Similar Intracellular Location and Stimulus Reactivity,but Differential Mobility of Tailless (Vicia faba) and Tailed Forisomes (Phaseolus vulgaris) in Intact Sieve Tubes

Sieve elements of legumes contain forisomes—fusiform protein bodies that are responsible for sieve-tube occlusion in response to damage or wound signals. Earlier work described the existence of tailless and tailed forisomes. This study intended to quantify and compare location and position of tailless (in Vicia faba) and tailed (in Phaseolus vulgaris) forisomes inside sieve elements and to assess their reactivity and potential mobility in response to a remote stimulus. Location (distribution within sieve elements) and position (forisome tip contacts) of more than altogether 2000 forisomes were screened in 500 intact plants by laser scanning confocal microscopy in the transmission mode. Furthermore, we studied the dispersion of forisomes at different locations in different positions and their positional behaviour in response to distant heat shocks. Forisome distribution turned out to be species-specific, whereas forisome positions at various locations were largely similar in bushbean (Phaseolus) and broadbean (Vicia). In general, the tailless forisomes had higher dispersion rates in response to heat shocks than the tailed forisomes and forisomes at the downstream (basal) end dispersed more frequently than those at the upstream end (apical). In contrast to the tailless forisomes that only oscillate in response to heat shocks, downstream-located tailed forisomes can cover considerable distances within sieve elements. This displacement was prevented by gentle rubbing of the leaf (priming) before the heat shock. Movement of these forisomes was also prohibited by Latrunculin A, an inhibitor of actin polymerization. The apparently active mobility of tailed forisomes gives credence to the idea that at least the latter forisomes are not free-floating, but connected to other sieve-element structures.     

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.     

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose

Introduction

Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model.

Methods

Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-β1 (TGF-β1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material.

Results

Non-stimulated and especially TGF-β1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 μm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-β1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited.

Conclusions

The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.     

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.     

A tiered approach to estimate inventory data and impacts of chemical products and mixtures

Purpose

Mixtures of organic chemicals are a part of virtually all life cycles, but LCI data exist for only relatively few chemicals. Thus, estimation methods are required. However, these are often either very time-consuming or deliver results of low quality. This article compares existing and new methods in two scenarios and recommends a tiered approach of different methods for an efficient estimation of the production impacts of chemical mixtures.

Methods

Four approaches to estimate impacts of a large number of chemicals are compared in this article: extrapolation from existing data, substitution with generic datasets on chemicals, molecular structure-based models (MSMs, in this case the Finechem tool), and using process-based estimation methods. Two scenarios were analyzed as case studies: soft PVC plastic and a tobacco flavor, a mixture of 20 chemicals.

Results

Process models have the potential to deliver the best estimations, as existing information on production processes can be integrated. However, their estimation quality suffers when such data are not available and they are time-consuming to apply, which is problematic when estimating large numbers of chemicals. Extrapolation from known to unknown components and use of generic datasets are generally not recommended. In both case studies, these two approaches significantly underestimated the impacts of the chemicals compared to the process models. MSMs were generally able to estimate impacts on the same level as the more complex process models. A tiered approach using MSMs to determine the relevance of individual components in mixtures and applying process models to the most relevant components offered a simpler and faster estimation process while delivering results on the level of most process models.

Conclusions

The application of the tiered combination of MSMs and process models allows LCA practitioners a relatively fast and simple estimation of the LCIA results of chemicals, even for mixtures with a large number of components. Such mixtures previously presented a problem, as the application of process models for all components was very time-consuming, while the existing, simple approaches were shown to be inadequate in this study. We recommend the tiered approach as a significant improvement over previous approaches for estimating LCA results of chemical mixtures.     

Sex, not genotype, determines recombination levels in mice

Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.     

Methods for the isolation of water-borne Escherichia coli O157

AIMS: To develop improved methods for the detection of Escherichia coli O157 from water and sediments. METHODS AND RESULTS: The effects of different broth enrichment media (unsupplemented tryptic soya broth, tryptic soya broth with antibiotics, and gram-negative broth), incubation durations (5 and 24 hrs), incubation temperatures (37 and 44.5 degrees C) and the use of immunomagnetic separation (IMS) on the sensitivity of E. coli O157 detection were evaluated on artificially and naturally-contaminated water and sediment samples. The sensitivity of recovery of E. coli O157 from samples was dependent upon the media composition, temperature duration of incubation and the use of IMS. CONCLUSION: Use of high temperature (44.5 degrees C) incubation for 24 hrs in unsupplemented tryptic soya broth and the use of IMS improved the sensitivity of E. coli O157 culture from water and sediment samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods described can be used to increase the sensitivity of E. coli O157 detection from water and sediments.