Definable equilibrium states in the folding of human prion protein

The role of conformational intermediates in the conversion of prion protein from its normal cellular form (PrP(C)) to the disease-associated "scrapie" form (PrP(Sc)) remains unknown. To look for such intermediates in equilibrium conditions, we have examined the unfolding transitions of PrP(C), primarily using the chemical denaturant guanidine hydrochloride (GuHCl). When the protein conformation is assessed by NMR, there is a gradual shift of NMR signals in the regions between residues 125-146 and 186-196. The denaturant dependence of these shifts shows that in aqueous solution the native and locally unfolded conformations are both significantly populated. Following this shift, there is the major unfolding transition to generate a substantially unfolded population. However, analysis of NMR chemical shift and intensity changes shows that there is persistent structure in the molecule well beyond this major cooperative unfolding transition. Residual structure within this state is extensive and encompasses the majority of the secondary structure elements found in the native state of the protein.     

Managing water resources for crop production

Increasing crop production to meet the food requirements of the world''s growing population will put great pressure on global water resources. Given that the vast freshwater resources that are available in the world are far from fully exploited, globally there should be sufficient water for future agricultural requirements. However, there are large areas where low water supply and high human demand may lead to regional shortages of water for future food production. In these arid and semi-arid areas, where water is a major constraint on production, improving water resource management is crucial if Malthusian disasters are to be avoided. There is considerable scope for improvement, since in both dryland and irrigated agriculture only about one-third of the available water (as rainfall, surface, or groundwater) is used to grow useful plants. This paper illustrates a range of techniques that could lead to increased crop production by improving agricultural water use efficiency. This may be achieved by increasing the total amount of water available to plants or by increasing the efficiency with which that water is used to produce biomass. Although the crash from the Malthusian precipice may ultimately be inevitable if population growth is not addressed, the time taken to reach the edge of the precipice could be lengthened by more efficient use of existing water resources. <br>     

A simple method for measurement of the haemoglobin binding capacity of canine haptoglobin

The potential of a series of related compounds to induce haemolytic anaemia in dogs highlighted the need for a reliable and sensitive technique to identify changes in plasma haptoglobin concentration. An indirect method established for human samples was adapted for use with canine plasma. This method measures the haemoglobin binding capacity of plasma, which is directly proportional to the functional haptoglobin concentration. The efficacy of this technique was investigated on samples taken from Beagle dogs which had previously received small quantities of water intravenously. A substantial reduction in haemoglobin binding capacity was recorded before other haematological parameters were significantly affected. It was concluded that measurement of haemoglobin binding capacity by this method provides a valid reflection of haptoglobin status in the dog.     

A1603P and K1617del,Mutations in β-Cardiac Myosin Heavy Chain that Cause Laing Early-Onset Distal Myopathy,Affect Secondary Structure and Filament Formation In Vitro and In Vivo

Over 20 mutations in β-cardiac myosin heavy chain (β-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase–light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged β-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged β-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease.     

The presence of <Emphasis Type="Italic">Zygina</Emphasis> sp. and <Emphasis Type="Italic">Puccinia myrsiphylli</Emphasis> reduces survival and influences oviposition of <Emphasis Type="Italic">Crioceris</Emphasis> sp.

A leaf beetle, Crioceris sp. (Coleoptera: Chrysomelidae), was introduced into Australia as a biological control agent of bridal creeper (Asparagus asparagoides L. Druce) during October 2002. Rearing of Crioceris sp. is labour intensive therefore all releases of Crioceris sp. have been under 1000 individuals, which may be too low to ensure establishment if high mortality and high competition with other agents occurs. The aim of this study is to understand how the presence of two well-established biocontrol agents, a rust fungus (Puccinia myrsiphylli (Thuem) Wint [Basidiomycota: Uredinales]) and a leafhopper (Zygina sp. [Hemiptera: Cicadellidae]), might influence Crioceris sp. establishment. Crioceris sp. neonate larvae were placed on bridal creeper plants with or without the leafhopper and/or rust. The number of larvae that pupated was reduced by 38 and 65% in the presence of the rust fungus and leafhopper, respectively and by 45% in the presence of both agents. As the area infected by the rust increased the area damaged by the leafhopper decreased. The rust appeared to be negatively impacted by the presence of the leafhopper. In a second experiment, female Crioceris sp. adults were given a choice between uninfested bridal creeper plants and those infested with the rust or the leafhopper. The females preferred to lay their eggs on plants without leafhoppers but did not seem to be deterred by the presence of the rust. Consequently, the performance and impact of Crioceris sp. on bridal creeper may be reduced if populations overlap with the other biocontrol agents in the field.     

