receptor.     

Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Soda consumption and overweight status of 2-year-old mexican-american children in california

Objective: The prevalence of overweight in United States children, 2 to 5 years old, has increased 2‐fold since 1975, with the highest prevalence in Mexican Americans. The objective of this study was to determine the association between current soda consumption and overweight in 2‐year‐old Mexican‐American children. Research Methods and Procedures: The Center for the Health Assessment of Mothers and Children of Salinas study is a longitudinal study of the health of low‐income Latino pregnant women and their children living in the Salinas Valley, CA. Six hundred pregnant women were enrolled (October 1999 to October 2000), and their children were followed until 2 years of age. This cross‐sectional analysis includes the 354 children who completed the 2‐year follow‐up interview. Standing height (centimeters) and weight (grams) were measured at 2 years. Overweight was defined as ≥95th percentile of the sex‐specific BMI for each child's age. Results: Fifty‐five (15.5%) children were overweight. Over half (56%) reported consuming any soda in the last week. After covariate adjustment, compared with no soda consumption, <1 soda/d was not related to overweight (adjusted odds ratio, 0.97; 95% confidence interval, 0.47, 1.99), but ≥1 soda/d was significantly associated with overweight (adjusted odds ratio, 3.39; 95% confidence interval, 1.43, 8.07), and the test for trend was significant (p = 0.02). Discussion: At 2 years of age, the prevalence of overweight among the Center for the Health Assessment of Mothers and Children of Salinas cohort is higher than the national prevalence estimate for Mexican‐American 2‐ to 5‐year‐old children and is significantly associated with current soda consumption. Interventions to reduce consumption of soda in young Mexican‐American children should be considered.     

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.     

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.     

Signal transduction in endothelial cells by the angiogenesis inhibitor histidine-rich glycoprotein targets focal adhesions

Histidine-rich glycoprotein (HRGP) is an abundant heparin-binding plasma protein. We have shown that a fragment released from the central histidine/proline-rich (His/Pro-rich) domain of HRGP blocks endothelial cell migration in vitro and vascularization and growth of murine fibrosarcoma in vivo. The minimal active HRGP domain exerting the anti-angiogenic effect was recently narrowed down to a 35 amino acid peptide, HRGP330, derived from the His/Pro-rich domain of HRGP. By use of a signal transduction antibody array representing 400 different signal transduction molecules, we now show that HRGP and the synthetic peptide HRGP330 specifically induce tyrosine phosphorylation of focal adhesion kinase and its downstream substrate paxillin in endothelial cells. HRGP/HRGP330 treatment of endothelial cells induced disruption of actin stress fibers, a process reversed by treatment of cells with the FAK inhibitor geldanamycin. In addition, VEGF-mediated endothelial cell tubular morphogenesis in a three-dimensional collagen matrix was inhibited by HRGP and HRGP330. In contrast, VEGF-induced proliferation was not affected by HRGP or HRGP330, demonstrating the central role of cell migration during tube formation. In conclusion, our data show that HRGP targets focal adhesions in endothelial cells, thereby disrupting the cytoskeletal organization and the ability of endothelial cells to assemble into vessel structures.     

Sexual conflict over floral receptivity

In flowering plants, the onset and duration of female receptivity vary among species. In several species the receptive structures wilt upon pollination. Here we explore the hypothesis that postpollination wilting may be influenced by pollen and serve as a general means to secure paternity of the pollen donor at the expense of female fitness. Taking a game-theoretical approach, we examine the potential for the evolution of a pollen-borne wilting substance, and for the coevolution of a defense strategy by the recipient plant. The model without defense predicts an evolutionarily stable strategy (ESS) for the production of wilting substance. The ESS value is highest when pollinator visiting rates are intermediate and when the probability that pollen from several donors arrives at the same time is low. This finding has general implications in that it shows that male traits to secure paternity also can evolve in species, such as plants, where mating is not strictly sequential. We further model coevolution of the wilting substance with the timing of stigma receptivity. We assume that pollen-receiving plants can reduce the costs induced by toxic pollen by delaying the onset of stigmatic receptivity. The model predicts a joint ESS, but no female counter-adaptation when the wilting substance is highly toxic. This indicates that toxicity affects the probability that a male manipulative trait stays beneficial (i.e., not countered by female defense) over evolutionary time. We discuss parallels to male induced changes in female receptivity known to occur in animals and the role of harm for the evolution of male manipulative adaptations.     

Choosing negative examples for the prediction of protein-protein interactions

The protein-protein interaction networks of even well-studied model organisms are sketchy at best, highlighting the continued need for computational methods to help direct experimentalists in the search for novel interactions. This need has prompted the development of a number of methods for predicting protein-protein interactions based on various sources of data and methodologies. The common method for choosing negative examples for training a predictor of protein-protein interactions is based on annotations of cellular localization, and the observation that pairs of proteins that have different localization patterns are unlikely to interact. While this method leads to high quality sets of non-interacting proteins, we find that this choice can lead to biased estimates of prediction accuracy, because the constraints placed on the distribution of the negative examples makes the task easier. The effects of this bias are demonstrated in the context of both sequence-based and non-sequence based features used for predicting protein-protein interactions.     

Affiliative and Aggressive Behavior in a Group of Female Somali Wild Ass (Equus africanus somalicus)

We observed a group of three young female Somali wild asses to develop an ethogram of social behavior in the first phase of a longer term study of social, sexual, and maternal/infant behavior. The most unexpected finding was the frequency and extent of aggressive interactions, which included Charge, Drive, Neck Wrestle, Head Butt, and Body Slam, behaviors previously reported only for males of other equid species. The overall frequency of aggressive behavior was higher than that of affiliative behavior (84±16.5 vs. 32±5.5, P=0.03), yet no injuries occurred. The dyadic directionality of aggressive behavior suggested a dominance hierarchy, a feature not previously reported for either wild ass or domestic donkeys. The aggression observed may be an accurate representation of the behavior of this species, or their relatively young ages, or their recent transfer from their natal group through quarantine and into a new enclosure may have heightened agonistic tendencies. Further studies will determine whether with time their aggressive behavior becomes more intense or dissipates with maturity. Zoo Biol 31:87;–97, 2012. © 2011 Wiley Periodicals, Inc.