Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

LIMaS: the JAVA-based application and database for microarray experiment tracking

Microarrays allow monitoring of gene expression for tens of thousands of genes in parallel and are being used routinely to generate huge amounts of valuable data. Handling and analysis of such data are becoming major bottlenecks in the utilization of the technology. To enable the researcher to interpret the results postanalysis, we have developed a laboratory information management system for microarrays (LIMaS) with an n-tier Java front-end and relational database to record and manage large-scale expression data preanalysis. This system enables the laboratory to replace the paper trail with an efficient and fully customizable interface giving it the ability to adapt to any working practice, e.g., handling many resources used to form many products (chaining of resources). The ability to define sets of activities, resources, and workflows makes LIMaS MIAME-supportive.LIMaS is available for download at (http://www.mgu.har.mrc.ac.uk/microarray.)     

Ecology drives intragenomic conflict over menopause

Menopause is the transition from reproductive to non‐reproductive life well before natural death. Rather than involving a smooth, rapid change, it is normally preceded by a long period of erratic hormonal fluctuation that is accompanied by a plethora of unpleasant symptoms. Here, we (1) suggest that this turbulent period owes to conflict, between a woman's maternally inherited (MI) and paternally inherited (PI) genes, over the trade‐off between reproduction and communal care; (2) perform a theoretical analysis to show that this conflict is resolved either through silencing or fluctuating expression of one of the genes; (3) highlight which of the symptoms preceding menopause may result from antagonistic co‐evolution of MI and PI genes; (4) argue that ecological differences between ancestral human populations may explain the variability in menopause among different ethnic groups; (5) discuss how these insights may be used to inform family planning and cancer risk assessment based on a woman's ancestral background.     

Effects of seed ingestion and herbivory by waterfowl on seedling establishment: a field experiment with wigeongrass Ruppia maritima in Doñana,south-west Spain

The ingestion of seeds by vertebrates usually affects the viability and/or germination rate of seeds. Increases in germination rate following passage through the vertebrate gut have often been assumed to be favourable for seedling survival and plant fitness, but this assumption has never been tested experimentally. Given that numbers of herbivorous waterfowl are higher in winter in Mediterranean wetlands, herbivory pressure there will be higher for early growing plants. In a factorial experiment we investigated the effects of seed ingestion by ducks (shoveler, Anas clypeata) on the survival of wigeongrass Ruppia maritima seedlings in the field in Doñana (south-west Spain), under differing exposures to herbivory by waterfowl and fish. We planted ingested and non-ingested seeds in December, using exclosures to protect half of them from herbivores. When they were protected inside exclosures, there was no difference between ingested and non-ingested seeds in the number of plants that survived until June-July. However, fewer plants survived from ingested seeds when exposed to natural levels of herbivory because they were exposed for longer than plants germinating from non-ingested seeds. In conclusion, increases in germination rate after ingestion are not necessarily beneficial for the plant, and the final outcome depends on complex interactions with other factors such as herbivore abundance.     

GM crops: science, politics and communication

As the public debate in Europe about genetically modified (GM) crops heats up and the trade row between the United States and the European Union over GM food escalates, what better time to examine the issues with an international group of experts (Box 1). Their views are diverse, but they all agree that we need more impartial communication, less propaganda and an effective regulatory regime that is based on a careful case-by-case consideration of GM technology. It seems that GM crops are here to stay, so let us hope that these requirements are met and that the developing nations that perhaps have the most to gain from this technology can start to reap its benefits.     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

The application of fluorescence spectroscopy to organic matter characterisation in drinking water treatment

Key to effective disinfection byproduct (DBP) management is source water control and management, and more specifically, organic matter (OM) control and management. However, the content and character of OM in source waters is spatially and temporally variable, and the prediction of its composition is challenging. Water treatment companies require adequate analytical techniques for OM characterisation to maintain the operation of the water supply and treatment systems adjusted to constantly changing environmental conditions. There is a requirement, therefore, for an improved understanding of OM composition and character in source water, how that composition and character varies with flow conditions, and how this impacts on drinking water treatment. This paper demonstrates that fluorescence spectroscopy offers a potential alternative to other analytical methods of OM characterisation. The advantages of fluorescence include rapid, sensitive and selective characterisation of OM, no sample pre-treatment, small sample volume, and the potential for on-line monitoring incorporation. Fluorescence can provide useful information on OM reactivity and treatability together with an indication of the OM sources (allochthonous or autochthonous). The paper discusses a body of literature which has identified relationships between fluorescence spectra and OM physico-chemical properties (i.e. degree of hydrophobicity, microbial content), has applied fluorescence spectroscopy to characterise the changes in OM upon disinfection, and has related the fluorescence properties to DBP formation. Further work is required in the robust management of data arising from fluorescence spectroscopy analysis and, in particular, Excitation Emission Matrices. Consideration must be given as to how the data might best be employed to greatest effect on a routine basis at WTW.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.