Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis.

The retinoblastoma susceptibility gene (Rb) participates in controlling the G1/S-phase transition, presumably by binding and inactivating E2F transcription activator family members. Mouse embryonic fibroblasts (MEFs) with no, one, or two inactivated Rb genes were used to determine the specific contributions of Rb protein to cell cycle progression and gene expression. MEFs lacking both Rb alleles (Rb-/-) entered S phase in the presence of the dihydrofolate reductase inhibitor methotrexate. Two E2F target genes, dihydrofolate reductase and thymidylate synthase, displayed elevated mRNA and protein levels in Rb- MEFs. Since absence of functional Rb protein in MEFs is sufficient for S-phase entry under growth-limiting conditions, these data indicate that the E2F complexes containing Rb protein, and not the Rb-related proteins p107 and p130, may be rate limiting for the G1/S transition. Antineoplastic drugs caused accumulation of p53 in the nuclei of both Rb+/+ and Rb-/- MEFs. While p53 induction led to apoptosis in Rb-/- MEFs, Rb+/- and Rb+/+ MEFs underwent cell cycle arrest without apoptosis. These results reveal that diverse growth signals work through Rb to regulate entry into S phase, and they indicate that absence of Rb protein produces a constitutive DNA replication signal capable of activating a p53-associated apoptotic response.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.

WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block.

Intrinsic transcript cleavage activity of RNA polymerase.

The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage constitutes the second enzymological activity of RNA polymerase along with polymerization/pyrophosphorolysis of RNA, whereas the Gre proteins merely enhance this intrinsic property.

Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.

Studies of the temperature-sensitive cdc37-1 mutant of Saccharomyces cerevisiae suggest that Cdc37 is required for passage through the G1 phase of the cell cycle, but its precise function is not known. We have investigated the role of Cdc37 in the regulation of the cyclin-dependent protein kinase Cdc28. We find that G1 arrest in the cdc37-1 mutant is accompanied by a decrease in the Cdc28 activity associated with the G1 cyclin Cln2. This defect appears to be caused by a decrease in the binding of Cdc28 and Cln2. cdc37-1 mutants also exhibit a defect in the binding and activation of Cdc28 by the mitotic cyclin Clb2. Thus Cdc37 may be a regulator that is required for the association of Cdc28 with multiple cyclins.

Overexpression of the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor beta 1.

One of the more intriguing aspects of transforming growth factor beta 1 (TGF beta 1) is its ability to function as both a mitogenic factor for certain mesenchymal cells and a potent growth inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/2 fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-2B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the growth-inhibitory response to TGF beta 1. Mouse keratinocytes (BALB/MK), which are normally growth-arrested by TGF beta 1, are rendered insensitive to the growth-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced growth arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced growth inhibition.

Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated oncogene.

Conditional oncogene expression in transgenic mice is of interest for studying the oncoprotein requirements during tumorigenesis and for deriving cell lines that can be induced to undergo growth arrest and enhance their differentiated functions. We utilized the bacterial tetracycline (Tet)-resistance operon regulatory system (tet) from Tn10 of Escherichia coli to control simian virus 40 (SV40) large tumor (T) antigen (TAg) gene expression and to generate conditionally transformed pancreatic beta cells in transgenic mice. A fusion protein containing the tet repressor (tetR) and the activating domain of the herpes simplex virus protein VP16, which converts the repressor into a transcription activator, was produced in beta cells of transgenic mice under control of the insulin promoter. In a separate lineage of transgenic mice, the TAg gene was introduced under control of a tandem array of tet operator sequences and a minimal promoter, which by itself is not sufficient for gene expression. Mice from the two lineages were then crossed to generate double-transgenic mice. Expression of the tetR fusion protein in beta cells activated TAg transcription, resulting in the development of beta-cell tumors. Tumors arising in the absence of Tet were cultured to derive a stable beta-cell line. Cell incubation in the presence of Tet led to inhibition of proliferation, as shown by decreased BrdUrd and [3H]thymidine incorporation. The Tet derivative anhydrotetracycline showed a 100-fold stronger inhibition compared with Tet. When administered in vivo, Tet efficiently inhibited beta-cell proliferation. These findings indicate that transformed beta cells selected for growth during a tumorigenesis process in vivo maintain a dependence on the continuous presence of the TAg oncoprotein for their proliferation. This system provides an approach for generation of beta-cell lines for cell therapy of diabetes as well as conditionally transformed cell lines from other cell types of interest.

