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Biomedical subjects

N R Brown

Publications and source records attributed to N R Brown.

At least 19 recordsLinked to original sources
PubMed2001

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.

Animals↗
PubMed2001

Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2.

The CDK-interacting protein phosphatase KAP dephosphorylates phosphoThr-160 (pThr-160) of the CDK2 activation segment, the site of regulatory phosphorylation that is essential for kinase activity. Here we describe the crystal structure of KAP in association with pThr-160-CDK2, representing an example of a protein phosphatase in complex with its intact protein substrate. The major protein interface between the two molecules is formed by the C-terminal lobe of CDK2 and the C-terminal helix of KAP, regions remote from the kinase-activation segment and the KAP catalytic site. The kinase-activation segment interacts with the catalytic site of KAP almost entirely via the phosphate group of pThr-160. This interaction requires that the activation segment is unfolded and drawn away from the kinase molecule, inducing a conformation of CDK2 similar to the activated state observed in the CDK2/cyclin A complex.

Amino Acid Sequence↗
PubMed2001

Seeds aren't anchors.

Exposure to a few task-relevant numerical facts (seed facts) often improves subsequent numerical estimates. We performed two experiments to investigate the mechanism that produces these seeding effects. In Experiment 1, participants estimated national populations; in Experiment 2, they estimated between-city distances. In both, items were selected so that the actual value of the seed facts (SA) was, on average, below participants' initial estimates for those items (S1) and above the initial estimates for the transfer items (T1). Given this configuration, the anchoring position predicts that the postseeding transfer estimates should be greater than the preseeding transfer estimates (T2 > T1), whereas the feedback/induction position predicts the opposite (T2 < T1). In both experiments, the latter pattern of results emerged, supporting the conclusion that seeds aren't anchors.

Adult↗
PubMed2000

Reasoning about geography.

To understand the nature and etiology of biases in geographical judgments, the authors asked people to estimate latitudes (Experiments 1 and 2) and longitudes (Experiments 3 and 4) of cities throughout the Old and New Worlds. They also examined how people's biased geographical judgments change after they receive accurate information ("seeds") about actual locations. Location profiles constructed from the pre- and postseeding location estimates conveyed detailed information about the representations underlying geography knowledge, including the subjective positioning and subregionalization of regions within continents; differential seeding effects revealed between-region dependencies. The findings implicate an important role for conceptual knowledge and plausible-reasoning processes in tasks that use subjective geographical information.

Adult↗
PubMed2000

Updating geographical knowledge: principles of coherence and inertia.

In 2 experiments, the authors investigated how representations of global geography are updated when people learn new location information about individual cities. Participants estimated the latitude of cities in North America (Experiment 1) and in the Old and New Worlds (Experiment 2). After making their first estimates, participants were given information about the latitudes of 2 cities and asked to make a second set of estimates. Both the first and second estimates revealed evidence for psychologically distinct geographical subregions that were coordinated, in an ordinal sense, across the Atlantic Ocean. Further, the second estimates were affected by the nature of the physical adjacency between regions (e.g., the southern U.S. and Mexico) and by accurate location information about distant, but coordinated, subregions (e.g., the southern U.S. and Mediterranean Europe). The data provide support for a framework for making geographical estimates in which people strike a balance between 2 principles: the need to keep their knowledge base coherent, and the inertial tendency to resist changing the knowledge base unless it is necessary to maintain coherence.

Cognition↗
PubMed2000

Conservation of heterochromatin protein 1 function.

Heterochromatin represents a cytologically visible state of heritable gene repression. In the yeast, Schizosaccharomyces pombe, the swi6 gene encodes a heterochromatin protein 1 (HP1)-like chromodomain protein that localizes to heterochromatin domains, including the centromeres, telomeres, and the donor mating-type loci, and is involved in silencing at these loci. We identify here the functional domains of swi6p and demonstrate that the chromodomain from a mammalian HP1-like protein, M31, can functionally replace that of swi6p, showing that chromodomain function is conserved from yeasts to humans. Site-directed mutagenesis, based on a modeled three-dimensional structure of the swi6p chromodomain, shows that the hydrophobic amino acids which lie in the core of the structure are critical for biological function. Gel filtration, gel overlay experiments, and mass spectroscopy show that HP1 proteins can self-associate, and we suggest that it is as oligomers that HP1 proteins are incorporated into heterochromatin complexes that silence gene activity.

