Refilling of a hydraulically isolated embolized xylem vessel: model calculations

When they are hydraulically isolated, embolized xylem vessels can be refilled, while adjacent vessels remain under tension. This implies that the pressure of water in the refilling vessel must be equal to the bubble gas pressure, which sets physical constraints for recovery. A model of water exudation into the cylindrical vessel and of bubble dissolution based on the assumption of hydraulic isolation is developed. Refilling is made possible by the turgor of the living cells adjacent to the refilling vessel, and by a reflection coefficient below 1 for the exchange of solutes across the interface between the vessel and the adjacent cells. No active transport of solutes is assumed. Living cells are also capable of importing water from the water-conducting vessels. The most limiting factors were found to be the osmotic potential of living cells and the ratio of the volume of the adjacent living cells to that of the embolized vessel. With values for these of 1.5 MPa and 1, respectively, refilling times were in the order of hours for a broad range of possible values of water conductivity coefficients and effective diffusion distances for dissolved air, when the xylem water tension was below 0.6 MPa and constant. Inclusion of the daily pattern for xylem tension improved the simulations. The simulated gas pressure within the refilling vessel was in accordance with recent experimental results. The study shows that the refilling process is physically possible under hydraulic isolation, while water in surrounding vessels is under negative pressure. However, the osmotic potentials in the refilling vessel tend to be large (in the order of 1 MPa). Only if the xylem water tension is, at most, twice atmospheric pressure, the reflection coefficient remains close to 1 (0.95) and the ratio of the volume of the adjacent living cells to that of the embolized vessel is about 2, does the osmotic potential stay below 0.4 MPa.     

Integrin alpha v beta 6 is an RGD-dependent receptor for coxsackievirus A9

Coxsackievirus A9 (CAV9), a member of the Enterovirus genus of Picornaviridae, is a common human pathogen and is one of a significant number of viruses containing a functional arginine-glycine-aspartic acid (RGD) motif in one of their capsid proteins. Previous studies identified the RGD-recognizing integrin alpha(v)beta(3) as its cellular receptor. However, integrin alpha(v)beta(6) has been shown to be an efficient receptor for another RGD-containing picornavirus, foot-and-mouth disease virus (FMDV). In view of the similarity in sequence context of the RGD motifs in CAV9 and FMDV, we investigated whether alpha(v)beta(6) can also serve as a receptor for CAV9. We found that CAV9 can bind to purified alpha(v)beta(6) and also to SW480 cells transfected with beta(6) cDNA, allowing expression of alpha(v)beta(6) on their surface, but it cannot bind to mock-transfected cells. In addition, a higher yield of CAV9 was obtained in beta(6)-expressing cells than in mock-transfected cells. There was no similar enhancement in infection with an RGD-less CAV9 mutant. We also found beta(6) on the surface of GMK cells, a cell line which CAV9 infects efficiently by an RGD-dependent mechanism. Significantly, this infection is blocked by an antibody to alpha(v)beta(6), while this antibody did not block the low level of infection by the RGD-less mutant. Thus, integrin alpha(v)beta(6) is an RGD-dependent receptor for CAV9 and may be important in natural CAV9 infections.     

Training for reduction in laboratory animal use

Reduction, refinement and replacement of laboratory animals wherever possible, are the guiding principles of the Federation of European Laboratory Animal Science Associations (FELASA) education guidelines. Of these, reduction is probably the least understood. Reduction and refinement are dependent upon each other. In the reduction "axis", there is a window of appropriate numbers of animals; too few and the experiment will lack power--too many and animals will be wasted. Reduction must be understood as the appropriate number of animals required for each experiment. The refinement "axis" is more straightforward. Better welfare is always desirable. Any factor can interfere with a study in two ways. If it changes the mean, this may not be serious, because it should do so in all groups. If it causes a change in variation, then this is far more troublesome, because this is bound to alter the appropriate number of animals for an experiment. Scientists are definitely concerned about the variation of the characters that they are working with. It is obvious that changes in variation may be study-specific, which makes the formulation of overall guidelines difficult. Indeed, instead of trying to assess the impact of housing and procedures on every possible character, it could be more productive to look at the effects of welfare indicators on variation, with the understanding that low variation here is likely to be reflected by low variation in most other characters, while aiming to achieve the most uniform welfare of the animals in the study.     