Male/Female Manifestations of Narcissistic Personality and Behavioral Disorders.

Specifically, this paper will address the following topics : 1. The history of psychoanalytic thinking onnarcissism will be discussed, leading up to more recent ideas on the narcissistic personality disorder. 2. Drawing on historical and current ideas, an integrated definition of the narcissistic personality disorder will be presented and elaborated upon, including an examination of differing male/female narcissistic compensatory styles. 3. To foster an understanding of the development of narcissistic defenses and of differing gender styles of defense, various theories relating to gender differences in the narcissistic personality disorder will be explored: self/object relations theories, Kohut's theory on narcissism, psychosexual development theories, behavioral manifestation theories, and bodily development theories.4. The role of preoedipal development, as it relates to the formation of the male/female narcissistic personality disorder, will be examined. This section of the paper will propose that, in the narcissistic personality disorder, a pathological arrest occurred during the second or third year of life in response to trauma experienced at that time. The degree and timing of the trauma and the degree of structuralization preceding the trauma all contribute to the rigidity of the narcissistic disorder and the severity of the pathology. 5. The role of oedipal development in the male/female narcissistic personality disorder will be discussed. This discussion will address the intrapsychic configurations which arise during the oedipal period, after narcissistic defenses have solidified during the preoedipal years. While narcissism can be seen as a developmental line, with narcissistic defenses arising at any time during development, this paper will focus primarily on defenses which arise during the separation/individuation phases of development.

Warrants from the governor and chief justice of New York, 1703-1704

Copies of warrants and writs concerning public unrest caused by an attempt to survey lands on Long Island in 1702.

Paris, France, 1589-1643 (Time Series Map 7 of 8) (Raster Image)

This layer is a georeferenced raster image from the historic paper map series entitled: Lutece ... plan de la ville de Paris ..., par M.L.C.D.L.M. ; A. Coquart, delineavit et sculp. It was published by Jean & Pierre Cot in 1705. Scale [ca. 1:10,000]. This image is of map 7 entitled: Septiême plan de la ville de Paris son acroissement [sic] et ses embelissemens sous Henry III. et Louis XIII. depuis 1589 jusqu'en 1643: tiré des lettres patentes ou arrest du conseil qui ont ordonné les ouvrages des devis et marchez faits avec les entrepreneurs et levé sur les lieux ou ils ont été construits, et ou la plus grand partie subsistent encore. The map represents Paris, 1589 to 1643. Map in French.The image inside the map neatline is georeferenced to the surface of the earth and fit to the European Datum 1950, Universal Transverse Mercator (UTM) Zone 31N projected coordinate system. All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, index maps, legends, or other information associated with the principal map. This map shows features such as drainage, towns and villages, roads, built-up areas and selected buildings, fortification, ground cover, and more. Relief shown by hachures. Includes index, text, and notes.This layer is part of a selection of digitally scanned and georeferenced historic maps from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of originators, ground condition dates, scales, and map purposes.

Europe’s most wanted? Recalibrating Trust in the European Arrest Warrant System. CEPS Paper Liberty and Security in Europe 55/21 March 2013

This paper assesses the uses and misuses in the application of the European Arrest Warrant (EAW) system in the European Union. It examines the main quantitative results of this extradition system achieved between 2005 and 2011 on the basis of the existing statistical knowledge on its implementation at EU official levels. The EAW has been anchored in a high level of ‘mutual trust’ between the participating states’ criminal justice regimes and authorities. This reciprocal confidence, however, has been subject to an increasing number of challenges resulting from its practical application, presenting a dual conundrum: 1. Principle of proportionality: Who are the competent judicial authorities cooperating with each other and ensuring that there are sufficient impartial controls over the necessity and proportionality of the decisions on the issuing and execution of EAWs? 2. Principle of division of powers: How can criminal justice authorities be expected to handle different criminal judicial traditions in what is supposed to constitute a ‘serious’ or ‘minor’ crime in their respective legal settings and ‘who’ is ultimately to determine (divorced from political considerations) when is it duly justified to make the EAW system operational? It is argued that the next generation of the EU’s criminal justice cooperation and the EAW need to recognise and acknowledge that the mutual trust premise upon which the European system has been built so far is no longer viable without devising new EU policy stakeholders’ structures and evaluation mechanisms. These should allow for the recalibration of mutual trust and mistrust in EU justice systems in light of the experiences of the criminal justice actors and practitioners having a stake in putting the EAW into daily effect. Such a ‘bottom-up approach’ should be backed up with the best impartial and objective evaluation, an improved system of statistical collection and an independent qualitative assessment of its implementation. This should be placed as the central axis of a renewed EAW framework which should seek to better ensure the accountability, impartial (EU-led) scrutiny and transparency of member states’ application of the EAW in light of the general principles and fundamental rights constituting the foundations of the European system of criminal justice cooperation.

The End of the Transitional Period for Police and Criminal Justice Measures Adopted before the Lisbon Treaty: Who monitors trust in the European Criminal Justice area? CEPS Liberty and Security in Europe No. 74, December 2014

This study examines the legal and political implications of the forthcoming end of the transitional period for the measures in the fields of police and judicial cooperation in criminal matters, as set out in Protocol 36 to the EU Treaties. This Protocol limits some of the most far-reaching innovations introduced by the Treaty of Lisbon over EU cooperation on Justice and Home Affairs for a period of five years after the entry into force of the Treaty of Lisbon (until 1 December 2014), and provides the UK with special ‘opt out/opt-in’ possibilities. The study focuses on the meaning of the transitional period for the wider European Criminal Justice area. The most far-reaching change emerging from the end of this transition will be the expansion of the European Commission and Luxembourg Court of Justice scrutiny powers over Member States’ implementation of EU criminal justice law. The possibility offered by Protocol 36 for the UK to opt out and opt back in to pre-Lisbon Treaty instruments poses serious challenges to a common EU area of justice by further institutionalising ‘over-flexible’ participation in criminal justice instruments. The study argues that in light of Article 82 TFEU the rights of the defence are now inextricably linked to the coherency and effective operation of the principle of mutual recognition of criminal decisions, and calls the European Parliament to request the UK to opt in EU Directives on suspects procedural rights as condition for the UK to ‘opt back in’ measures like the European Arrest Warrant.

Management of floating thrombus in the aortic arch

OBJECTIVE Floating aortic thrombus is an underrecognized source of systemic emboli and carries a life-threatening risk of stroke when located in the aortic arch. Optimal treatment is not established in available guidelines. We report our experience in managing floating thrombi in the aortic arch. METHODS Consecutive patients diagnosed with a floating aortic arch thrombus at a tertiary referral center between January 2008 and December 2014 were reviewed. Perioperative and midterm outcomes were assessed. RESULTS Ten patients (8 female) with a median age of 56 years (range, 47-82 years) were identified. Eight patients presented with a symptomatic embolic event, and 2 patients were asymptomatic. One patient presenting with stroke due to embolic occlusion of all supra-aortic vessels died 2 days after admission. Three patients (2 asymptomatic and 1 unfit for surgery) were treated conservatively by anticoagulation, leading to thrombus resolution in 2 patients. In the third patient, the thrombus persisted despite anticoagulation, resulting in recurrent embolic events. The remaining 6 patients underwent open thrombectomy of the aortic arch during deep hypothermic circulatory arrest. All patients treated by surgery had an uneventful postoperative course with no recurrent thrombus or embolic event during follow-up. Median follow-up of all patients was 17 months (range, 11-89 months). CONCLUSIONS Floating aortic arch thrombus is a dangerous source of systemic emboli. Surgical removal of the thrombus is easy to perform and followed by good clinical results. Conservative treatment with anticoagulation may be considered in asymptomatic, inoperable or high-risk patients.

Site report: Lexington, Kentucky - field test of combined speed, alcohol, and safety belt enforcement strategies. Final report.

National Highway Traffic Safety Administration, Washington, D.C.

An analysis of ASAP impact on the judicial system. SD:ASAP analytic study no. 4 - 1976. Final report.

National Highway Traffic Safety Administration, Office of Driver and Pedestrian Programs, Washington, D.C.