Alternative protein conformations: yeast iso-1-cytochrome c and heme crevice dynamics

The field of protein biochemistry has been dominated by the dogma that a protein sequence yields a 3-dimensional structure important for a singular function. More modern insights are beginning to demonstrate that proteins are not static structures. Rather, proteins undergo numerous conformational fluctuations yielding an ensemble of conformational populations. Conformational change can result in changed or altered protein function. Small or large energetic barriers existing between conformers regulate the ease with which a protein can sample alternative conformations. In the dissertation work presented here, alternative conformations of yeast iso-1-cytochrome c are investigated with particular emphasis on heme crevice loop dynamics. The heme crevice loop, or O-loop D, is a highly conserved, dynamic region. Conformational changes in O-loop D lead to altered electron transfer and peroxidase activity in cytochrome c (Cytc). As Cytc participates in both the electron transport chain and functions as a peroxidase during apoptosis, it is important to understand how this conformational change is regulated. Within O-loop D we investigate the effects of a trimethyllysine to alanine mutation and a destabilizing leucine to alanine mutation at residues 72 and 85, respectively, on heme crevice dynamics. Residue 72 plays an important role in regulating access to alternative heme crevice conformers. Of particular interest, residue 72 plays a role in regulating access to a peroxidase capable conformer of Cytc, a function of Cytc during the early stages of apoptosis. We have also solved the structure of the first monomeric Cytc structure in a peroxidase capable conformer, as well as, a dimeric Cytc structure with CYMAL-6 protruding into the interior of the heme cavity, in a manner potentially similar to the Cytc/cardiolipin interaction

Switch II Region in Gαi1: Specificity for Ric-8A

On the cell surface are G protein coupled receptors that bind to agonists, causing activation of intracellular G proteins, by catalyzing exchange of GTP for GDP at the G protein alpha subunit (Ga). G proteins are also activated by guanine nucleotide exchange factors (GEF) inside of the cell; these include Ric-8A and Ric-8B. GTP-bound Ga can stimulate the activity of intracellular enzymes. For example, Gas activates adenylyl cyclase, while Gai1 inhibits the activity of this enzyme. Biochemical studies have shown that Ric-8A is a GEF towards Gai1 whereas its isoform Ric-8B acts on Gas. Previous studies in our laboratory and others have shown that a region in Gαi1 called switch II binds to Ric-8A. In this study, we test the hypothesis that differences in amino acid sequence between Gai1 and Gas in switch II are responsible for the ability of these G proteins to discriminate between Ric-8A and Ric-8B. The switch regions of Gai1 and Gas differ in only three amino acids. We predict that, by mutating these amino acids in Gai1 to their corresponding residues in Gas, affinity for Ric-8A will be impaired. Single mutation primers (S206D, K209R and H213Q) were made, transformed and amplified through a polymerase chain reaction (PCR). Once mutant plasmid was expressed in E. coli cells, it is purified, and a tryptophan fluorescence assay is performed. This assay technique detects changes in the fluorescence of tryptophan 211, a side chain in switch II that is sensitive to the exchange of GTP for GDP. Our research sheds light on how mutants in Gai1 in the switch II region plays important role in specificity of binding for Ric-8A

New archaeointensity data from Italy and geomagnetic field intensity variation in the Italian Peninsula

We present new archaeointensity results from three Italian kilns situated at Ascoli Satriano, Vagnari and Fontanetto Po obtained with the Thellier modified by Coe double heating method. These data complement the directional results previously published. All sites are dated on the basis of archaeological information and/or thermoluminescence dating. The results are corrected for the anisotropy of the thermoremanent magnetization and the cooling rate effects. The new data are compared with previously published archaeointensity data from Italy and nearby countries within 900 km radius from Viterbo. An initial data set including archaeointesity data mainly coming from Italy, France, Switzerland, Czech Republic, Slovakia, Hungary, Greece and Bulgaria has been compiled. After the application of strict selection criteria, the most reliable data have been used for the calculation of a preliminary Italian intensity secular variation (SV) curve for the last 3000 yr. The new curve covers the 300 BC–400 AD and 1200–1900 AD periods. It is established by means of sliding windows of 200 yr shifted by 100 yr. The lack of reliable data for the 1000–200 BC and 400–1200 AD time intervals does not permit the calculation of a continuous curve. Clearly, more high-quality archaeointensity data from Italy and Europe are still needed to draw a robust intensity SV curve for the Italian Peninsula that could be used for archaeomagnetic dating in combination with the directional data

Full-vector archaeomagnetic dating of a medieval limekiln at Pinilla Del Valle site (Madrid, Spain)

