The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

Growth and dislocation studies of β-HMX

Background: The defect structure of organic materials is important as it plays a major role in their crystal growth properties. It also can play a subcritical role in “hot-spot” detonation processes of energetics and one such energetic is cyclotetramethylene-tetranitramine, in the commonly used beta form (β-HMX). Results: The as-grown crystals grown by evaporation from acetone show prismatic, tabular and columnar habits, all with {011}, {110}, (010) and (101) faces. Etching on (010) surfaces revealed three different types of etch pits, two of which could be identified with either pure screw or pure edge dislocations, the third is shown to be an artifact of the twinning process that this material undergoes. Examination of the {011} and {110} surfaces show only one type of etch pit on each surface; however their natural asymmetry precludes the easy identification of their Burgers vector or dislocation type. Etching of cleaved {011} surfaces demonstrates that the etch pits can be associated with line dislocations. All dislocations appear randomly on the crystal surfaces and do not form alignments characteristic of mechanical deformation by dislocation slip. Conclusions: Crystals of β-HMX grown from acetone show good morphological agreement with that predicted by modelling, with three distinct crystal habits observed depending upon the supersaturation of the growth solution. Prismatic habit was favoured at low supersaturation, while tabular and columnar crystals were predominant at higher super saturations. The twin plane in β-HMX was identified as a (101) reflection plane. The low plasticity of β-HMX is shown by the lack of etch pit alignments corresponding to mechanically induced dislocation arrays. On untwinned {010} faces, two types of dislocations exist, pure edge dislocations with b = [010] and pure screw dislocations with b = [010]. On twinned (010) faces, a third dislocation type exists and it is proposed that these pits are associated with pure screw dislocations with b = [010]

Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits

Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC- gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC- beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC- beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway

26th Annual Midwest/Midsouth Estate Planning Institute

Materials from the 26th Annual Midwest/Midsouth Estate Planning Institute held by UK/CLE in July 1999

Measurement of the Target-Normal Single-Spin Asymmetry in Quasi-Elastic Scattering from the Reaction 3^3He↑(e,e′)^\uparrow(e,e^\prime)

We report the first measurement of the target single-spin asymmetry, $A_y$, in quasi-elastic scattering from the inclusive reaction $^3$He$^{\uparrow}(e,e^\prime)$ on a $^3$He gas target polarized normal to the lepton scattering plane. Assuming time-reversal invariance, this asymmetry is strictly zero for one-photon exchange. A non-zero $A_y$ can arise from the interference between the one- and two-photon exchange processes which is sensitive to the details of the sub-structure of the nucleon. An experiment recently completed at Jefferson Lab yielded asymmetries with high statistical precision at $Q^{2}=$ 0.13, 0.46 and 0.97 GeV$^{2}$. These measurements demonstrate, for the first time, that the $^3$He asymmetry is clearly non-zero and negative with a statistical significance of (8-10)$\sigma$. Using measured proton-to-$^{3}$He cross-section ratios and the effective polarization approximation, neutron asymmetries of $-$(1-3)% were obtained. The neutron asymmetry at high $Q^2$ is related to moments of the Generalized Parton Distributions (GPDs). Our measured neutron asymmetry at $Q^2=0.97$ GeV$^2$ agrees well with a prediction based on two-photon exchange using a GPD model and thus provides a new, independent constraint on these distributions

