Functional and biophysical analysis of the C-terminus of the CGRP-receptor; a family B GPCR

G-protein coupled receptors (GPCRs) typically have a functionally important C-terminus which, in the largest subfamily (family A), includes a membrane-parallel eighth helix. Mutations of this region are associated with several diseases. There are few C-terminal studies on the family B GPCRs and no data supporting the existence of a similar eighth helix in this second major subfamily, which has little or no sequence homology to family A GPCRs. Here we show that the C-terminus of a family B GPCR (CLR) has a disparate region from N400 to C436 required for CGRP-mediated internalization, and a proximal region of twelve residues (from G388 to W399), in a similar position to the family A eighth helix, required for receptor localization at the cell surface. A combination of circular and linear dichroism, fluorescence and modified waterLOGSY NMR spectroscopy (SALMON) demonstrated that a peptide mimetic of this domain readily forms a membrane-parallel helix anchored to the liposome by an interfacial tryptophan residue. The study reveals two key functions held within the C-terminus of a family B GPCR and presents support for an eighth helical region with striking topological similarity to the nonhomologous family A receptor. This helix structure appears to be found in most other family B GPCRs.

Synthesis and cytotoxity of oxysterols:studies on the A,B ring polyoxygenated sterols

Oxysterols (OS), the polyoxygenated sterols, represent a class of potent regulatory molecules for important biological actions. Cytotoxicity of OS is one of the most important aspects in studies of OS bioactivities. However, studies, the structure-activity relationship (SAR) study in particular, have been hampered by the limited availability of structurally diverse OS in numbers and amounts. The aim of this project was to develop robust synthetic methods for the preparation of polyhydroxyl sterols, thereof, evaluate their cytotoxicity and establish structure-activity relationship. First, we found hydrophobicity of the side chain is essential for 7-HC's cytotoxicity, and a limited number of hydroxyl groups and a desired configuration on the A, B ring are required for a potent cytotoxicity of an OS, after syntheses and tests of a number of 7-HC's analogues against cancer cell lines. Then polyoxygenation of cholesterol A, B rings was explored. A preparative method for the synthesis of four diastereomerically pure cholest-4-en-3,6-diols was developed. Epoxidation on these cholest-4-en-3,6-diols showed that an allyl group exerts an auxiliary role in producing products with desired configuration in syntheses of the eight diastereomerically pure 45-epoxycholestane-3,6-diols. Reduction of the eight 45-epoxycholestane-3,6-diols produced all eight isomers of the cytotoxic 5α-acholestane 3β,5,6β-triol (CT) for the first time. Epoxide ring opening with protic or Lewis acids on the eight 45-epoxycholestane-3,6-diols are carefully studied. The results demonstrated a combination of an acid and a solvent affected the outcomes of a reaction dramatically. Acyl group participation and migration play an important role with numbers of substrates under certain conditions. All the eight 4,5-trans cholestane- 3,4,5,6-tetrols were synthesised through manipulation of acyl participation. Furthermore these reaction conditions were tested when a number of cholestane-3,4, 5,6,7-pentols and other C3-C7 oxygenated sterols were synthesised for the first time. Introduction of an oxygenated functional group through cholest-2-ene derivatives was studied. The elimination of 3-(4-toluenesulfonate) esters showed the interaction between the existing hydroxyls or acyls with the reaction centre often resulted in different products. The allyl oxidation, epoxidation and Epoxide ring opening reactions are investigated with these cholest-2-enes.

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.

The Gilles de la Tourette Syndrome-Quality of Life Scale for children and adolescents (C&A-GTS-QOL):development and validation of the Italian version

