Biomedical subjects

Fiona Simpson

Publications and source records attributed to Fiona Simpson.

18 recordsLinked to original sources

PubMed2006

A rapidly evolving secretome builds and patterns a sea shell.

BACKGROUND: Instructions to fabricate mineralized structures with distinct nanoscale architectures, such as seashells and coral and vertebrate skeletons, are encoded in the genomes of a wide variety of animals. In mollusks, the mantle is responsible for the extracellular production of the shell, directing the ordered biomineralization of CaCO3 and the deposition of architectural and color patterns. The evolutionary origins of the ability to synthesize calcified structures across various metazoan taxa remain obscure, with only a small number of protein families identified from molluskan shells. The recent sequencing of a wide range of metazoan genomes coupled with the analysis of gene expression in non-model animals has allowed us to investigate the evolution and process of biomineralization in gastropod mollusks. RESULTS: Here we show that over 25% of the genes expressed in the mantle of the vetigastropod Haliotis asinina encode secreted proteins, indicating that hundreds of proteins are likely to be contributing to shell fabrication and patterning. Almost 85% of the secretome encodes novel proteins; remarkably, only 19% of these have identifiable homologues in the full genome of the patellogastropod Lottia scutum. The spatial expression profiles of mantle genes that belong to the secretome is restricted to discrete mantle zones, with each zone responsible for the fabrication of one of the structural layers of the shell. Patterned expression of a subset of genes along the length of the mantle is indicative of roles in shell ornamentation. For example, Has-sometsuke maps precisely to pigmentation patterns in the shell, providing the first case of a gene product to be involved in molluskan shell pigmentation. We also describe the expression of two novel genes involved in nacre (mother of pearl) deposition. CONCLUSION: The unexpected complexity and evolvability of this secretome and the modular design of the molluskan mantle enables diversification of shell strength and design, and as such must contribute to the variety of adaptive architectures and colors found in mollusk shells. The composition of this novel mantle-specific secretome suggests that there are significant molecular differences in the ways in which gastropods synthesize their shells.

Animal Structures↗

PubMed2006

Visualisation of macropinosome maturation by the recruitment of sorting nexins.

We report that phosphoinositol-binding sorting nexin 5 (SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

Antigens, Surface↗

PubMed2006

Identification and analysis of novel genes expressed in the mouse embryonic facial primordia.

Craniofacial anomalies are a common feature of human congenital dysmorphology syndromes, suggesting that genes expressed in the developing face are likely to play a wider role in embryonic development. To facilitate the identification of genes involved in embryogenesis, we previously constructed an enriched cDNA library by subtracting adult mouse liver cDNA from that of embryonic day (E)10.5 mouse pharyngeal arch cDNA. From this library, 273 unique clones were sequenced and known proteins binned into functional categories in order to assess enrichment of the library (1). We have now selected 31 novel and poorly characterised genes from this library and present bioinformatic analysis to predict proteins encoded by these genes, and to detect evolutionary conservation. Of these genes 61% (19/31) showed restricted expression in the developing embryo, and a subset of these was chosen for further in silico characterisation as well as experimental determination of subcellular localisation based on transient transfection of predicted full-length coding sequences into mammalian cell lines. Where a human orthologue of these genes was detected, chromosomal localisation was determined relative to known loci for human congenital disease.

Animals↗

PubMed2006

Murine embryonic expression of the gene for the UV-responsive protein p15(PAF).

In a screen for genes expressed in the embryonic mouse facial primordia, we identified the gene sequence annotated as KIAA0101, which has previously been shown to encode a novel proliferating cell nuclear antigen (PCNA)-interacting protein named p15(PAF). We have since demonstrated that this protein also interacts in a complex with the tumour suppressor product p33ING1b, and that overexpression results in a decrease in UV-induced cell death. Although available data suggest widespread or ubiquitous expression in the adult, here we report highly restricted expression of the p15(PAF) gene in a spatio-temporal manner during mouse embryogenesis. Major sites of expression include the facial prominences, limbs, somites, brain, spinal cord and hair follicles. Based on the nature of its interacting partners, p15(PAF) is proposed to play a role in tumorigenesis. Our data also suggest a role in embryonic development, consistent with findings that a wide range of tumours result from aberrant activity of key developmental pathways.

Animals↗

PubMed2006

Evolutionary conservation and murine embryonic expression of the gene encoding the SERTA domain-containing protein CDCA4 (HEPP).

