The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells

Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.     

High-resolution molecular epidemiology and evolutionary history of HIV-1 subtypes in Albania

Background

HIV-1 epidemic in Western Europe is largely due to subtype B. Little is known about the HIV-1 in Eastern Europe, but a few studies have shown that non-B subtypes are quite common. In Albania, where a recent study estimated a ten-fold increase of AIDS incidence during the last six years, subtype A and B account for 90% of the know infections.

Methodology/Principal Findings

We investigated the demographic history of HIV-1 subtype A and B in Albania by using a statistical framework based on coalescent theory and phylogeography. High-resolution phylogenetic and molecular clock analysis showed a limited introduction to the Balkan country of subtype A during the late 1980s followed by an epidemic outburst in the early 1990s. In contrast, subtype B was apparently introduced multiple times between the mid-1970s and mid-1980s. Both subtypes are growing exponentially, although the HIV-1A epidemic displays a faster growth rate, and a significantly higher basic reproductive number R₀. HIV-1A gene flow occurs primarily from the capital Tirane, in the center of the country, to the periphery, while HIV-1B flow is characterized by a balanced exchange between center and periphery. Finally, we calculated that the actual number of infections in Albania is at least two orders of magnitude higher than previously thought.

Conclusions/Significance

Our analysis demonstrates the power of recently developed computational tools to investigate molecular epidemiology of pathogens, and emphasize the complex factors involved in the establishment of HIV-1 epidemics. We suggest that a significant correlation exists between HIV-1 exponential spread and the socio-political changes occurred during the Balkan wars. The fast growth of a relatively new non-B epidemic in the Balkans may have significant consequences for the evolution of HIV-1 epidemiology in neighboring countries in Eastern and Western Europe.     

DNA polymerase lambda from calf thymus preferentially replicates damaged DNA

A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.     

Properties of endogenous β-glucosidase of a Saccharomyces cerevisiae strain isolated from Sicilian musts and wines

Three hundred sixty-one yeast strains (80 of which ascribable to Saccharomyces cerevisiae) were isolated from Sicilian musts and wines with the purpose of looking for β-glucosidase (βG, EC 3.2.1.21) activity. Of these, the AL 41 strain had highest endogenous βG activity and was identified as belonging to the species S. cerevisiae by biochemical and molecular methods. This enzyme was subsequently characterized. It had optimum effect at pH 3.5–4.0, whilst optimum temperature was 20 °C, compatible with typical wine-cellar conditions; it was not inhibited by ethanol, at concentrations of 12–14%, or fructose and glucose. The βG was also characterised in terms of the kinetic parameters K_m (2.55 mM) and V_max (1.71 U mg⁻¹ of protein). Finally, it remained stable for at least 35 days in model solutions of must and wine.     

Task partitioning in swarms of robots: an adaptive method for strategy selection

Task partitioning is the decomposition of a task into two or more sub-tasks that can be tackled separately. Task partitioning can be observed in many species of social insects, as it is often an advantageous way of organizing the work of a group of individuals. Potential advantages of task partitioning are, among others: reduction of interference between workers, exploitation of individuals?? skills and specializations, energy efficiency, and higher parallelism. Even though swarms of robots can benefit from task partitioning in the same way as social insects do, only few works in swarm robotics are dedicated to this subject. In this paper, we study the case in which a swarm of robots has to tackle a task that can be partitioned into a sequence of two sub-tasks. We propose a method that allows the individual robots in the swarm to decide whether to partition the given task or not. The method is self-organized, relies on the experience of each individual, and does not require explicit communication between robots. We evaluate the method in simulation experiments, using foraging as testbed. We study cases in which task partitioning is preferable and cases in which it is not. We show that the proposed method leads to good performance of the swarm in both cases, by employing task partitioning only when it is advantageous. We also show that the swarm is able to react to changes in the environmental conditions by adapting the behavior on-line. Scalability experiments show that the proposed method performs well across all the tested group sizes.     

Light controls stamen elongation via cryptochromes,phytochromes and COP1 through HY5 and HYH

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 – oppositely to ARF8 – directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far‐red light receptors PHYA/PHYB. In conclusion, different light qualities – sequentially perceived by specific photoreceptors – and the downstream COP1–HY5/HYH module finely tune auxin‐induced stamen elongation and thus male fertility.