Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).

Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma.

High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse.

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.

Signals for endocytosis and for basolateral and lysosomal sorting are closely related in a number of membrane proteins, suggesting similar sorting mechanisms at the plasma membrane and in the trans-Golgi network (TGN). We tested the hypothesis that basolateral membrane proteins are transported to the cell surface via endosomes for the asialoglycoprotein receptor H1. This protein was tagged with a tyrosine sulfation site (H1TS) to allow specific labeling with [35S]sulfate in the TGN. Madin-Darby canine kidney cells expressing H1TS were pulse-labeled and chased for a period of time insufficient for labeled H1TS to reach the cell surface. Upon homogenization and gradient centrifugation, fractions devoid of TGN were subjected to immunoisolation of compartments containing mannose 6-phosphate receptor, which served as an endosomal marker. H1TS in transit to the cell surface was efficiently coisolated, whereas a labeled secretory protein and free glycosaminoglycan chains were not. This indicates an indirect pathway for the asialoglycoprotein receptor to the plasma membrane via endosomes and has important implications for protein sorting in the TGN and endosomes.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Reduction of tobacco smoke components yield in commercial cigarette brands by addition of HUSY, NaY and Al-MCM-41 to the cigarette rod

The effect of two zeolites, HUSY, NaY and a mesoporous synthesized Al-MCM-41 material on the smoke composition of ten commercial cigarettes brands has been studied. Cigarettes were prepared by mixing the tobacco with the three powdered materials, and the smoke obtained under the ISO conditions was analyzed. Up to 32 compounds were identified and quantified in the gas fraction and 80 in the total particulate matter (TPM) condensed in the cigarettes filters and in the traps located after the mouth end of the cigarettes. Al-MCM-41 is by far the best additive, providing the highest reductions of the yield for most compounds and brands analyzed. A positive correlation was observed among the TPM and nicotine yields with the reduction obtained in nicotine, CO, and most compounds with the three additives. The amount of ashes in additive free basis increases due to the coke deposited on the solids, especially with Al-MCM-41. Nicotine is reduced with Al-MCM-41 by an average of 34.4% for the brands studied (49.5% for the brand where the major reduction was obtained and 18.5 for the brand behaving the worst). CO is reduced by an average of 18.6% (ranging from 10.3 to 35.2% in the different brands).

Comparison of PRC and Vietnam's Responses to the Elimination of U.S. Textile and Apparel Quotas: Economic and Cultural Perspectives. ACES Working Paper No. 1, 2007

International trade in textiles and apparel has, as of January 1, 2005, been set free from the very intricate Multi-Fiber textile and apparel quota Arrangement (MFA). This event has raised many uncertainties about the new international trade climate and has placed enormous pressure on China as the expected clear cut beneficiary of this liberalization.' Other countries considered to be major contenders include Vietnam which also has a large population employed in the textile and apparel (T&A) sector. Since the old quota system had provided a certain degree of market certainty to competing T&A producers, will the new free trade environment lead to a shake out where mass producers with large economies of scale dominate the new reality? The removal of T&A quotas will create opportunities for Vietnam and China along with other developing countries, but it will also expose them to additional competition from each other. The outcome of this competition will depend on the demand in the US, the ability of the exporting countries to differentiate their exports and on their ability to transfer additional resources to expand domestic output in the direction of the new 'free market signals' and away from rent seeking objectives. Obviously, exporting countries that adjust to this new environment quickly will improve their competitiveness, and will be the new beneficiaries of a quota free international trade in textiles and apparel. This paper attempts to shed some light on the differences and similarities in the responses of Chinese and Vietnamese T&A sectors to this new environment. It first focuses on the demand side attempting to determine whether or not Chinese and Vietnamese T&A items, formally under quota control, are substitutes or compliments. On the supply side, the paper focuses on institutional differences between each country's T&A sectors, the different domestic government policies that have contributed to their growth and the unique cultural differences which will determine the future progress in each country's T&A sectors.

The EU Counter-Terrorism Policy Responses to the Attacks in Paris: Towards an EU Security and Liberty Agenda. CEPS Liberty and Security in Europe No. 81/February 2015

This paper examines the main EU-level initiatives that have been put forward in the weeks following the attacks in Paris in January 2015, which will be discussed in the informal European Council meeting of 12 February 2015. It argues that a majority of these proposals predated the Paris shootings and had until that point proved contentious as regards their efficacy, legitimacy and lawfulness. The paper finds that EU counterterrorism responses raise two fundamental challenges: A first challenge is posed to the freedom of movement, Schengen and EU citizenship. Priority is being given to the expanded use of large-scale surveillance and systematic monitoring of all travellers including EU citizens, which stands in contravention of Schengen and the free movement principle. A second challenge concerns EU democratic rule of law. Current pressures calling for the adoption of measures such as the EU Passenger Name Record challenge the scrutiny roles held by the European Parliament and the Court of Justice of the EU on counterterrorism measures in a post-Lisbon Treaty setting. The paper proposes that the EU adopts a new European Agenda on Security and Liberty based on an EU security (criminal justice-led) cooperation model that is firmly anchored in current EU legal principles and rule of law standards. This model would call for ‘less is more’ concerning the use, processing and retention of data by police and intelligence communities. Instead, it would pursue better and more accurate use of data meeting the quality standards of evidence in criminal judicial proceedings.

