Synchronization of cell populations in G1/S and G2/M phases of the cell cycle.

The method described in the following chapter utilizes a double thymidine block (an inhibitor of DNA synthesis) followed by treatment of cells with nocodazole (a mitotic inhibitor) to obtain large cell populations at distinct phases of the cell cycle. Treatment with double thymidine results in a G1/S-phase arrested cell population, and the use of flow cytometry allows progression of the cells through the cell cycle to be monitored. Flow cytometry enables the calculation of timings for collection of cells at distinct cell cycle phases from G1/S (following treatment with thymidine) through to G2/M (owing to the presence of nocodazole).

The mammalian cell cycle: an overview.

In recent years, we have witnessed major advances in our understanding of the mammalian cell cycle and how it is regulated. Normal mammalian cellular proliferation is tightly regulated at each phase of the cell cycle by the activation and deactivation of a series of proteins that constitute the cell cycle machinery. This review article describes the various phases of the mammalian cell cycle and focuses on the cell cycle regulatory molecules that act at each stage to ensure normal cellular progression.

Analysis of void volumes in proteins and application to stability of the p53 tumour suppressor protein.

We have developed a new method for the analysis of voids in proteins (defined as empty cavities not accessible to solvent). This method combines analysis of individual discrete voids with analysis of packing quality. While these are different aspects of the same effect, they have traditionally been analysed using different approaches.

Bayesian estimation of ancestral character states on phylogenies.

Biologists frequently attempt to infer the character states at ancestral nodes of a phylogeny from the distribution of traits observed in contemporary organisms. Because phylogenies are normally inferences from data, it is desirable to account for the uncertainty in estimates of the tree and its branch lengths when making inferences about ancestral states or other comparative parameters. Here we present a general Bayesian approach for testing comparative hypotheses across statistically justified samples of phylogenies, focusing on the specific issue of reconstructing ancestral states.

Is it functional? Report on the first BSPR/EBI meeting on functional proteomics.

This report describes the first scientific meeting of the British Society for Proteome Research (BSPR), which was organised jointly with the European Bioinformatics Institute (EBI) and held in July 2004. The focus of the conference was functional proteomics with an emphasis on possible clinical application. The main subjects described here are: the need to simplify samples, the use of biological fluids versus tissue, consideration of biological and experimental variation and the creation of databases to achieve meaningful functional analysis.

The influence of G protein subtype on agonist action at D2 dopamine receptors.

In previous studies, we have shown that agonists influence the ability of D2 dopamine receptors to couple to G proteins and here we extend this work. The human D2Short dopamine receptor and a natural polymorphism of this D(2Short)(Ser311Cys), have been studied by co-expressing the receptors in insect cells with Gbeta1gamma2 and either Galpha(o), Galpha(i1), Galpha(i2) or Galpha(i3) G protein subunits. These preparations have been used to study the G protein coupling profiles of the two receptors and the influence of agonists.

A comparative study of the effects of metal contamination on Collembola in the field and in the laboratory.

We examined the species diversity and abundance of Collembola at 32 sampling points along a gradient of metal contamination in a rough grassland site (Wolverhampton, England), formerly used for the disposal of metal-rich smelting waste. Differences in the concentrations of Cd, Cu, Pb and Zn between the least and most contaminated part of the 35 metre transect were more than one order of magnitude. A gradient of Zn concentrations from 597 to 9080 microg g(-1) dry soil was found.

Biodiversity of Collembola in urban soils and the use of Folsomia candida to assess soil 'quality'.

The effects of metal contamination on natural populations of Collembola in soils from five sites in the Wolverhampton area (West Midlands, England) were examined. Analysis revealed that metal concentrations were elevated above background levels at all sites. One location in particular (Ladymoor, a former smelting site) was highly contaminated with Cd, Cu, Pb and Zn at more than 20 times background levels.

A role for the thiol isomerase protein ERP5 in platelet function.

