Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin

Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.     

Functional analysis of the minor subunits of S fimbrial adhesin (SfaI) in pathogenic Escherichia coli

S fimbrial adhesins I and II (SfaI and II), produced by extraintestinal Escherichia coli pathogens that cause urinary tract infections (UTI) and newborn meningitis (NBM), respectively, mediate bacterial adherence to sialic acid-containing glycoprotein receptors present on host epithelial cells and extracellular matrix. The S fimbrial adhesin complexes consist of four proteins: SfaI-A, the major subunit protein and the minor subunit proteins SfaI-G, SfaI-S and SfaI-H. Sialic acid-specific binding is mediated by the minor subunit protein SfaI-S. In order to determine whether the minor subunit proteins SfaI-G, -S and -H play a role in the modulation of adherence and the degree of fimbriation, a trans-complementation system was developed. A non-adhesive E. coli K-12 derivative, harbouring the sfaI-A gene but lacking sfaI-G, -S and -H, was transformed with sfaI-G, -S or -H. Only SfaI-S was able to increase the degree of fimbriation and to confer adhesion properties on the recombinant E. coli K-12 strains. Amino acid residues in SfaI-S that are involved in modulation of fimbriation as well as in receptor recognition were localized by random and site-directed mutagenesis.     

Transient expression of β-glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun

Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.     

Elucidating the Population Histories and Transmission Dynamics of Papillomaviruses Using Phylogenetic Trees

Using gene genealogies constructed from gene sequence data, we show that both the mucosal and cutaneous papillomaviruses (PV)—supergroups A and B—appear to have been transmitted through susceptible populations faster than exponentially. The data and methods involved (1) examining the PV database for phylogenetic signal in an L1 open reading frame (ORF) fragment and an E1 ORF segment, (2) demonstrating that the same two fragments have evolved in a way consistent with a molecular clock, and (3) applying methods of phylogenetic tree analysis that test different scenarios for the dynamics of viral transmission within populations. The results indicate increases in PV populations of both supergroups A and B in the recent past. This form of the increases, which fit a null model of population growth with an exponent increasing in time, is compatible with the fact that human populations have grown at a faster than exponential rate, thus increasing the numbers of susceptible hosts for HPVs. There are, however, indications that the population of supergroup A has now stopped increasing in size. Received: 4 June 1996 / Accepted: 12 August 1996     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

An information‐theoretic approach to estimating the composite genetic effects contributing to variation among generation means: Moving beyond the joint‐scaling test for line cross analysis

The pace and direction of evolution in response to selection, drift, and mutation are governed by the genetic architecture that underlies trait variation. Consequently, much of evolutionary theory is predicated on assumptions about whether genes can be considered to act in isolation, or in the context of their genetic background. Evolutionary biologists have disagreed, sometimes heatedly, over which assumptions best describe evolution in nature. Methods for estimating genetic architectures that favor simpler (i.e., additive) models contribute to this debate. Here we address one important source of bias, model selection in line cross analysis (LCA). LCA estimates genetic parameters conditional on the best model chosen from a vast model space using relatively few line means. Current LCA approaches often favor simple models and ignore uncertainty in model choice. To address these issues we introduce Software for Analysis of Genetic Architecture (SAGA), which comprehensively assesses the potential model space, quantifies model selection uncertainty, and uses model weighted averaging to accurately estimate composite genetic effects. Using simulated data and previously published LCA studies, we demonstrate the utility of SAGA to more accurately define the components of complex genetic architectures, and show that traditional approaches have underestimated the importance of epistasis.     

Measuring the influence of unmanned aerial vehicles on Adélie penguins

Unmanned aerial vehicles (UAVs) have become a useful tool in polar research. While their performance is already proven, little is known about their impact on wildlife. To assess the disturbance caused on the penguins, flights with a UAV were conducted over an Adélie penguin (Pygoscelis adeliae) colony. Vertical and horizontal flights were performed between 10 and 50 m in altitude. Penguins’ reactions were video-recorded, and the behavioural response was used to indicate the level of disturbance. During any flight mode, disturbance increased immediately after takeoff and remained elevated at all altitudes between 20 and 50 m. When the UAV descended below 20 m, the disturbance increased further with almost all individuals being vigilant. Only at these low altitudes, vertical flights caused an even higher level of disturbance than horizontal ones. Repetitions of horizontal overflights showed no short-term habituation occurring. Since the results are only valid for the specific UAV model used, we recommend a more extensive approach with different UAV specifications. As the highest flight altitudes already caused detectable but not subjectively visible responses, we also recommend to regard subjective impressions of disturbance with caution.