The European Union in the World — A Community of Values

These are momentous times in Europe. The Euro has been successfully introduced, the enlargement negotiations are approaching their climax, and the European Convention (“Convention”) is moving towards the drafting of a constitution for a new, continent-wide political entity. At the same time, unrest is manifest, particularly in two areas. On the one hand, many of our citizens, and not just the political elites, are dissatisfied with Europe\u27s performance on the world stage and are concerned about the maintenance of peace and security within the Union. In these areas they would like to see a strengthened, more effective entity-- “more Europe.” On the other hand, their disenchantment with the long reach of European Union (“EU” or “Union”) regulation in the first pillar area of economic policy is growing. The feeling of loss of local control over their destiny and a vague feeling of potential loss of identity within an ever more centralized polity is palpable. Here, they want “less Europe.” In the outside world, change is also the order of the day. The ice-sheet of bipolarity, which overlaid and hid the complexity of international relations during the Cold War, is breaking up at an ever-increasing speed and revealing a world in which two paradigms are competing to become the underlying ordering principles for the new century. The traditional paradigm of interacting Nation States, each pursuing its own separate interests, with alliances allowing the small to compete with the large, is alive and well, and its proponents like Machiavelli or Churchill continue to be in vogue in the literature of international relations and the rhetoric of world leaders. At the same time, there is a school of thought which points to the growing economic and ecological interdependence of our societies and the necessity for new forms of global governance to complement national action. It is also becoming abundantly clear that the concept of a “Nation State” is often a fiction, positing as it does an identity between the citizens of a State and the members of a culturally homogenous society. For both reasons, the concept of the Nation State as the principal actor on the world stage, is called into question. The experience of the Union with the sharing of State sovereignty is clearly related to the second paradigm and also to the EU\u27s firm support for the development of the United Nations (“U.N.”) as well as other elements of multilateral governance. It would hardly be wise to suggest that any foreign policy, and certainly not that of the EU, should be based only on this paradigm. Given the recurrent threats to security, which seem to be part of the human condition expressed by some as the “inevitability of war”--the defense of territorial integrity; action against threats of aggression; and resistance to crimes against humanity such as genocide--the ability to conduct a security policy based much more on the old paradigm of interacting interests will continue to be required. That the EU needs to develop such a capability will be taken here as a given. Such a crisis-management capability will be essential to the Union, but will be distinguished here from the more long-term elements of foreign policy, which can be thought of as being designed to reduce the need for crisis management in the context of a security policy to a minimum. The crisis-management area of policy will not be treated further here. The thesis of this Essay is that the same set of political concepts can serve as a guide to the future internal development of the EU and as the basis of such a long-term foreign policy. Furthermore, it suggests that neither should be seen in terms of the balancing of interests but rather, as the expression of a small list of fundamental values. The list is as follows: (1) the rule of law as the basis for relations between members of society; (2) the interaction between the democratic process and entrenched human rights in political decision-making; (3) the operation of competition within a market economy as the source of increasing prosperity; (4) the anchoring of the principle of solidarity among all members of society alongside that of the liberty of the individual; (5) the adoption of the principle of sustainability of all economic development; and (6) the preservation of separate identities and the maintenance of cultural diversity within society. These values can be seen as the answer to the question posed both, by citizens of the Union and by our fellow citizens of the world: “What does the EU stand for?” In exploring these values we should, however, remember that in the real world there will be occasions on which Realpolitik will intrude and the interest-based paradigm will prevail

Estimating the Incidence of Typhoid Fever and Other Febrile Illnesses in Developing Countries

To measure the incidence of typhoid fever and other febrile illnesses in Bilbeis District, Egypt, we conducted a household survey to determine patterns of health seeking among persons with fever. Then we established surveillance for 4 months among a representative sample of health providers who saw febrile patients. Health providers collected epidemiologic information and blood (for culture and serologic testing) from eligible patients. After adjusting for the provider sampling scheme, test sensitivity, and seasonality, we estimated that the incidence of typhoid fever was 13/100,000 persons per year and the incidence of brucellosis was 18/100,000 persons per year in the district. This surveillance tool could have wide applications for surveillance for febrile illness in developing countries

Smoking in cars in England: a study of school students in an English city

Background Exposure to secondhand smoke is associated with an increased risk of adverse health effects among children. Although smoking in the home is an established major source of exposure, less is known about rules on smoking in cars. Methods In a survey including a sample of secondary school students in Nottingham (UK) in 2012, participants were asked whether smoking was allowed in the family car, and how often the respondent travelled in a car in which smoking was allowed. Rules on smoking in cars were investigated in relation to socio-demographic variables and whether children had ever smoked themselves using logistic regression. Results Of 4,190 students aged 11–16 who provided data, approximately 12% reported that smoking was allowed in their family car and 35% that they travelled in a car where smoking was allowed at least sometimes. Absence of smoke free rules in the family car was more likely to be reported by children from more disadvantaged families, if parents and friends were smokers and if smoking was allowed in the main home. These factors, and having a sibling who smokes, were also independently associated with an increased risk of travelling in a car in which smoking was allowed at least sometimes. Respondents who were not protected from secondhand smoke in the car were also more likely to have ever smoked (adjusted odds ratio 1.59, 95% CI 1.18-2.14). Conclusions Absence of smoke free rules in a family car and travelling in a car where smoking was allowed was relatively common among secondary school students, was strongly related to social disadvantage and a higher risk of smoking experimentation. Measures to prevent such exposure are therefore indicated