Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury.

Arterial injury induces a series of proliferative, vasoactive, and inflammatory responses that lead to vascular proliferative diseases, including atherosclerosis and restenosis. Although several factors have been defined which stimulate this process in vivo, the role of specific cellular gene products in limiting this response is not well understood. The p21 cyclin-dependent kinase inhibitor affects cell cycle progression, senescence, and differentiation in transformed cells, but its expression in injured blood vessels has not been investigated. In this study, we report that p21 protein is induced in porcine arteries following balloon catheter injury and suggest that p21 is likely to play a role in limiting arterial cell proliferation in vivo. Vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle. Following injury to porcine arteries, p21 gene product was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Direct gene transfer of p21 using an adenoviral vector into balloon injured porcine arteries inhibited the development of intimal hyperplasia. Taken together, these findings suggest that p21, and possibly related cyclin-dependent kinase inhibitors, may normally regulate cellular proliferation following arterial injury, and strategies to increase its expression may prove therapeutically beneficial in vascular diseases.

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.

Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.

Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.

The role of voice input for human-machine communication.

Optimism is growing that the near future will witness rapid growth in human-computer interaction using voice. System prototypes have recently been built that demonstrate speaker-independent real-time speech recognition, and understanding of naturally spoken utterances with vocabularies of 1000 to 2000 words, and larger. Already, computer manufacturers are building speech recognition subsystems into their new product lines. However, before this technology can be broadly useful, a substantial knowledge base is needed about human spoken language and performance during computer-based spoken interaction. This paper reviews application areas in which spoken interaction can play a significant role, assesses potential benefits of spoken interaction with machines, and compares voice with other modalities of human-computer interaction. It also discusses information that will be needed to build a firm empirical foundation for the design of future spoken and multimodal interfaces. Finally, it argues for a more systematic and scientific approach to investigating spoken input and performance with future language technology.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: a potential pathologic role.

Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaque-infiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.

Agouti regulation of intracellular calcium: role in the insulin resistance of viable yellow mice.

Several dominant mutations at the agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the agouti gene product mediates these effects is unclear. Since intracellular Ca2+ is believed to play a role in mediating insulin action and dysregulation of Ca2+ flux is observed in diabetic animals and humans, we examined the status of intracellular Ca2+ in mice carrying the dominant agouti allele, viable yellow (Avy). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca2+]i) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the agouti gene product is capable of inducing increased [Ca2+]i in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca2+]i.

International financial regulation: A role for the Eurozone? EU Diplomacy Paper 08/2012, October 2012

The deepest financial crisis to strike the global economy since the Great Depression has unceremoniously called into question the very foundations of the Western economic model. The liberalisation of capital flows and the growing internationalisation of financial markets outpaced global regulatory and supervisory efforts. The repercussions of the financial crisis have given new dynamism to the reform of financial regulation both globally and within the European Union (EU). The Eurozone, by way of its own failings, has emerged as a stronger conceptual and legitimate entity since the onset of the crisis, but to what extent does this equate to a greater external role, in particular in the reform of international financial regulation? This paper argues that the Eurozone is currently not in a position to play an important role in the reform of international financial regulation, as it is a weak actor in the context of the EU financial architecture, which is still largely characterised by differing national regimes, a prevailing influence from the UK and fragmented external representation. The key finding from this study is that internal tensions in the EU are at the very heart of the Eurozone’s difficulties in playing a role in the reform of international financial regulation. Surmounting these tensions is a pre-requisite for the Eurozone if it is to overcome its structural weakness in international financial politics. However, the implications of such evolutions to the Eurozone, as an entity, and to European integration are far-reaching.

The Role of Market Mechanisms in a Post-2020 Climate Change Agreement. CEPS Special Report No. 87/May 2014

This special report is intended to serve as a background briefing document for the European Climate Platform seminar on Carbon Markets in the 2015 Agreement: Role and Architecture, but also raises issues of more enduring relevance in the wider debate about market mechanisms and the next climate change agreement. The paper looks at the relationship between the carbon market and a new climate change agreement, to be finalised in Paris in 2015. It tries to answer two key questions: does the carbon market have a role to play in a post-2020 agreement, and what is the role of a post-2020 agreement in the creation and operation of a carbon market? Introduction. The world has changed in many ways since 1997 when the Kyoto Protocol was adopted, along some critical axes, both from an economic and emissions points of view. Moreover, and this cannot be quantified, the appetite for global governance, especially for an agreement with such far-reaching implications as a climate change agreement, has diminished considerably. This paper looks at the relationship between the carbon market and a new climate change agreement, to be finalised in Paris in 2015. It tries to answer two key questions: does the carbon market have a role to play in a post-2020 agreement, and what is the role of a post-2020 agreement in the creation and operation of a carbon market?

Improving the role of equity crowdfunding in Europe's capital markets. Bruegel Policy Contribution Issue 2014/09, August 2014

Crowdfunding is a growing phenomenon that encompasses several different models of financing for business or other ventures. Despite the hype, equity crowdfunding is still the smallest part of the crowdfunding market. Because of its legal framework, Europe has been at the forefront of equity crowdfunding market development. Equity crowdfunding is more complex than other forms of crowdfunding and requires proper checks and balances if it is to provide a viable channel for financial intermediation in the seed and early-stage market in Europe. It is important to explore this new channel of funding for young and innovative firms given the critical role these start-ups can play job creation and economic growth in Europe. We assess the potential role of equity crowdfunding in the overall seed and early-stage financing market and highlight the potential risks of equity crowdfunding. We describe the current state of play in this nascent industry, considering both the innovations introduced by market operators and existing regulation. Currently in Europe there is a patchwork of national legal frameworks related to equity crowdfunding and this should be addressed in a harmonised way.

Keeping health high on the EU agenda: role for economic governance? EPC Policy Brief, 7 May 2015

In an attempt to get Europe out of the economic crisis and establish right conditions for growth, the EU coordinates and monitors member states’ economic and budgetary policies via a system called the European Semester. As member states’ spending on the health sector accounts for 10% of GDP and is expected to grow, it is no wonder that an increasing emphasis has been paid to sustainability of health systems – an area that is traditionally considered as a national competence. In this Policy Brief, Annika Hedberg and Martina Morosi reflect on the strengths and weaknesses of the European Semester and country-specific recommendations in promoting more sustainable and efficient health systems in Europe, and why the EU must continue to play a role in encouraging member states to value health and improve their spending on health.

Towards better health outcomes - What role for pharmaceutical innovation? EPC Policy Brief, 10 December 2015

From the current refugee crisis to ageing populations, and from rising healthcare prices to patients’ rising expectations: demands on European health systems continue to increase. Delivering health efficiently and ensuring the long-term sustainability of healthcare in the face of reduced public budgets requires new thinking – and there is a role for pharmaceuticals as well. Building on the series of discussions organised under Transformations, this Policy Brief focuses on the specific role of medicines and pharmaceutical innovation in improving health outcomes. It considers the state of play of drug innovation, from the development to the deployment of medicines, and the measures needed to make it deliver more for society and the economy, while ensuring that patients in Europe can have access to innovative and safe solutions.

Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.