Formation and rearrangement of disulfide bonds during the correct folding of nascent proteins is modulated by a family of enzymes known as thiol isomerases, which include protein disulfide isomerase (PDI), endoplasmic reticulum protein 5 (ERP5), and ERP57. Recent evidence supports an alternative role for this family of proteins on the surface of cells, where they are involved in receptor remodeling and recognition. In platelets, blocking PDI with inhibitory antibodies inhibits a number of platelet activation pathways, including aggregation, secretion, and fibrinogen binding.

Evaluation of malacosporean life cycles through transmission studies.

Myxozoans, belonging to the recently described Class Malacosporea, parasitise freshwater bryozoans during at least part of their life cycle, but no complete malacosporean life cycle is known to date. One of the 2 described malacosporeans is Tetracapsuloides bryosalmonae, the causative agent of salmonid proliferative kidney disease. The other is Buddenbrockia plumatellae, so far only found in freshwater bryozoans.

A phylogenetic mixture model for detecting pattern-heterogeneity in gene sequence or character-state data.

We describe a general likelihood-based 'mixture model' for inferring phylogenetic trees from gene-sequence or other character-state data. The model accommodates cases in which different sites in the alignment evolve in qualitatively distinct ways, but does not require prior knowledge of these patterns or partitioning of the data. We call this qualitative variability in the pattern of evolution across sites "pattern-heterogeneity" to distinguish it from both a homogenous process of evolution and from one characterized principally by differences in rates of evolution.

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12.

It is known that Escherichia coli K-12 is cryptic (Phn-) for utilization of methyl phosphonate (MePn) and that Phn+ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn+ variants are present at the surprisingly high frequency of >10(-2) in K-12 strains.

Mechanisms of agonist action at D2 dopamine receptors.

In this study, we investigated the biochemical mechanisms of agonist action at the G protein-coupled D2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding by full and partial agonists was determined at different concentrations of [35S]GTPgammaS (0.1 and 10 nM) and in the presence of different concentrations of GDP.

The phylogeography of salmonid proliferative kidney disease in Europe and North America.

Salmonid proliferative kidney disease (PKD) is caused by the myxozoan Tetracapsuloides bryosalmonae. Given the serious and apparently growing impact of PKD on farmed and wild salmonids, we undertook a phylogeographic study to gain insights into the history of genealogical lineages of T. bryosalmonae in Europe and North America, and to determine if the global expansion of rainbow trout farming has spread the disease.

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin alpha 2 beta 1.

Background: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets.

Objectives: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line.

Rapid parallel expression in E. Coli and insect cells: analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV).

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells.

Platelet adhesion signalling and the regulation of thrombus formation.

Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin alpha(2)beta(1). These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.

CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and Gi proteins.

The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with Gi, Go and Gq species. Interaction with Gi leads to G protein activation, whereas Gq does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2).

Hemokinins and endokinins.

The mammalian tachykinins are a family of peptides that, until recently, has included substance P (SP), neurokinin A and neurokinin B. Since, the discovery of a third preprotachykinin gene ( TAC4), the number of tachykinins has more than doubled to reveal several species-divergent peptides. This group includes hemokinin-1 (HK-1) in mouse and rat, endokinin-1 (EK-1) in rabbit, and EKA, EKB, human HK-1 (hHK-1) and hHK(4-11) in humans.

Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein.

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection.

A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides.

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.

Phylogenomics of eukaryotes: impact of missing data on large alignments.

Resolving the relationships between Metazoa and other eukaryotic groups as well as between metazoan phyla is central to the understanding of the origin and evolution of animals. The current view is based on limited data sets, either a single gene with many species (e.g.

Isolation of haloarchaea that grow at low salinities.

Summary Archaea, the third domain of life, were long thought to be limited to environmental extremes. However, the discovery of archaeal 16S rRNA gene sequences in water, sediment and soil samples has called into question the notion of Archaea as obligate extremophiles. Until now, none of these novel Archaea has been brought into culture, a critical step for discovering their ecological roles.