Cdca4 (Hepp) was originally identified as a gene expressed specifically in hematopoietic progenitor cells as opposed to hematopoietic stem cells. More recently, it has been shown to stimulate p53 activity and also lead to p53-independent growth inhibition when overexpressed. We independently isolated the murine Cdca4 gene in a genomic expression-based screen for genes involved in mammalian craniofacial development, and show that Cdca4 is expressed in a spatio-temporally restricted pattern during mouse embryogenesis. In addition to expression in the facial primordia including the pharyngeal arches, Cdca4 is expressed in the developing limb buds, brain, spinal cord, dorsal root ganglia, teeth, eye and hair follicles. Along with a small number of proteins from a range of species, the predicted CDCA4 protein contains a novel SERTA motif in addition to cyclin A-binding and PHD bromodomain-binding regions of homology. While the function of the SERTA domain is unknown, proteins containing this domain have previously been linked to cell cycle progression and chromatin remodelling. Using in silico database mining we have extended the number of evolutionarily conserved orthologues of known SERTA domain proteins and identified an uncharacterised member of the SERTA domain family, SERTAD4, with orthologues to date in human, mouse, rat, dog, cow, Tetraodon and chicken. Immunolocalisation of transiently and stably transfected epitope-tagged CDCA4 protein in mammalian cells suggests that it resides predominantly in the nucleus throughout all stages of the cell cycle.

Amino Acid Sequence↗

PubMed2006

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1.

Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.

Animals↗

PubMed2006

Early enteral nutrition in the critically ill: do we need more evidence or better evidence?

PURPOSE OF REVIEW: Nutritional support of the critically ill is accepted as a standard of care. Recommendations for early enteral nutrition are based on reasonable evidence but only 50% of eligible patients receive enteral nutrition within 48 h of admission to the intensive care unit. The purpose of this review is to determine how recent developments advance the current state of knowledge. RECENT FINDINGS: The benefits of early enteral nutrition are supported by two recent evidence-based guidelines initiatives. Early (< 48 h after intensive care unit admission) enteral nutrition may decrease hospital discharge mortality by 8-12% (grade B+ evidence-based recommendation). Five recent level II clinical trials, two of which contain major methodological flaws, are consistent with this recommendation. SUMMARY: Higher levels of evidence, demonstrating benefit to a patient-oriented outcome, are more likely to change practice. The addition of more level II trials to a meta-analysis composed of level II trials may not convince clinicians to change practice. A level I trial (equivalent to a phase III Food and Drug Administration licensing trial) is required to convince more clinicians to provide early enteral nutrition. A level I trial would also provide an important opportunity to validate changes in disease-oriented outcomes (measures of nutritional status) against improvements in patient-oriented outcomes.

Animals↗

PubMed2005

The PCNA-associated factor KIAA0101/p15(PAF) binds the potential tumor suppressor product p33ING1b.

The KIAA0101/p15(PAF)/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21(WAF). PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15(PAF) fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15(PAF) protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15(PAF) and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15(PAF) in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15(PAF) and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15(PAF) forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.

Animals↗

PubMed2005

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking.

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.

3T3-L1 Cells↗

PubMed2005

Randomization and allocation concealment: a practical guide for researchers.

Although the randomized controlled trial is the most important tool currently available to objectively assess the impact of new treatments, the act of randomization itself is often poorly conducted and incompletely reported. The primary purpose of randomizing patients into treatment arms is to prevent researchers, clinicians, and patients from predicting, and thus influencing, which patients will receive which treatments. This important source of bias can be eliminated by concealing the upcoming allocation sequence from researchers and participants. Although there are many approaches to randomization that are known to effectively conceal the randomization sequence, the use of sequentially numbered, opaque sealed envelopes (SNOSE) is both cheap and effective. The purpose of this tutorial is to describe a step-by-step process for the preparation of SNOSE. We will outline how to prepare SNOSE to preserve allocation concealment in a trial that (a) uses unrestricted (simple) randomization, (b) stratifies randomization on one factor, (c) uses permuted blocks and, and (d) is conducted at more than 1 study site.

Humans↗

PubMed2005

Characterization of Rab23, a negative regulator of sonic hedgehog signaling.

The hedgehog signaling pathway is indispensable in embryogenesis, being responsible for the development of a wide array of vertebrate organs. Given its importance in embryogenesis, the precise regulation of hedgehog signaling is crucial. Aberrant activation of this pathway in postnatal life has been associated with a number of tumor types, reinforcing the role of developmental signaling pathways in tumorigenesis. The small GTPase Rab23 acts as a negative regulator of the hedgehog signaling pathway, most notably in the vertebrate neural system. By analogy with studies of other Rab proteins, analysis of the localization of wild-type and constitutively active and inactive forms of Rab23 provides the potential to shed light on the role of Rab23 at the cellular level. We previously produced expression constructs encoding these proteins for analysis in mammalian cell cultures at both the light and the electron microscopy level. This revealed that both wild-type and active Rab23 localizes to the plasma membrane and to endocytic vesicles (T. M. Evans et al. [2003] Traffic4, 869-884). We describe the methods used to design and make the Rab23 expression constructs, and to assess their localization relative to key hedgehog pathways and endocytic markers in both transiently and stably transfected cell cultures.

Amino Acid Sequence↗

PubMed2005

A novel hook-related protein family and the characterization of hook-related protein 1.

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains.

Amino Acid Sequence↗

PubMed2005

A novel mammalian retromer component, Vps26B.