Therapeutic aerosols in children.

The use and variety of drugs administered to children as inhaled aerosols is increasing, but little is known about how much drug reaches the lung and how it is distributed there in different age groups. In this article the reasons for measuring aerosol deposition in children are discussed and the potential methods for doing this described. Of the methods available, only the use of radiolabelled aerosols gives accurate information on total lung deposition and distribution. The potential risk of the radiation exposure required for these measurements varies with the age of the child but seems to be small. Properly designed studies are expected to clarify the factors affecting lung deposition in children and identify methods of inhalation associated with efficient and predictable delivery of the drug. Measurements of radioaerosol deposition may therefore be justified in children when this information is expected to lead to improvements in the effectiveness or safety of their treatment.     

Structural analysis of anti-DR1 allorecognition by using DR1/H-2Ek hybrid molecules. Influence of the beta 2-domain correlates with CD4 dependence.

A segmental analysis of the key regions of HLA-DR1 that control T cell allorecognition was performed by using a series of transfected cell lines expressing the products of recombinant DRB/H-2Eb genes, paired with either DR alpha or H-2E alpha. Four of eight human T cell clones tolerated substitution of the H-2E alpha chain, but only one clone showed any response to the DR alpha/H-2E beta k dimer. Both the membrane-proximal and the membrane-distal domains of the beta-chain played an important part in stimulating these clones. The response of four of eight clones was markedly inhibited by substitution of the H-2E beta 2 for the DR beta 2 domain. This inhibition showed a complete correlation with the sensitivity of the clones to inhibition by anti-CD4 mAb. Taken together, these results suggest that the interaction site for CD4 may include residues on the beta 2-domain. Introduction of H-2Ek sequence into either half of the beta 1-domain led to a complete loss of response by all but two of the clones. This is consistent with these clones having dual specificity for exposed DR1-specific polymorphisms and for DR1-bound peptides. The pattern of response of one of the clones suggested that indirect conformational effects on the alpha 1-domain may also contribute to the influence of the amino-terminal half of the beta 1-domain on T cell recognition. In the presence of H-2E alpha, this clone responded more strongly when the amino-terminal half of the beta 1-domain was of H-2Ek rather than DR1 sequence. This implies that species matching of the floor of the beta 1-domain with the alpha-chain is more important than the presence of the alpha-chain of the parental species.     

Host protein interactions with enteropathogenic Escherichia coli (EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization

Enteropathogenic Escherichia coli (EPEC) cause infantile diarrhoea and are characterized by their ability to produce attaching and effacing lesions on the surface of intestinal epithelial cells. EPEC employ a filamentous type III secretion system to deliver effector molecules that subvert mammalian cell function to generate actin- and cytokeratin-rich pedestals beneath adherent bacteria. Tir is a major effector protein that is delivered to the plasma membrane of the eukaryotic cell where it acts as the receptor for the bacterial adhesin intimin. Host cell proteins that are recruited to the site of intimate attachment include focal adhesion and cytoskeletal proteins that contribute to pedestal formation. We have used Tir as bait in a yeast two-hybrid screen to identify the protein 14-3-3tau as a binding partner. 14-3-3 proteins are a family of adaptor proteins that modulate protein function in all eukaryotic cells. Here we demonstrate that the tau isoform (also known as theta) of 14-3-3 can bind specifically to Tir in a phosphorylation-independent manner, and that the interaction occurs during the infection process by co-immunoprecipitation of the partners from infected HeLa cell extracts. 14-3-3tau is recruited to the site of the pedestal (3 h after infection) and can decorate attached EPEC in the later stages of the infection process (5-7 h). Pedestal formation can be impaired by depletion of cellular 14-3-3tau using small interfering RNAs. This study indicates a direct functional role for the 14-3-3tau:Tir interaction and is the first to demonstrate the association of a host protein with the surface of EPEC.