Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Role of the private sector in elective surgery in England and Wales, 1986.

From a sample of 19,000 treatment episodes at 183 of the 193 independent hospitals with operating facilities in England and Wales that were open in 1986 it is estimated that 287,000 residents of England and Wales had elective surgery as inpatients in 1986 (an increase of 77% since 1981) and 72,000 as day cases. From 1985 Hospital In-Patient Enquiry data it was estimated that a further 36,000 similar elective inpatient treatments were undertaken in NHS pay beds (a decrease of 38%) and 21,000 as day cases. Overall, an estimated 16.7% of all residents of England and Wales who had non-abortion elective surgery as inpatients were treated in the private sector, as were 10.5% of all day cases. An estimated 28% of all total hip joint replacements were done privately, and in both the North West and South West Thames regions the proportion of inpatients treated privately for elective surgery was 31%. It is concluded that mainly for reasons of available manpower private sector activity may not be able to grow much more without arresting or reversing the growth of the NHS, in which case some method of calculating NHS resource allocation which takes account of the local strength of the private sector will be needed.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

Comparison of the activity of short stay independent hospitals in England and Wales, 1981 and 1986.

From a sample of 19,000 treatment episodes at 183 of the 193 independent hospitals with operating facilities in England and Wales that were open during 1986 it is estimated that 404,000 inpatients were treated in 1986 (an increase of 48% since 1981) and 99,000 day cases (an increase of 112%). It was found that the procedure most commonly performed was abortion, though this made up only 19% of the total caseload in 1986 compared with 30% in 1981, otherwise the case mix in 1986 was similar to that in 1981. Fewer patients came from overseas in 1986 than in 1981, but the distribution by age and sex remained the same, with three quarters of the patients aged between 15 and 65. The estimated bed occupancy in the independent hospitals in 1986 was less than 60% nationally and only 52% in the Thames regions. It is concluded that in these five years the nature of the independent hospital sector changed little, and in 1986 the activity still consisted largely of routine cold elective surgery for people of working age, and the regional differences in admission rates to independent hospitals were nearly as great as in 1981.     

Structural diversity of bacterial flagellar motors

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.     

Architecture and assembly of the Gram‐positive cell wall

The bacterial cell wall is a mesh polymer of peptidoglycan – linear glycan strands cross‐linked by flexible peptides – that determines cell shape and provides physical protection. While the glycan strands in thin ‘Gram‐negative’ peptidoglycan are known to run circumferentially around the cell, the architecture of the thicker ‘Gram‐positive’ form remains unclear. Using electron cryotomography, here we show that Bacillus subtilis peptidoglycan is a uniformly dense layer with a textured surface. We further show it rips circumferentially, curls and thickens at free edges, and extends longitudinally when denatured. Molecular dynamics simulations show that only atomic models based on the circumferential topology recapitulate the observed curling and thickening, in support of an ‘inside‐to‐outside’ assembly process. We conclude that instead of being perpendicular to the cell surface or wrapped in coiled cables (two alternative models), the glycan strands in Gram‐positive cell walls run circumferentially around the cell just as they do in Gram‐negative cells. Together with providing insights into the architecture of the ultimate determinant of cell shape, this study is important because Gram‐positive peptidoglycan is an antibiotic target crucial to the viability of several important rod‐shaped pathogens including Bacillus anthracis, Listeria monocytogenes, and Clostridium difficile.     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells