Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.     

Brief communication: Tissue isotopic enrichment associated with growth depression in a pig: Implications for archaeology and ecology

Stressors such as fasting or poor diet quality are thought to potentially alter the nitrogen and carbon isotopic values of animal tissues. In this study, we demonstrate an inverse correlation between growth rate and multiple tissue enrichment of δ¹⁵N, δ¹³C, and, to a lesser degree, δ¹⁸O in a juvenile pig. A more complex pattern is observed with respect to tissue δD and growth rate. The observed association between growth rate and tissue isotopic fractionation has important implications for paleodietary and migratory reconstructions of archaeological populations that may have been affected by famine, malnutrition, seasonal variation in food availability, and/or other factors that can affect childhood growth rates. Am J Phys Anthropol 2010. © 2010 Wiley‐Liss, Inc.     

Studies on Bacillus stearothermophilus. Part 1. Transformation of progesterone to a new metabolite 9,10-seco-4-pregnene-3,9,20-trione

When Bacillus stearothermophilus, a thermophilic bacterium isolated from the Kuwaiti desert, was incubated with exogenous progesterone for 24 h, three monohydroxylated metabolites were produced. 20α-Hydroxyprogesterone was the major metabolite produced in 60.8 relative percentage yield. The other two monohydroxylated metabolites were identified as 6β-hydroxyprogesterone and the rare 6α-hydroxyprogesterone in 21.0 and 13.6 relative percentage yields, respectively. A new metabolite 9,10-seco-4-pregnene-3,9,20-trione was isolated in 3.7 relative percentage yield. All metabolites were purified by preparative TLC and HPLC followed by their identification using ¹H, ¹³C NMR and other spectroscopic data.     

Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes

Type I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter- and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so-called ‘DEAD box’ proteins; these conserved sequences may be associated with the ATP-dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.     

Skull and limb morphology differentially track population history and environmental factors in the transition to agriculture in Europe

The Neolithic transition in Europe was a complex mosaic spatio-temporal process, involving both demic diffusion from the Near East and the cultural adoption of farming practices by indigenous hunter–gatherers. Previous analyses of Mesolithic hunter–gatherers and Early Neolithic farmers suggest that cranial shape variation preserves the population history signature of the Neolithic transition. However, the extent to which these same demographic processes are discernible in the postcranium is poorly understood. Here, for the first time, crania and postcranial elements from the same 11 prehistoric populations are analysed together in an internally consistent theoretical and methodological framework. Results show that while cranial shape reflects the population history differences between Mesolithic and Neolithic lineages, relative limb dimensions exhibit significant congruence with environmental variables such as latitude and temperature, even after controlling for geography and time. Also, overall limb size is found to be consistently larger in hunter–gatherers than farmers, suggesting a reduction in size related to factors other than thermoregulatory adaptation. Therefore, our results suggest that relative limb dimensions are not tracking the same demographic population history as the cranium, and point to the strong influence of climatic, dietary and behavioural factors in determining limb morphology, irrespective of underlying neutral demographic processes.     

Removal of Viruses from Human Intravenous Immune Globulin by 35 nm Nanofiltration

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.     

Two trypanosome-specific proteins are essential factors for 5S rRNA abundance and ribosomal assembly in Trypanosoma brucei

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-amino-acid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a higher-molecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.