Amino Acid Sequence↗
PubMed2000

Estimating the frequency of nonevents: the role of recollection failure in false recognition.

Participants studied lists of multiply presented converging associates (e.g., bed, dream, pillow, etc.) and were timed as they estimated how often they saw list items, related foils (e.g., blanket), and nonpresented critical items (SLEEP). Average number of repetitions (few [3] vs. many [6]) and repetition variability (fixed vs. variable) were manipulated between subjects. Participants responded more slowly to critical items (3.18 sec) than to list items (2.45 sec) or foils (2.22 sec). In addition, critical-item judgments of frequency (JOFs) were about as large as list-item JOFs, and false recognition (i.e., nonzero JOFs) of critical items was most likely in the few-fixed condition (96%) and least likely in the many-fixed condition (74%). These findings suggest that people can use recollection failure--the absence of an anticipated recollective experience, coupled with strong familiarity--to distinguish critical items from list items and that recollection failure is weighted most heavily when people expect familiar probes to access episodic information.

Adult↗
PubMed1999

Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity.

We have prepared phosphorylated cyclin-dependent protein kinase 2 (CDK2) for crystallization using the CDK-activating kinase 1 (CAK1) from Saccharomyces cerevisiae and have grown crystals using microseeding techniques. Phosphorylation of monomeric human CDK2 by CAK1 is more efficient than phosphorylation of the binary CDK2-cyclin A complex. Phosphorylated CDK2 exhibits histone H1 kinase activity corresponding to approximately 0.3% of that observed with the fully activated phosphorylated CDK2-cyclin A complex. Fluorescence measurements have shown that Thr160 phosphorylation increases the affinity of CDK2 for both histone substrate and ATP and decreases its affinity for ADP. By contrast, phosphorylation of CDK2 has a negligible effect on the affinity for cyclin A. The crystal structures of the ATP-bound forms of phosphorylated CDK2 and unphosphorylated CDK2 have been solved at 2.1-A resolution. The structures are similar, with the major difference occurring in the activation segment, which is disordered in phosphorylated CDK2. The greater mobility of the activation segment in phosphorylated CDK2 and the absence of spontaneous crystallization suggest that phosphorylated CDK2 may adopt several different mobile states. The majority of these states are likely to correspond to inactive conformations, but a small fraction of phosphorylated CDK2 may be in an active conformation and hence explain the basal activity observed.

Adenosine Triphosphate↗
PubMed1999

The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases.

Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.

Amino Acid Motifs↗
PubMed1998

Strategies for estimating behavioural frequency in survey interviews.

When people answer survey questions of the form "During the past month, how many times did you...?" their responses provide valuable data for researchers and policy makers. Yet the way respondents produce their answers to these "behavioural frequency questions" is not well understood. This article demonstrates that survey respondents can use an array of distinct estimation strategies, depending on what information is available in their memories. The kind of event information that people use is related to factors such as the regularity of occurrence, similarity of one episode to the next, and frequency. In a study conducted as a telephone survey, respondents' verbal reports and response-time patterns indicate that they usually answer behavioural frequency questions by either retrieving and counting episodes, retrieving or estimating rates of occurrence, or converting a general impression of frequency into a numerical quantity. The third strategy should be of particular concern to survey researchers because respondents provide a quantitative estimate without any relevant numerical knowledge. The set of strategies and the factors that influence their use are integrated into a statistical model that could help survey practitioners to improve data quality and memory researchers to broaden their perspective.

Behavior↗
PubMed1998

Forward telescoping: the question matters.

Forward telescoping, the reporting or dating of events as being more recent than they actually were, is often observed in surveys and produces inaccurate data. We believe that some forward telescoping occurs when the question format allows people to respond without extensive retrieval of temporal information concerning the target events. We collected two types of data. The first, the type usually collected by survey researchers, involved visits to medical doctors. As is common in survey research, the actual dates of the events were not verifiable. The second type involved students' participation in laboratory research studies. Here, the actual dates were verifiable. We demonstrate that modifying the questions asked produced differences in the amount of forward telescoping in participants' responses.

Data Collection↗
PubMed1997

The cyclin box fold: protein recognition in cell-cycle and transcription control.

Regulation of both the cell cycle and gene transcription is essential for orderly progression of cell growth and division. Recent results on the structures of two cyclins, cyclin A and cyclin H, and two transcription factor mediator proteins, TFIIB and the A pocket region of the retinoblastoma tumour suppressor protein (Rb), show that they share domains with a strikingly similar alpha-helical topology, despite remote sequence identity.

