Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The preparation and properties of ficin chemically attached to carboxymethylcellulose

1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed.     

Peter Chalmers Mitchell and antiwar evolutionism in Britain during the Great War

Conclusions It may be concluded that Mitchell's peace evolutionism incorporated most of the features of the cooperationist and Novicovian traditions. He questioned the conflict paradigm that underpinned biological militarism, and reinforced a holistic and more peaceful model of nature by reference to the emerging discipline of ecology. His restrictionist objections to the deterministic tendencies of much prevailing biosocial thought combined philosophical with biological arguments to assert that human history was sui generis, based upon the unique development of human consciousness and the cultural transmission of knowledge. Mitchell's opposition to biological militarism reflected Victorian anxieties about the legitimacy of evolutionary ethics. However, he introduced an innovatory note, linked to the modernist intellectual milieu of the time, when he put objections to the use of analogy on the grounds (1) that the Darwinist paradigm had not been properly established, and (2) that scientific laws themselves were uncertain and subjective.The first objection related to the bitter controversies that racked the biological world in the 1900s when mutation theory thrust the Darwinian concept of natural selection into temporary disrepute. In this respect Mitchell encoutered continuing Darwinist orthodoxy, not least from peace biology itself, while confusion was added by his personal devotion to Darwinism and his sociopolitical suspicion of Mendelian hereditarianism. The later triumph of a new Darwinian synthesis under men like R. A. Fisher made Mitchell's criticisms seem outmoded. In the second respect, Mitchell's attack on the primacy of naturalistic science echoed the epistemology of the new physics and movements such as German neo-Kantianism. However, positivism was still deeply embedded in Britain, indeed enjoying a resurgence from the last decade of the nineteenth century.⁷⁹ Mitchell's critique of the Darwinist version of it seems to have been too novel and puzzling to influence a generation still convinced of the soundness of the science.Mitchell made more impact when he put his objections to the use of analogy on the grounds of professional methodology. As a naturalist, he could argue:It is impossible to make correct comparisons even between an insect and spider, two creatures so closely allied that only zoologists would separate them, unless we could trace the qualities of the insect and of the spider respectively down to their common ancestor, and in so doing we should almost certainly lose all that made the comparison interesting and significant, and be left with little more than the qualities common to all protoplasm..., It is quite true that the whole web of life is in physical and physiological community, but considerations drawn from any part of it require so much modification before they can be applied to any other part, that they become merely verbal.⁸⁰ This type of criticism was to have a more lasting heritage. Chalmers Mitchell is worth remembering as an articulate early spokesman of a persistent, if often embattled, modern tradition that has resisted interpretations of human nature and history based upon genetic determinants or immutable biological laws, or upon the use of animal analogies to generalize too freely about human aggression and war.     

Structural development of the shoot and root systems of two winter wheat cultivars, Triticum aestivum L.

The structural development of the stems and basal anchorageroots of Galahad and Hereward winter wheat cultivars (Triticumaestivum L.) were investigated and related to their mechanicalfunction. Stem and root morphology, anatomy and mechanical propertieswere examined from tillering (March) up to maturity (August),together with plant weight distribution. This allowed us tocalculate a ‘factor of safety’ against root andstem failure throughout development. As the plants grew taller the stem and the anchorage ‘coronalroots’ increased in bending strength countering the increasingmechanical demands. The bending strength, in turn, was correlatedwith the amount of lignified material around the stem and rootperimeter. Structural development ceased by ear emergence, whenthe plant was at its tallest, but because the ear weight continuedto rise the ‘self-weight’ moment pushing the plantover continued to increase. This meant that the ‘safetyfactors’ of both cultivars against both root and stemmechanical failure decreased throughout development. In bothcultivars the safety factors against root failure were lowerthan for stem failure, and Galahad had lower factors of safetythan Hereward. All these findings were consistent with resultsof field trials; failure tends to occur late in development,during grain filling, and is localized to the root system, whilstGalahad is more prone to lodging than Hereward. The pattern of mechanical development of winter wheat seemsto be one which would maximize its reproductive success, maintainingits structural integrity especially early in development whileinvesting in a minimum of structural material. Key words: Safety factor, anchorage, lodging, biomechan-ics, structural development     

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.     

Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation.

The transforming function of human papillomavirus type 16 (HPV16) E7 has been shown to depend on activities additional to the ability to bind RB. In this paper we describe two further properties of E7 which may also contribute to transformation, an association with a histone H1 kinase at the G2/M phase of the cell cycle and an ability to bind the RB-related protein p107. The region of E7 identified previously as important for RB binding was found to be involved in the association with the kinase and complex formation with p107, although analysis of E7 point mutants within this region revealed a difference in the precise sequence requirement for RB and p107 binding. Association with the kinase activity correlated with the ability to bind RB, but the restriction of the kinase association to the G2/M phase of the cell cycle implies that this activity might not be directly mediated by RB binding. Since kinase-binding-deficient E7 mutants are also transformation defective, this may represent an independent function of E7 which plays a role in the G2/M phase of the cell cycle.     

Prostaglandin synthetase activity from human rheumatoid synovial tissue and its inhibition by non-steroidal anti-inflammatory drugs.

1. Prostaglandin synthetase activity was found in a microsomal fraction from human rheumatoid synovia. 2. The microsomes produced PGE2 and a small amount of PGF2 when incubated with arachidonic acid. 3. The pH optimum of the enzyme from this source was similar to that found with microsomal preparations from rabbit renal medullae and bovine seminal vesicles. 4. The enzyme was inhibited in vitro by the non-steroidal anti-inflammatory drugs flurbiprofen, indomethacin and aspirin in the same rank order of potency as prostaglandin synthetase from other tissues.     

A Modified RNA-Seq Approach for Whole Genome Sequencing of RNA Viruses from Faecal and Blood Samples

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.     

The discovery of the benzazepine class of histamine H3 receptor antagonists

This Letter describes the discovery of a novel series of H₃ receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H₃ receptor antagonists based on the benzazepine core. Employing an H₃ driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.