A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors

A novel sensor chip for use in surface plasmon resonance (SPR) biosensors has been developed to capture vesicles which may contain membrane-bound receptors. Sulforhodamine-containing vesicles were shown by fluorescence microscopy to be immobilized intact on the sensor chip. Binding of cholera toxin to captured vesicles containing ganglioside GM(1) was demonstrated using SPR, and the derived kinetic and affinity constants were similar to literature values. Biotinylated vesicles captured on the sensor chip were used to bind streptavidin and then biotinylated ss-DNA. The hybridization of complementary ss-DNA to the immobilized ss-DNA was then analyzed using SPR. The values obtained were similar to those obtained for an identical interaction analyzed using a commercially available streptavidin-containing sensor chip. Binding of vancomycin-group antibiotics to captured vesicles containing a bacterial cell wall mucopeptide analogue was demonstrated. No binding of the bacterial endotoxin Cry1A(c) to captured vesicles containing its cell surface receptor could be demonstrated.     

Surface roughness parameters as predictors of anchorage strength in bone: a critical analysis

The surface roughness of a bone implant was defined parametrically. The values of the parameters defining the surface were varied. Some traditionally used surface roughness parameters were calculated. By means of a theoretical model the bone-implant interfacial shear strength was estimated. No simple correlation between the values of the surface roughness parameters and the estimated interfacial shear strength was found. It was concluded that the value of the traditional surface roughness parameters as predictors of interfacial shear strength is limited. If however a change of the surface topography of an implant is restricted to scale a positive correlation was found between the theoretical interfacial shear strength and some surface roughness parameters. It is suggested that the bone-implant interfacial shear strength in the general case be estimated by means of strength analyses based upon a study of the size, shape and density of the individual elements constituting the rough surface.     

Arrest of chlorophyll accumulation prior to anthocyanin formation in Euphorbia pulcherrima

Euphorbia pulcherrima Klotz plants exposed to short days (11 h light/13 h dark) for a period of eight weeks, developed flowers and a red canopy consisting of bracts and few true leaves. Plants maintained for three weeks under short day conditions, failed to produce flower primordia or a red canopy in the following 5 weeks in continuous light. Between the 4th and 5th short day week, the youngest leaves began to accumulate anthocyanin and turned red while the apical meristems differentiated into flower primordia. Chlorophyll accumulation ceased at the onset of anthocyanin synthesis and the protein content per unit leaf area declined. mRNA for glutamyl-tRNA^Glu synthetase (EC 6.1.1.17) and glutamyl-tRNA^Glu reductase also declined during this period. Western blot analysis revealed a loss of glutamyl-tRNA^Glu reductase, glutamate 1-semialdehyde (EC 5.4.3.8) and the Mg-chelatase subunits, Olive and CH42, in the last 2 to 4 weeks of the photoperiod.     

Biomanipulation as an Application of Food-Chain Theory: Constraints, Synthesis, and Recommendations for Temperate Lakes

The aim of this review is to identify problems, find general patterns, and extract recommendations for successful biomanipulation. An important conclusion is that the pelagic food chain from fish to algae may not be the only process affected by a biomanipulation. Instead, this process should be viewed as the “trigger” for secondary processes, such as establishment of submerged macrophytes, reduced internal loading of nutrients, and reduced resuspension of particles from the sediment. However, fish reduction also leads to a high recruitment of young-of-the-year (YOY) fish, which feed extensively on zooplankton. This expansion of YOY the first years after fish reduction is probably a major reason for less successful biomanipulations. Recent, large-scale biomanipulations have made it possible to update earlier recommendations regarding when, where, and how biomanipulation should be performed. More applicable recommendations include (1) the reduction in the biomass of planktivorous fish should be 75% or more; (2) the fish reduction should be performed efficiently and rapidly (within 1–3 years); (3) efforts should be made to reduce the number of benthic feeding fish; (4) the recruitment of YOY fish should be reduced; (5) the conditions for establishment of submerged macrophytes should be improved; and (6) the external input of nutrients (phosphorus and nitrogen) should be reduced as much as possible before the biomanipulation. Recent biomanipulations have shown that, correctly performed, the method also achieves results in large, relatively deep and eutrophic lakes, at least in a 5-year perspective. Although repeated measures may be necessary, the general conclusion is that biomanipulation is not only possible, but also a relatively inexpensive and attractive method for management of eutrophic lakes, and in particular as a follow-up measure to reduced nutrient load. Received 14 April 1998; accepted 31 August 1998     

A1M Ameliorates Preeclampsia-Like Symptoms in Placenta and Kidney Induced by Cell-Free Fetal Hemoglobin in Rabbit