Archaeomagnetic dating based on the full geomagnetic field vector was carried out on a limekiln excavated at Pinilla del Valle archaeological site (Madrid, Spain). The limekiln tradition in this area is largely documented by historical sources for recent centuries but the date of the studied kiln's last use was unknown. The combination of mean archaeomagnetic directional and absolute archaeointensity results shows that the kiln was last used between ad 1296 and 1413, in good agreement with two independent radiocarbon dates. This study provides the first archaeomagnetic evidence that the local limekiln tradition dates back to at least late medieval times. Furthermore, the inclusion of these data in the Iberian secular variation curve and geomagnetic field models will help to improve the archaeomagnetic dating technique.Spanish Ministry of Economy and Competitiveness, MINECO and the European Regional Development Fund (projects CGL2012-38481 and CGL2012-32149). AGacknowledges partial financial support given by PAPIIT IN-105214

The production and consumption activities relating to the celebrity artist

This paper considers the impact of the celebrity artist on the associated production and consumption activities. It also considers the role which entrepreneurial marketing plays in helping to create the celebrity artist aura. The artist Thomas Kinkade is used to illustrate how this occurs in practice. Here, authenticity and nostalgia dimensions are also influential factors. Underpinning these relationships are the roles played out by the media, including communication of celebrity artist identity, and the catalysing of its commodification within the celebrity artist brandscape. An enduring celebrity brand results due to the market creation activities of the celebrity artist. A conceptual model is developed which synthesises the factors behind the production and consumption of the celebrity artist which can stimulate further research. This paper provides innovative insight into the world of the celebrity artist by interrogating the market making and shaping devices behind successful production and consumption practices

Parts, Wholes, and Context in Reading: A Triple Dissociation

Research in object recognition has tried to distinguish holistic recognition from recognition by parts. One can also guess an object from its context. Words are objects, and how we recognize them is the core question of reading research. Do fast readers rely most on letter-by-letter decoding (i.e., recognition by parts), whole word shape, or sentence context? We manipulated the text to selectively knock out each source of information while sparing the others. Surprisingly, the effects of the knockouts on reading rate reveal a triple dissociation. Each reading process always contributes the same number of words per minute, regardless of whether the other processes are operating

Association of HIV infection with distribution and viral load of HPV types in Kenya: a survey with 820 female sex workers

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) and HIV are each responsible for a considerable burden of disease. Interactions between these infections pose substantial public health challenges, especially where HIV prevalence is high and HPV vaccine coverage low.</p> <p>Methods</p> <p>Between July 2005 and January 2006, a cross-sectional community-based survey in Mombasa, Kenya, enrolled female sex workers using snowball sampling. After interview and a gynaecological examination, blood and cervical cytology samples were taken. Quantitative real-time PCR detected HPV types and viral load measures. Prevalence of high-risk HPV was compared between HIV-infected and -uninfected women, and in women with abnormal cervical cytology, measured using conventional Pap smears.</p> <p>Results</p> <p>Median age of the 820 participants was 28 years (inter-quartile range [IQR] = 24-36 years). One third of women were HIV infected (283/803; 35.2%) and these women were y more likely to have abnormal cervical cytology than HIV-negative women (27%, 73/269, versus 8%, 42/503; <it>P </it>< 0.001). Of HIV-infected women, 73.3% had high-risk HPV (200/273) and 35.5% had HPV 16 and/or 18 (97/273). Corresponding figures for HIV-negative women were 45.5% (229/503) and 15.7% (79/503). After adjusting for age, number of children and condom use, high-risk HPV was 3.6 fold more common in HIV-infected women (95%CI = 2.6-5.1). Prevalence of all 15 of the high-risk HPV types measured was higher among HIV-infected women, between 1.4 and 5.5 fold. Median total HPV viral load was 881 copies/cell in HIV-infected women (IQR = 33-12,110 copies/cell) and 48 copies/cell in HIV-uninfected women (IQR = 6-756 copies/cell; <it>P </it>< 0.001). HPV 16 and/or HPV 18 were identified in 42.7% of LSIL (32/75) and 42.3% of HSIL (11/26) lesions (<it>P </it>= 0.98). High-risk HPV types other than 16 and 18 were common in LSIL (74.7%; 56/75) and HSIL (84.6%; 22/26); even higher among HIV-infected women.</p> <p>Conclusions</p> <p>HIV-infected sex workers had almost four-fold higher prevalence of high-risk HPV, raised viral load and more precancerous lesions. HPV 16 and HPV 18, preventable with current vaccines, were associated with cervical disease, though other high-risk types were commoner. HIV-infected sex workers likely contribute disproportionately to HPV transmission dynamics in the general population. Current efforts to prevent HIV and HPV are inadequate. New interventions are required and improved implementation of existing strategies.</p