Amino Acid Sequence↗
PubMed1997

Context memory and the selection of frequency estimation strategies.

When people estimate event frequency, they sometimes retrieve and count event instances. This study demonstrates a direct relation between the use of these enumeration-based strategies and the contents of memory. In 3 experiments, participants studied target-context word pairs, estimated presentation frequency for target words, and recalled context words. Study time, target-context relatedness, and study-phase instructions were manipulated, producing large differences in memory for context words. When context memory was best, estimation time increased sharply with presentation frequency, and the steepness of this estimation time-presentation frequency function decreased with context memory. These results indicate that enumeration was common only when context memory was good, that encoding factors determine how frequency is represented, and that the contents of memory restrict strategy selection.

Humans↗
PubMed1995

The crystal structure of cyclin A.

BACKGROUND: Eukaryotic cell cycle progression is regulated by cyclin dependent protein kinases (CDKs) whose activity is regulated by association with cyclins and by reversible phosphorylation. Cyclins also determine the subcellular location and substrate specificity of CDKs. Cyclins exhibit diverse sequences but all share homology over a region of approximately 100 amino acids, termed the cyclin box. From the determination of the structure of cyclin A, together with results from biochemical and genetic analyses, we can identify which parts of the cyclin molecular may contribute to cyclin A structure and function. RESULTS: We have solved the crystal structure, at 2.0 A resolution, of an active recombinant fragment of bovine cyclin A, cyclin A-3, corresponding to residues 171-432 of human cyclin A. The cyclin box has an alpha-helical fold comprising five alpha helices. This fold is repeated in the C-terminal region, although this region shares negligible sequence similarity with the cyclin box. CONCLUSIONS: Analysis of residues that are conserved throughout the A, B, and E cyclins identifies two exposed clusters of residues, one of which has recently been shown to be involved in the association with human CDK2. The second cluster may identify another site of cyclin A-protein interaction. Comparison of the structure of the unbound cyclin with the structure of cyclin A complexed with CDK2 reveals that cyclin A does not undergo any significant conformational changes on complex formation. Threading analysis shows that the cyclin-box fold is consistent with the sequences of the transcription factor TFIIB and other functionally related proteins. The structural results indicate a role for the cyclin-box fold both as a template for the cyclin family and as a generalised adaptor molecule in the regulation of transcription.

Algorithms↗
PubMed1993

Studies of the active site of m-calpain and the interaction with calpastatin.

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.

Amino Acid Sequence↗
PubMed1993

Metrics and mappings: a framework for understanding real-world quantitative estimation.

Estimation is influenced by a variety of processes: application of heuristics, domain-specific reasoning, and intuitive statistical induction, among them. In this article, we propose the metrics and mapping framework to account for how these processes are integrated to generate estimates. This framework identifies 2 types of information as critical: knowledge of distributional properties (metric knowledge) and knowledge of relative status of individual entities within the distribution (mapping knowledge). Heuristics and domain-specific knowledge are both viewed as cues that contribute to mapping knowledge; intuitive statistical induction is viewed as providing cues to metric properties. Results of 4 experiments illustrate the framework's usefulness for integrating these types of information and for predicting when people emphasize heuristics and when they emphasize domain-specific knowledge.

Adolescent↗
PubMed1992

The role of availability in the estimation of national populations.

In two parallel experiments, conducted 17 months apart, we examined the relations among population estimates, availability, and media coverage of the 100 countries with populations of 4 million or more. The results were consistent with the main hypothesis, that availability influences population estimates. Specifically, we found that (1) rated knowledge of each country (our measure of availability) was strongly correlated with estimated population for that country; (2) rated knowledge was more strongly correlated with estimated population than were other plausible predictors, such as land area; (3) a strong partial correlation between rated knowledge and estimated population remained even after true population and true land area were partialed out; and (4) longitudinal changes in rated knowledge predicted longitudinal changes in estimated population. Also as hypothesized, media coverage was positively related to availability and to population estimates. Further, we found that population estimates, rated knowledge, and amount of media coverage that each country received were very stable over the year-and-a-half period. These results led us to consider when availability is most likely to be relied upon heavily in quantitative estimation and how it may be integrated with other knowledge to derive quantitative estimates.

Adult↗