BACKGROUND: Gilles de la Tourette syndrome (GTS) is a chronic childhood-onset neuropsychiatric disorder with a significant impact on patients' health-related quality of life (HR-QOL). Cavanna et al. (Neurology 2008; 71: 1410-1416) developed and validated the first disease-specific HR-QOL assessment tool for adults with GTS (Gilles de la Tourette Syndrome-Quality of Life Scale, GTS-QOL). This paper presents the translation, adaptation and validation of the GTS-QOL for young Italian patients with GTS. METHODS: A three-stage process involving 75 patients with GTS recruited through three Departments of Child and Adolescent Neuropsychiatry in Italy led to the development of a 27-item instrument (Gilles de la Tourette Syndrome-Quality of Life Scale in children and adolescents, C&A-GTS-QOL) for the assessment of HR-QOL through a clinician-rated interview for 6-12 year-olds and a self-report questionnaire for 13-18 year-olds. RESULTS: The C&A-GTS-QOL demonstrated satisfactory scaling assumptions and acceptability. Internal consistency reliability was high (Cronbach's alpha > 0.7) and validity was supported by interscale correlations (range 0.4-0.7), principal-component factor analysis and correlations with other rating scales and clinical variables. CONCLUSIONS: The present version of the C&A-GTS-QOL is the first disease-specific HR-QOL tool for Italian young patients with GTS, satisfying criteria for acceptability, reliability and validity. © 2013 - IOS Press and the authors. All rights reserved.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.

Assessing the effectiveness of five-year mechanical engineering technology programs of junior colleges in Taiwan, R.O.C.: An application of the CIPP evaluation model

The purpose of this study was to evaluate the mechanical engineering technology curriculum effectiveness at the junior college in Taiwan by using the CIPP evaluation model. The study concerned the areas of the curriculum, curriculum materials, individualized instruction, support services, teaching effectiveness, student achievement, and job performance. A descriptive survey method was used with questionnaires for data collection from faculty, students, graduates, and employers.^ All categories of respondents tended to agree that the curriculum provides appropriate occupational knowledge and skills. Students, graduates, and faculty tended to be satisfied with the curriculum; faculty tended to be satisfied with student achievement; graduates tended to be satisfied with their job preparation; and employers were most satisfied with graduates' job performance.^ Conclusions were drawn in the context, input, process, and product of the CIPP model. In Context area: Students were dissatisfied with curriculum flexibility in students characteristics. Graduates were dissatisfied with curriculum design for student's adaptability in new economic and industrial conditions; practicum flexibility in student characteristics; and course overlap. Both students and graduates were dissatisfied with practicum credit hours. Both faculty and students were dissatisfied with the number of required courses.^ In Input area: Students, faculty, and graduates perceived audiovisuals and manipulative aids positively. Faculty and students perceive CAI implementation positively. Students perceived textbooks negatively.^ In Process area: Faculty, students, and graduates perceived all support service negatively. Faculty tended to perceive the ratios of graduates who enter advanced study and related occupation, and who passed the professional skills certification, negatively. Students tended to perceive teaching effectiveness in terms of instructional strategies, the quality of instruction, overall suitability, and receivable, negatively. Graduates also tended to identify the instructional strategies as a negative perception. Faculty and students perceived curriculum objectives and practicum negatively. Both faculty and students felt that instructors should be more interested in making the courses a useful learning experience.^ In Product area: Employers were satisfied with graduates' academic preparation and job performance, adaptability, punctuality, and their ability to communicate, cooperate, and meet organization needs. Graduates were weak in terms of equipment familiarity and supervisory ability.^ In sum, the curriculum of the five-year mechanical engineering technology programs of junior college in Taiwan has served adequately up to this time in preparing a work force to enter industry. It is now time to look toward the future and adapt the curriculum and instruction for the future needs of this high-tech society. ^

An aquatic risk assessment of ethion and bromacil inputs to the C-25 Canal and Indian River Lagoon

Pesticide monitoring in St. Lucie County by various local, state and federal agencies has indicated consistent residues of several pesticides, including ethion and bromacil. Although pesticides have long been known to pose a threat to non-target species and much background monitoring has been done, no pesticide aquatic risk assessment has been done in this geographical area. Several recognized United States Environmental Protection Agency (USEPA) methods of quantifying risk are employed here to include hazard quotients (HQ) and probabilistic modeling with sensitivity analysis. These methods are employed to characterize potential impacts to aquatic biota of the C-25 Canal and the Indian River Lagoon (in St. Lucie County, Florida) based on current agricultural pesticide use and drainage patterns. The model used in the analysis incorporates available physical-chemical property data, local hydrology, ecosystem information, and pesticide use practices. HQ's, probabilistic distributions, and field sample analyses resulted in high levels of concern (LOCs), which usually indicates a need for regulatory action, including restrictions on use, or cancellation. ^

