Molecular analysis of programmed cell death during senescence in Arabidopsis thaliana and Brassica oleracea: cloning broccoli LSD1, Bax inhibitor and serine palmitoyltransferase homologues

This study was undertaken to characterize the programmed cell death (PCD) processes that occur during detached and natural on-plant senescence and correlate them with the expression of putative regulatory genes that may be involved in the process. DNA fragmentation and TUNEL analysis of broccoli florets showed that DNA was processed into fragments of approximately 180 bp after 48 h of harvest-induced tissue senescence. Characteristic laddering patterns were also visible in Arabidopsis leaves undergoing natural on-plant senescence and during detached senescence. Several recently isolated plant proteins have been assigned a PCD role, for example, the zinc finger containing protein, LSD1 (lesion simulating disease); Bax inhibitor (BI); and serine palmitoyltransferase (SPT), an enzyme in the sphingolipid signalling pathway. Two cDNAs encoding each of these proteins were isolated from broccoli (BoBI-1, BoBI-2, BoLSD1, BoLSD2, BoSPT1, BoSPT2), and the mRNAs increased during harvest-induced senescence in floret tissue. Expression of the Arabidopsis homologues (AtBI-1, AtLSD1, AtSPT1) were also characterized during detached leaf senescence in Arabidopsis leaves. AtBI-1 expression was constitutively expressed during detached senescence, AtLSD1 expression remained constitutively low, and AtSPT1 expression increased during detached senescence.     

Binding of prion proteins to lipid membranes

A key molecular event in prion diseases is the conversion of the normal cellular form of the prion protein (PrPC) to an aberrant form known as the scrapie isoform, PrPSc. Under normal physiological conditions PrPC is attached to the outer leaflet of the plasma membrane via a GPI-anchor. It has been proposed that a direct interaction between PrP and lipid membranes could be involved in the conversion of PrPC to its disease-associated corrupted conformation, PrPSc. Recombinant PrP can be refolded into an alpha-helical structure, designated alpha-PrP isoform, or into beta-sheet-rich states, designated beta-PrP isoform. The current study investigates the binding of recombinant PrP isoforms to model lipid membranes using surface plasmon resonance spectroscopy. The binding of alpha- and beta-PrP to negatively charged lipid membranes of POPG, zwitterionic membranes of DPPC, and model raft membranes composed of DPPC, cholesterol, and sphingomyelin is compared at pH 7 and 5, to simulate the environment at the plasma membrane and within endosomes, respectively. It is found that PrP binds strongly to lipid membranes. The strength of the association of PrP with lipid membranes depends on the protein conformation and pH, and involves both hydrophobic and electrostatic lipid-protein interactions. Competition binding measurements established that the binding of alpha-PrP to lipid membranes follows a decreasing order of affinity to POPG>DPPC>rafts.     

pp60v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.     

Controlled investigation of deaths from asthma in hospitals in the North East Thames region.

One hundred and thirty deaths definitely or potentially due to asthma occurring in hospitals in the North East Thames region over one year were identified from death certificates and Hospital Activity Analysis records. Thirty five of these deaths were considered after independent assessment to have been directly due to asthma. Control patients who left hospital alive after acute asthma attacks were selected and matched with cases for sex, age, and hospital. Management was compared in the two groups. Inadequate monitoring, including failure to monitor arterial blood gas values, and inadequate use of nebulised beta agonists occurred significantly more often in fatal cases. Use of sedation, inadequate treatment with steroids, exposure to potentially toxic doses of aminophylline, and inadequate clinical assessment were more common in cases than controls, but not significantly so. Failure to institute artificial ventilation contributed to seven deaths. Assessors considered important defects in management to have occurred in 83% (29/35) of the cases and 40% (14/35) of the controls. Nevertheless, most of the hospital deaths (19/35) were considered not to have been preventable. Eight other deaths in the region were attributed to the complications of asthma or its treatment. Three of these were associated with gastrointestinal bleeding and one with perforation of a duodenal ulcer. Before considering policies aimed at speeding admission to hospital of patients with acute attacks of asthma it is crucial that the general standard of hospital care offered to all patients with asthma should be improved.     

Synthesis and characterization of 8-methoxy-2'- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2'-deoxyadenosine within DNA.

The synthesis of 8-methoxy-2'-deoxyadenosine (moA) protected at N6 as an N,N-dimethylformamidine derivative and incorporation of the modified nucleoside into oligodeoxynucleotides via the phosphoramidite method are described. UV thermal denaturation studies were conducted on duplexes containing moA:G, moA:C and moA:T base pairs to determine the thermodynamic stability of duplexes containing moA relative to their adenosine (A)-containing counterparts. In the case of moA:G base pairs the effect of moA substitution is sequence dependent. In A:G mismatch-containing sequences, which have been shown by structural characterization to have a syn conformational preference at the glycosidic bond of A, moA substitution results in stabilization of the duplex. In contrast, in sequences where the A in the A:G mismatch has been shown to prefer the anti conformation moA substitution is destabilizing to the duplex. Thus moA may be a useful probe for investigating the conformational preferences of the N-glycosidic bond of adenosine within DNA. In addition, moA nucleoside is more resistant to acid-catalyzed depurination than previously described 8-bromo-2'-deoxyadenosine, allowing for facile incorporation into oligonucleotides via automated solid phase DNA synthesis.     

The effectivity of arbuscular mycorrhizal fungi from high input conventional and organic grassland and grass-arable rotations

The effectivity of arbuscular mycorrhizal spores in promoting growth of Allium ameloprasum L. cv. Musselburgh and Trifolium repens L. cv. Menna was tested for inocula from three soil series under long term organic or intensive, conventional grass and grass-arable rotations. For two soil series, Allium responses to inocula from soils recently converted to organic fanning were also assessed. Finally, Trifolium root fragments were used to inoculate Allium so as to evaluate responses to this inoculation procedure. Plants were sown into previously sterilised, matched soils from organic farms with no nutrient input. Mycorrhizal treatments generally increased growth for Allium. However, for Trifolium, infection decreased growth in the most fertile soil and gave an increase only in the least fertile. In the least fertile soil, inocula from organic farms were more effective than those from conventional farms. For Trifolium (all soils) and for Allium (least fertile soil), there was evidence of more efficient uptake of phosphorus in plants inoculated with spores from organic farms. The pattern of Allium response to inoculation with spores from conventional, conversion and organic sources was not consistent between soil type, but there was evidence of lower root infection for conversion compared with organic inocula and of a trend towards higher infectivity as the time period under organic management increased. Inoculating Allium with AMF root fragments produced a plant response similar to that obtained when spores were used, confirming that spore viability was not the sole factor influencing AMF effectivity in earlier experiments. Intensive farming practices may reduce the effectiveness of indigenous arbuscular mycorrhizal populations, particularly where fertiliser inputs are high and inherent fertility is low. This could have practical implications where high input farms are converted to organic management.     

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.