From Lampedusa to the Post-Stockholm Programme: Difficult European solidarity in the field of migration. European Policy Brief No. 24, March 2014

Solidarity is a founding principle of the European migration policy. To hold true, Member States must be faithful to their common commitment to European migration rules and implement fair burden sharing of the costs attached to border controls. However, solidarity among Member States appears altogether fragile and under threat, a situation that could jeopardise the founding principle of the free movement of persons in the European Union’s space. The recent solidarity crisis among Member States was solved by an increased externalisation of the European migration policy. Consequently, for the EU to live up to its values, it will have to prove itself generous towards third countries.

Enhancing the Prospects of the EU’s Deep and Comprehensive Free Trade Areas in the Mediterranean: Lessons from the Eastern Partnership. CEPS Commentary, 24 June 2015

In their March 2015 Consultation paper on the review of the European Neighbourhood Policy (ENP), the European Commission and the High Representative raised the question whether the Deep and Comprehensive Free Trade Areas (DCFTAs) “are the right objective for all or should more tailor-made alternatives be developed, to reflect differing interests and ambitions of some partners?” Such ambitious but complex trade agreements have now finally been signed with Ukraine, Moldova and Georgia, but they are still on the table for several Mediterranean ENP countries. Although these Mediterranean partners have a completely different political, economic and legal relationship with the EU, some important lessons can be drawn from the ‘Eastern DCFTA experience’. In particular, the DCFTA negotiators should avoid overly ambitious and ill-defined legislative approximation commitments and develop a comprehensive implementation strategy.

What is happening to the Schengen borders? CEPS Liberty and Security in Europe No. 86, December 2015

What is happening to the Schengen borders? Is Schengen in ‘crisis’? This paper examines the state of play in the Schengen system in light of the developments during 2015. It critically examines the assertion that Schengen is ‘in crisis’ and seeks to set the record straight on what has been happening to the intra-Schengen border-free and common external borders system. The paper argues that Schengen is here to stay and that reports about the reintroduction of internal border checks are exaggerated as they are in full compliance with the EU rule of law model laid down in the Schengen Borders Code and subject to scrutiny by the European Commission. It also examines the legal challenges inherent to police checks within the internal border areas as having an equivalent effect to border checks as well as the newly adopted proposal for a European Border and Coast Guard system. The analysis shows that the most far-reaching challenge to the current and future configurations of EU border policies relates to ensuring that they are in full compliance with fundamental human rights obligations to refugees, effective accountability and independent monitoring of the implementation of EU legal standards. This should be accompanied by a transparent and informed discussion on which ‘Schengen’ and which 'common European Border and Coast Guard Agency' we exactly want within current democratic rule of law and fundamental rights remits.

Free Movement of Persons: The Mirage of Social Security Schemes. Bruges European Economic Research Papers (BEER) 34/2015

The purpose of this paper is to address the issue of social security benefits that jobseekers, nationals of other Member State, residing in another Member States are in title to, as well as the economic implications of free movement of persons and labour market access. Consequently, it aims to disentangle between labour mobility welfare effects and “benefit tourism” looking in particular at the United Kingdom social security system and analysing the policy framework currently in place that governs the free movement of people across the European Union Member States.

Effects of Interspecific Gene Flow on the Phenotypic Variance-Covariance Matrix in Lake Victoria Cichlids

Quantitative genetics theory predicts adaptive evolution to be constrained along evolutionary lines of least resistance. In theory, hybridization and subsequent interspecific gene flow may however rapidly change the evolutionary constraints of a population and eventually change its evolutionary potential, but empirical evidence is still scarce. Using closely related species pairs of Lake Victoria cichlids sampled from four different islands with different levels of interspecific gene flow, we tested for potential effects of introgressive hybridization on phenotypic evolution in wild populations. We found that these effects differed among our study species. Constraints measured as the eccentricity of phenotypic variance-covariance matrices declined significantly with increasing gene flow in the less abundant species for matrices that have a diverged line of least resistance. In contrast we find no such decline for the more abundant species. Overall our results suggest that hybridization can change the underlying phenotypic variance-covariance matrix, potentially increasing the adaptive potential of such populations.

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.