Preeclampsia is one of the most serious pregnancy-related diseases and clinically manifests as hypertension and proteinuria after 20 gestational weeks. The worldwide prevalence is 3-8% of pregnancies, making it the most common cause of maternal and fetal morbidity and mortality. Preeclampsia lacks an effective therapy, and the only “cure” is delivery. We have previously shown that increased synthesis and accumulation of cell-free fetal hemoglobin (HbF) in the placenta is important in the pathophysiology of preeclampsia. Extracellular hemoglobin (Hb) and its metabolites induce oxidative stress, which may lead to acute renal failure and vascular dysfunction seen in preeclampsia. The human endogenous protein, α₁-microglobulin (A1M), removes cell-free heme-groups and induces natural tissue repair mechanisms. Exogenously administered A1M has been shown to alleviate the effects of Hb-induced oxidative stress in rat kidneys. Here we attempted to establish an animal model mimicking the human symptoms at stage two of preeclampsia by administering species-specific cell-free HbF starting mid-gestation until term, and evaluated the therapeutic effect of A1M on the induced symptoms. Female pregnant rabbits received HbF infusions i.v. with or without A1M every second day from gestational day 20. The HbF-infused animals developed proteinuria and a significantly increased glomerular sieving coefficient in kidney that was ameliorated by co-administration of A1M. Transmission electron microscopy analysis of kidney and placenta showed both intracellular and extracellular tissue damages after HbF-treatment, while A1M co-administration resulted in a significant reduction of the structural and cellular changes. Neither of the HbF-treated animals displayed any changes in blood pressure during pregnancy. In conclusion, infusion of cell-free HbF in the pregnant rabbits induced tissue damage and organ failure similar to those seen in preeclampsia, and was restored by co-administration of A1M. This study provides preclinical evidence supporting further examination of A1M as a potential new therapy for preeclampsia.     

CD103+CD11b+ Dendritic Cells Induce Th17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.     

Experimental study on possible transmission of the bacterium <Emphasis Type="Italic">Erysipelothrix rhusiopathiae</Emphasis> to chickens by the poultry red mite, <Emphasis Type="Italic">Dermanyssus gallinae</Emphasis>

The vector potential of the poultry red mite, Dermanyssus gallinae De Geer (Acari: Dermanyssidae), in relation to chicken erysipelas was investigated under experimental conditions. Chickens were inoculated intramuscularly with the bacterium Erysipelothrix rhusiopathiae, and mites were allowed to feed on the inoculated chickens for 5 days. After 20 days of starvation, the mites were allowed to feed on healthy chickens to enable transmission of bacteria. Blood samples were collected from the birds and analysed for the presence of E. rhusiopathiae, and ELISA tests were performed for seropositivity. The internal presence of E. rhusiopathiae in the mites after feeding of inoculated birds was also investigated. It could not be demonstrated that mites take up and transmit E. rhusiopathiae under the experimental conditions described. However, since there are case reports as well as other in vitro studies indicating the potential of D. gallinae to act as a reservoir and potential vector for infections agents, we cannot exclude the possibility that the red poultry mite transmits E. rhusiopathiae between chickens under field conditions.     

