Separation and some properties of the major proteins of the human erythrocyte membrane

A fractionation procedure is described which allows the isolation of three major human erythrocyte membrane proteins. Their isolation involves three sequential extraction procedures followed by gel filtration in 1% sodium dodecyl sulphate and preparative gel electrophoresis. All three proteins can be isolated from a single preparation. One of the proteins is the erythrocyte sialoglycoprotein, for which no C- or N-terminal residues were found. The other two proteins, which have not previously been isolated, have subunit molecular weights of 74000 and 93000 and contain 9 and 7% carbohydrate respectively. These glycoproteins have blocked N-terminal residues and show similarities in their chemical properties. Preparations derived from blood-group O erythrocytes contain no N-acetylgalactosamine, but similar preparations from blood-group A erythrocytes do contain this sugar. These three proteins cannot easily be solubilized by gentle aqueous procedures and represent about half of the erythrocyte ;ghost' protein. They carry a large proportion of the cell-surface carbohydrate.     

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization

Background

Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization.

Methods

In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep.

Results

The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation.

Conclusion

Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.     

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the alpha-amylase AmyQ was provided with leucine-rich (high hydrophobicity) or alanine-rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine-rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse-chase labelling experiments. Remarkably, the alanine-rich signal peptide was functional in protein translocation only in E. coli. Cross-linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine-rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.     

Increased litterfall in tropical forests boosts the transfer of soil CO2 to the atmosphere

Aboveground litter production in forests is likely to increase as a consequence of elevated atmospheric carbon dioxide (CO(2)) concentrations, rising temperatures, and shifting rainfall patterns. As litterfall represents a major flux of carbon from vegetation to soil, changes in litter inputs are likely to have wide-reaching consequences for soil carbon dynamics. Such disturbances to the carbon balance may be particularly important in the tropics because tropical forests store almost 30% of the global soil carbon, making them a critical component of the global carbon cycle; nevertheless, the effects of increasing aboveground litter production on belowground carbon dynamics are poorly understood. We used long-term, large-scale monthly litter removal and addition treatments in a lowland tropical forest to assess the consequences of increased litterfall on belowground CO(2) production. Over the second to the fifth year of treatments, litter addition increased soil respiration more than litter removal decreased it; soil respiration was on average 20% lower in the litter removal and 43% higher in the litter addition treatment compared to the controls but litter addition did not change microbial biomass. We predicted a 9% increase in soil respiration in the litter addition plots, based on the 20% decrease in the litter removal plots and an 11% reduction due to lower fine root biomass in the litter addition plots. The 43% measured increase in soil respiration was therefore 34% higher than predicted and it is possible that this 'extra' CO(2) was a result of priming effects, i.e. stimulation of the decomposition of older soil organic matter by the addition of fresh organic matter. Our results show that increases in aboveground litter production as a result of global change have the potential to cause considerable losses of soil carbon to the atmosphere in tropical forests.     

An archaeal peptidase assembles into two different quaternary structures: A tetrahedron and a giant octahedron

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data. The internal organization of the PhTET1 particles reveals highly self-compartmentalized systems made of networks of access channels extended by vast catalytic chambers. The two edifices display aminopeptidase activity, and their organizations indicate substrate navigation mechanisms different from those described in other large peptidase complexes. Compared with the tetrahedron, the octahedron forms a more expanded hollow structure, representing a new type of giant peptidase complex. PhTET1 assembles into two different quaternary structures because of quasi-equivalent contacts that previously have only been identified in viral capsids.     

Identification and expression of the first nonmammalian amyloid-beta precursor-like protein APLP2 in the amphibian Xenopus laevis.

The Alzheimer's disease-linked amyloid-beta precursor protein (APP) belongs to a superfamily of proteins, which also comprises the amyloid-beta precursor-like proteins, APLP1 and APLP2. Whereas APP has been identified in both lower and higher vertebrates, thus far, APLP1 and 2 have been characterized only in human and rodents. Here we identify the first nonmammalian APLP2 protein in the South African claw-toed frog Xenopus laevis. The identity between the Xenopus and mammalian APLP2 proteins is approximately 75%, with the highest degree of conservation in a number of amino-terminal regions, the transmembrane domain and the cytoplasmic tail. Furthermore, amino acid residues known to be phosphorylated and glycosylated in mammalian APLP2 are conserved in Xenopus. The availability of the Xenopus APLP2 protein sequence allowed a phylogenetic analysis of APP superfamily members that suggested the occurrence of APP and preAPLP lineages with their separation predating the mammalian-amphibian split. As in mammals, Xenopus APLP2 mRNA was ubiquitously expressed and alternatively spliced forms were detected. However, the expression ratios between the mRNA forms in the various tissues examined were different between Xenopus and mammals, most prominently for the alternatively spliced forms containing the Kunitz protease inhibitor-coding region that were less abundantly expressed than the corresponding mammalian forms. Thus, the identification of APLP2 in Xenopus has revealed evolutionarily conserved regions that may help to delineate functionally important domains, and its overall high degree of conservation suggests an important role for this APP superfamily member.     

Structural biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

Molecular,genetic and evolutionary analysis of a paracentric inversion in Arabidopsis thaliana

Chromosomal inversions can provide windows onto the cytogenetic, molecular, evolutionary and demographic histories of a species. Here we investigate a paracentric 1.17‐Mb inversion on chromosome 4 of Arabidopsis thaliana with nucleotide precision of its borders. The inversion is created by Vandal transposon activity, splitting an F‐box and relocating a pericentric heterochromatin segment in juxtaposition with euchromatin without affecting the epigenetic landscape. Examination of the RegMap panel and the 1001 Arabidopsis genomes revealed more than 170 inversion accessions in Europe and North America. The SNP patterns revealed historical recombinations from which we infer diverse haplotype patterns, ancient introgression events and phylogenetic relationships. We find a robust association between the inversion and fecundity under drought. We also find linkage disequilibrium between the inverted region and the early flowering Col‐FRIGIDA allele. Finally, SNP analysis elucidates the origin of the inversion to South‐Eastern Europe approximately 5000 years ago and the FRI‐Col allele to North‐West Europe, and reveals the spreading of a single haplotype to North America during the 17th to 19th century. The ‘American haplotype’ was identified from several European localities, potentially due to return migration.     

Black flower coloration in wild Lisianthius nigrescens: its chemistry and ecological consequences

The major pigments responsible for the flower color within the black flowered Gentianaceae, Lisianthius nigrescens, were characterized by HPLC and chemical analyses HPLC analysis showed one major and one minor anthocyanin and 3 major and 3 minor flavone glycosides. The anthocyanins [delphinidin-3-O-rhamnol(1-6)galactoside and its 5-O-glucoside] comprised an extraordinary 24% of the dry weight of wild collected L. nigrescens corallas, and were accompanied in a 1:1 ratio by a range of apigenin and luteolin 8-C-glucosides and their 7-O-methyl ethers. The high levels of anthocyanins and flavones (and their co-pigmentation) is thought to account for the almost complete absorption of both UV and visible wavebands observed by reflectance photography.