Assessing the effectiveness of two-year banking and insurance technology programs of junior colleges in Taiwan, R.O.C.: An application of the Context Input Process Product (CIPP) evaluation model

The purpose of this descriptive study was to evaluate the banking and insurance technology curriculum at ten junior colleges in Taiwan. The study focused on curriculum, curriculum materials, instruction, support services, student achievement and job performance. Data was collected from a diverse sample of faculty, students, alumni, and employers. ^ Questionnaires on the evaluation of curriculum at technical junior colleges were developed for use in this specific case. Data were collected from the sample described above and analyzed utilizing ANOVA, T-Tests and crosstabulations. Findings are presented which indicate that there is room for improvement in terms of meeting individual students' needs. ^ Using Stufflebeam's CIPP model for curriculum evaluation it was determined that the curriculum was adequate in terms of the knowledge and skills imparted to students. However, students were dissatisfied with the rigidity of the curriculum and the lack of opportunity to satisfy the individual needs of students. Employers were satisfied with both the academic preparation of students and their on the job performance. ^ In sum, the curriculum of the two-year banking and insurance technology programs of junior college in Taiwan was shown to have served adequately preparing a work force to enter businesses. It is now time to look toward the future and adapt the curriculum and instruction for the future needs of the ever evolving high-tech society. ^

Gnosticism, Transformation, and the Role of the Feminine in the Gnostic Mass of the Ecclesia Gnostica Catholica (E.G.C.)

The Gnostic Mass of the Ecclesia Gnostica Catholica (E.G.C.) suggests a heterosexual gender binary in which the female Priestess seated on the altar as the sexual and fertile image of the divine feminine is directed by the male Priest’s activity, desire and speech. The apparent contradiction between the empowered individual and the polarized gender role was examined by comparing the ritual symbolism of the feminine with the interpretations of four Priestesses and three Priests (three pairs plus one). Findings suggest that the Priestess’ role in the Gnostic Mass is associated with channeling, receptivity, womb, cup, and fertility, while the Priest’s role is associated with enthusiasm, activity, phallus, lance, and virility. Despite this strong gender duality, the Priestesses asserted that their role was personally and spiritually empowering, and they maintained heterosexual and polarized gendered roles are necessary in a transformative ritual which ultimately reveals the godlike unified individual.

Comparison of Ba/Ca and d18OWATER as freshwater proxies: a multi-species core-top study on planktonic foraminifera from the vicinity of the Orinoco River mouth

Past river run-off is an important measure for the continental hydrological cycle and the as-sessment of freshwater input into the ocean. However, paleosalinity reconstructions applying different proxies in parallel often show offsets between the respective methods. Here, we compare the established foraminiferal Ba/Ca and d18OWATER salinity proxies for their capability to record the highly seasonal Orinoco freshwater plume in the eastern Caribbean. For this purpose we obtained a data set comprising Ba/Ca and d18OWATER determined on multiple spe-cies of planktonic foraminifera from core tops distributed around the Orinoco river mouth. Our findings indicate that interpretations based on either proxy could lead to different conclu-sions. In particular, Ba/Ca and d18OWATER diverge in their spatial distribution due to different governing factors. Apparently, the Orinoco freshwater plume is best tracked by Ba/Ca ratios of G. ruber (pink and sensu lato morphotypes), while d18OWATER based on the same species is more related to the local precipitation-evaporation balance overprinting the riverine freshwater contribution. Other shallow dwelling species (G. sacculifer, O. universa) show a muted response to the freshwater discharge, most likely due to their ecological and habitat prefer-ences. Extremely high Ba/Ca ratios recorded by G. ruber are attributed to Ba2+-desorption from suspended matter derived from the Orinoco. Samples taken most proximal to the freshwater source do not show pronounced Ba/Ca or d18OWATER anomalies. Here, the suspension loaded freshwater lid developing during maximum discharge suppresses foraminiferal populations. Both proxies are therefore biased towards dry season conditions at these sites, when surface salinity is only minimally reduced.

Esther Peterson, Executive Vice Chair of Women's Bureau in Washington, D.C. since 1920, speaks at U.S. department of Labor Conference in NYC

General note: Title and date provided by Bettye Lane.

U.S. Department of Labor conference in NYC. Dorothy Height (left), original member of Women's bureau in Washington, D.C. and Esther Peterson, Executive Vice Chair of Women's Bureau since 1920

General note: Title and date provided by Bettye Lane.

Size-related isotopic trends in Maastrichtian planktic foraminifera of DSDP Core 44-390A (Appendix B)

Stable isotope analyses were performed on ontogenetic dissections of four taxa of low latitude Late Cretaceous planktonic foraminifera from DSDP Hole 390A. The species studied include Planoglobulina acervulinoides, Planoglobulina multicamerata, Pseudoguembelina palpebra, and Racemiguembelina fructicosa. Delta18O and delta13C data indicate a deeper surface water paleohabitat for P. multicamerata than the other three taxa, and ontogenetic increases in delta18O values suggest all these taxa underwent vertical migrations from shallow to deeper surface waters. Changes in delta13C values through ontogeny include sharp increases in delta13C composition in the juvenile size intervals, a decrease in the rate of delta13C change through intermediate size intervals, and reversals to a negative trend in delta13C values in terminal size intervals. The intermediate and terminal growth changes in delta13C signals are similar to ontogenetic trends observed in some extant and Paleogene planktonic foraminifera and may result from decreasing metabolic rates through ontogeny or endosymbiont digestion prior to gametogenesis. The ontogenetic delta13C increases of 1.04?, 0.76?, 0.83?, and 0.77? in R. fructicosa, P. palpebra, P. acervulinoides, and P. multicamerata, respectively, may indicate the presence of photosymbionts. However, our review and critique of the current literature discussing photosymbiont effects on stable isotope values in living and fossil planktonic foraminifera suggests that conclusions regarding the presence of photosymbionts in fossil taxa may be more equivocal than previously thought.

(Table 3, page 280) Chemical composition of micronodules from a series of stations and sediment depths from Areas C, F, G and K in the Pacific Ocean from R/V Sonne Cruise SO06

Compositional data for coexisting manganese nodules, micronodules, sediments and pore waters from five areas in the equatorial and S.W. Pacific have been obtained. This represents the largest study of its type ever undertaken to establish the distribution of elements between the various phases within the sediment column. The composition of manganese nodules, micronodules and sediments (on a carbonate-free basis) shows marked differences between the equatorial high productivity zone and the low productivity region of the S.W. Pacific. In the case of the nodules, th is reflects an increased supply of transition elements (notably Ni, Cu and Zn) to the nodules as a result of the in situ dissolution of siliceous tests within the sediment column in the equatorial Pacific high productivity zone. Micronodules display similar, but somewhat different, compositions to those of the associated nodules in each area. Micronodule composition is therefore influenced by the same basic factors that control nodule composition, but is modified by dissolution of the micronodules in situ within the sediment column. Locally, as in the area immediately south of the Marquesas Fracture Zone, the micronodule population is contaminated by small, angular volcanic rock fragments; this leads to apparently anomalous micronodule compositions. Micronodules appear to be a transient feature in the sediment column, especially in the equatorial Pacific. Dissolution of micronodules in the sediment column therefore represents an important source of elements for the growth of manganese nodules in the equatorial Pacific. Sediment composition is markedly influenced by the carbonate content. On a carbonate-free basis, the sediments from the equatorial high productivity zone are quite distinct in composition from those in the S.W. Pacific. This reflects differences in the lithology of the sediments. In the Aitutaki Passage, the local influence of volcanoclastic material in sediment composition has been established. The major cations and anions in pore waters measured here show no major differences between equatorial and S.W. Pacific sediments. Silica is, however, higher in equatorial Pacific pore waters reflecting the dissolution of siliceous tests in these sediments.