Sliding window discretization: A new method for multiple band matching of bacterial genotyping fingerprints

Microbiologists have traditionally applied hierarchical clustering algorithms as their mathematical tool of choice to unravel the taxonomic relationships between micro-organisms. However, the interpretation of such hierarchical classifications suffers from being subjective, in that a variety of ad hoc choices must be made during their construction. On the other hand, the application of more profound and objective mathematical methods—such as the minimization of stochastic complexity—for the classification of bacterial genotyping fingerprints data is hampered by the prerequisite that such methods only act upon vectorized data. In this paper we introduce a new method, coined sliding window discretization, for the transformation of genotypic fingerprint patterns into binary vector format. In the context of an extensive amplified fragment length polymorphism (AFLP) data set of 507 strains from the Vibrionaceae family that has previously been analysed, we demonstrate by comparison with a number of other discretization methods that this new discretization method results in minimal loss of the original information content captured in the banding patterns. Finally, we investigate the implications of the different discretization methods on the classification of bacterial genotyping fingerprints by minimization of stochastic complexity, as it is implemented in the BinClass software package for probabilistic clustering of binary vectors. The new taxonomic insights learned from the resulting classification of the AFLP patterns will prove the value of combining sliding window discretization with minimization of stochastic complexity, as an alternative classification algorithm for bacterial genotyping fingerprints.     

Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.     

Experimental Helicobacter felis infection in transgenic mice expressing human group IIA phospholipase A2

BACKGROUND: Both various virulence factors of Helicobacter pylori and host factors influence the clinical outcome of H. pylori infection. In animal experiments with Helicobacter felis, large variations in the severity of disease have been observed between different mouse strains infected with a single isolate of H. felis. C57BL/6 J mouse strain that lacks the expression of group IIA phospholipase A2 has been shown to develop more severe gastric inflammation than other mouse strains. Thus, group IIA phospholipase A2 has been suggested to play a role in regulating inflammation in gastric mucosa. The aim of this study was to examine the possible role of group IIA phospholipase A2 in experimental Helicobacter infection. MATERIALS AND METHODS: Transgenic mice expressing human group IIA phospholipase A2 and their group IIA phospholipase A2 deficient nontransgenic C57BL/6 J littermates were infected with H. felis. The mice were killed 3, 8, and 19 weeks after inoculation of bacteria to determine the histopathological changes in gastric mucosa. RESULTS: The infected mice developed chronic inflammation in gastric mucosa. We found no differences in the colonization of bacteria between transgenic and nontransgenic mice. At 3 and 8 weeks, no difference was found in the severity of inflammation between the two groups. Nineteen weeks after the administration of bacteria the inflammation was more marked in nontransgenic than transgenic mice. Group IIA phospholipase A2 was expressed by in situ hybridization in the neck cells of the glandular stomach in transgenic mice. CONCLUSIONS: The results of the present study suggest that the endogenous expression of group IIA phospholipase A2 diminishes chronic inflammation in gastric mucosa in experimental H. felis infection in mice.     

Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A

The macrophage scavenger receptor macrophage receptor with a collagenous structure (MARCO) is expressed in mice by the marginal zone macrophages of the spleen and by macrophages of the medullary cords of lymph nodes, as well as the peritoneal macrophages. MARCO is a relative of scavenger receptor A (SR-A), the more widely expressed prototypic member of the scavenger receptor family. In the present study, we found that genetic ablation of MARCO leads to changes in the organization of the splenic marginal zone, and causes a significant reduction in the size of the resident peritoneal macrophage population, possibly due to changes in adhesion and migration capacity. In mice lacking both MARCO and SR-A these effects are even more apparent. During ontogeny, the appearance and organization of the MARCO-expressing cells in the spleen precedes the appearance of other receptors on macrophages in the marginal zone, such as SIGNR1 and Siglec-1. In the absence of MARCO, a clear delay in the organization of the marginal zone was observed. Similar findings were seen when the reappearance of the various subsets from precursors was studied after depleting macrophages from the adult spleen by a liposome treatment. When challenged with a pneumococcal polysaccharide vaccine, a T-independent type 2 Ag for which an intact marginal zone is crucial, the knockout mice exhibited a clearly impaired response. These findings suggest that both MARCO and SR-A, in addition to being important scavenger receptors, could be involved in the positioning and differentiation of macrophages, possibly through interaction with endogenous ligands.     

The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.     

Plant-mediated nitrous oxide emissions from beech (Fagus sylvatica) leaves

Nitrous oxide (N2O) emission estimates from forest ecosystems are based currently on emission measurements using soil enclosures. Such enclosures exclude emissions via tall plants and trees and may therefore underestimate the whole-ecosystem N2O emissions. Here, we measured plant-mediated N2O emissions from the leaves of potted beech (Fagus sylvatica) seedlings after fertilizing the soil with 15N-labelled ammonium nitrate (15NH4(15)NO3), and after exposing the roots to elevated concentrations of N2O. Ammonium nitrate fertilization induced N2O + 15N2O emissions from beech leaves. Likewise, the foliage emitted N2O after beech roots were exposed to elevated concentrations of N2O. The average N2O emissions from the fertilization and the root exposure experiments were 0.4 and 2.0 microg N m(-2) leaf area h(-1), respectively. Higher than ambient atmospheric concentrations of N2O in the leaves of the forest trees indicate a potential for canopy N2O emissions in the forest. Our experiments demonstrate the existence of a previously overlooked pathway of N2O to the atmosphere in forest ecosystems, and bring about a need to investigate the magnitude of this phenomenon at larger scales.