Induced root-secreted phenolic compounds as a belowground plant defense

Rhizosphere is the complex place of numerous interactions between plant roots, microbes and soil fauna. Whereas plant interactions with aboveground organisms are largely described, unravelling plant belowground interactions remains challenging. Plant root chemical communication can lead to positive interactions with nodulating bacteria, mycorriza or biocontrol agents or to negative interactions with pathogens or root herbivores. A recent study¹ suggested that root exudates contribute to plant pathogen resistance via secretion of antimicrobial compounds. These findings point to the importance of plant root exudates as belowground signalling molecules, particularly in defense responses. In our report,² we showed that under Fusarium attack the barley root system launched secretion of phenolic compounds with antimicrobial activity. The secretion of de novo biosynthesized t-cinnamic acid induced within 2 days illustrates the dynamic of plant defense mechanisms at the root level. We discuss the costs and benefits of induced defense responses in the rhizosphere. We suggest that plant defense through root exudation may be cultivar dependent and higher in wild or less domesticated varieties.Key words: root exudates, plant defense, t-cinnamic acid, fusarium, induced defensePlants grow and live in very complex and changing ecosystems. Because plants lack the mobility to escape from attack by pathogens or herbivores, they have developed constitutive and in addition inducible defenses that are triggered by spatiotemporally dynamic signaling mechanisms. These defenses counteract the aggressor directly via toxins or defense plant structures or indirectly by recruitment of antagonists of aggressors. Whereas induced defenses are well described in aboveground interactions, evidence of the occurrence of such mechanisms in belowground interactions remains limited. The biosynthesis of a defensive molecule could be both constitutive and inducible with a low level of a preformed pool (Fig. 1). In addition, upon encounter of an attacking organism, those levels could be induced to rise locally to a high level of active compound that is able to disarm the pathogen.^2,3 Only a few examples show that root exudates play a role in induced plant defense. Hairy roots of Ocimum basilicum secrete rosmarinic acid only when challenged by the pathogenic fungus Pythium ultimum.⁴ Wurst et al.⁵ reported on the induction of irridoid glycosides in root exudates of Plantago lanceolata in presence of nematodes. In vivo labelling experiments² with ¹³CO₂ showed the induction of phenolic compounds secreted by barley roots after Fusarium graminearum infection and the de novo biosynthesis of root secreted t-cinnamic acid within 2 days. These results show that the pool of induced t-cinnamic acid originated from both pre-formed and newly formed carbon pools (Fig. 1), highlighting a case of belowground induced defense inside and outside the root system.Open in a separate windowFigure 1Suggested mechanisms for the induction of root defense exudates in barley in response to Fusarium attack. Upon pathogen attack by Fusarium, the initial preformed pool of phenolic compounds is increased by the addition of inducible, de novo biosynthesized t-cinnamic acid. Both, the preformed pool and the de novo biosynthesized pool fuel the exudation of defense compounds from infected roots.The concept of fitness costs is frequently presented to explain the coexistence of both constitutive and induced defense.⁶ In the case of induced defense, resources are invested in defenses only when the plant is under attack. In the absence of an infection, plants can optimize allocation of their resources to reproduction and growth to compete with neighbours.⁷ Constitutive defenses are thought to be more beneficial when the probability of attack is high, whereas adjustable, induced defenses are more valuable to fight against an unpredictable pathogen. Non disturbed soil is a heterogeneous matrix where biodiversity is very high and patchy^8,9 and organism motility is rather restricted.¹⁰ As a consequence of the patchiness, belowground environment is expected to be favourable to selection for induced responses.¹¹ The absence of defense root exudates between two infections may form an unpredictable environment for soil pathogens and reduce the chance for adaptation of root attackers. Plants may also use escape strategies to reduce the effect of belowground pathogens. Henkes et al. (unpublished) showed that Fusarium-infected barley plants reduced carbon allocation towards infected roots within a day and increased allocation carbon to uninfected roots. These results illustrate how reallocation of carbon toward non infected root parts represents a way to limit the negative impact of root infection.We have demonstrated the potential of barley plants to defend themselves against soil pathogen by root exudation.² Even the barley cultivar ‘Barke’ used in our study, a modern cultivated variety, was able to launch defense machinery via exudation of antimicrobial compounds when infected by F. graminearum. We suggest that plant defense through root exudation might be cultivar dependent and perhaps higher in wild or less domesticated varieties. Taddei et al.¹² reported that constitutivelyproduced root exudates from a resistant Gladiolus cultivar inhibit spore germination of Fusarium oxysporum whereas root exudates from a susceptible cultivar do not affect F. oxysporum germination. Root exudates from the resistant cultivar contained higher amounts of aromaticphenolic compounds compared to the susceptible cultivar and these compounds may be responsible for the inhibition of spore germination. Metabolic profiling of wheat cultivars, ‘Roblin’ and ‘Sumai3’, respectively, susceptible and resistant to Fusarium Head Blight, showed that t-cinnamic acid was a discriminating factor responsible for resistance/defense function.¹³ Therefore it is likely that wild barley varieties hold higher defense capacities compare to cultivated varieties selected for high yield. In the future, plant breeders in organic and low-input farming could use root-system defense ability as new trait in varietal variation.     

Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice

Objective: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC₁-R, VPAC₂-R and PAC₁-R. Based on beneficial effects of VIP and VPAC₁-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice.Methods and results: Contrary to our hypothesis, administration of a VPAC₁-R agonist, (Ala^11,22,28)-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A^b, and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala^11,22,28)-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease.Conclusion: Despite beneficial effects in murine models of several inflammatory disorders, VPAC₁-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.     

Lost and found: the enigmatic large-billed reed warbler Acrocephalus orinus rediscovered after 139 years

We present compelling evidence of the continued existence of the large-billed reed warbler Acrocephalus orinus , hitherto known only from the unique type specimen collected in NW India 139 years ago. Morphological and genetic analyses of an unusual Acrocephalus warbler mist-netted south-west of Bangkok, Thailand, on 27 March 2006, confirmed its identity as A. orinus , and revealed that it was heterozygous at four out of eight microsatellite markers, indicating the continued existence of a viable population whose breeding and wintering areas are still unknown.