The mammalian retromer protein complex, which consists of three proteins--Vps26, Vps29, and Vps35--in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

Amino Acid Sequence↗

PubMed2005

A review of the true methodological quality of nutritional support trials conducted in the critically ill: time for improvement.

In this review we sought to appraise the true methodological quality of nutritional support studies conducted in critically ill patients and to compare these findings to the methodological quality of sepsis trials. An extensive literature search revealed 111 randomized controlled trials conducted in critically ill patients evaluating the impact of nutritional support interventions on clinically meaningful outcomes. Compared with sepsis trials, nutritional support studies were significantly less likely to use blinding (32 of 40 versus 35 of 111, P < 0.001) or present an intention-to-treat analysis (37 of 40 versus 64 of 111, P < 0.001). There was a trend toward the less frequent use of randomization methods that are known to maintain allocation concealment (12 of 40 versus 19 of 111, P = 0.10). Although nutritional support studies demonstrated a significant increase in the use of blinding after the publication of the CONSORT statement in 1996 (9 of 47 versus 26 of 64 post-CONSORT, P = 0.023), there were no improvements in other key areas. Previous publications have described the overall methodological quality of sepsis trials as "poor." Nutritional support studies were significantly worse than sepsis trials in all aspects of methodological quality, and there were few improvements noted over time. To detect important differences in clinically meaningful outcomes in critical care, the methodological quality of future studies must be improved.

Clinical Trials as Topic↗

PubMed2004

Parenteral vs. enteral nutrition in the critically ill patient: a meta-analysis of trials using the intention to treat principle.

OBJECTIVE: Controversy surrounds the use of parenteral nutrition in critical illness. Previous overviews used composite scales to identify high-quality trials, which may mask important differences in true methodological quality. Using a component-based approach this meta-analysis investigated the effect of trial quality on overall conclusions reached when standard enteral nutrition is compared to standard parenteral nutrition in critically ill patients. METHODS: An extensive literature search was undertaken to identify all eligible trials. We retrieved 465 publications, and 11 qualified for inclusion. Nine trials presented complete follow-up, allowing the conduct of an intention to treat analysis. RESULTS: Aggregation revealed a mortality benefit in favour of parenteral nutrition, with no heterogeneity. A priori specified subgroup analysis demonstrated the presence of a potentially important treatment-subgroup interaction between studies of parenteral vs. early enteral nutrition compared to parenteral vs. late enteral. Six trials with complete follow-up reported infectious complications. Infectious complications were increased with parenteral use. The I(2) measure of heterogeneity was 37.7%. CONCLUSIONS: Intention to treat trials demonstrated reduced mortality associated with parenteral nutrition use. A priori subgroup analysis attributed this reduction to trials comparing parenteral to delayed enteral nutrition. Despite an association with increased infectious complications, a grade B+ evidence-based recommendation (level II trials, no heterogeneity) can be generated for parenteral nutrition use in patients in whom enteral nutrition cannot be initiated within 24 h of ICU admission or injury.

Communicable Diseases↗

PubMed2004

Insulin and oleate promote translocation of inosine-5' monophosphate dehydrogenase to lipid bodies.

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of (32)P-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.

3T3-L1 Cells↗

PubMed2003

Efficient literature searching: a core skill for the practice of evidence-based medicine.

BACKGROUND: Efficient literature searching and the application of formal rules of evidence in evaluating the clinical literature are the two key skills defining the practice of evidence-based medicine. Although clinicians embrace the concepts of evidence-based medicine, most identify limited personal time as the major barrier towards its implementation into daily practice. Busy clinicians who practice evidence-based medicine identify systematic reviews and evidence-based clinical practice guidelines as very useful resources. METHODS: This review presents a simple, easy-to-follow, three-step searching strategy that emphasises the use of powerful new PubMed features that allow clinicians to retrieve high-quality systematic reviews, clinical practice guidelines and primary studies with a single mouse click. The overall effectiveness of the process is further improved by highlighting the major features of successful and unsuccessful literature searches. CONCLUSIONS: At the end of this tutorial the reader should be able to conduct efficient and effective literature searches that support clinical decision making in under 10 minutes.

Decision Making↗

PubMed2002

Electrical anisotropy below slow- and fast-moving plates: paleoflow in the upper mantle?

Upper mantle electrical conductivities can be explained by hydrogen diffusivity in hydrous olivine. Diffusivity enhances the conductivity of olivine anisotropically, making the a axis the most conductive of the three axes. Therefore, the hypothesis that plate motion induces lattice-preferred orientation of olivine can be tested with the use of long-period electromagnetic array measurements. Here, we compared electrical anisotropies below the slow-moving Fennoscandian and fast-moving Australian plates. The degree of olivine alignment is greater in the mantle below the Fennoscandian plate than below the Australian plate. This finding may indicate that convection rather than plate motion is the dominant deformation mechanism.

Journal Article↗