Retention of xenobiotics along the phloem path

Detached leaves of Cyclamen persicum Mill. can be used as a simple source-sink system. Phloem transport in the excised material was monitored by the noninvasive ¹¹C-technique. Assimilate movement stopped immediately when the petiole was cut off. However, within 20 min a recovery of transport was observed. The translocation rate in the detached leaf was only 13% of that in the intact plant. ¹⁴C-Xenobiotics and [³H]sucrose were injected into the upper petiole parenchyma (source). They moved downstream by a symplastic route. The stump of the petiole was inserted into a buffer solution containing ethylenediaminetetraacetic acid (sink). After 3 h, the distribution of sucrose and xenobiotics was determined in five subsequent segments of the petiole (path). The retention coefficient (r) was calculated from the ratio of radioactivity in the vascular bundle to that in the petiole parenchyma. The distribution along the vascular path was given by a geometric progression, whereas its constant was the transport coefficient (q). Values of r and q corresponded with the degree of phloem mobility and ambimobility. Four groups of compounds were classified: (i) acidic substances with log K_ow = — 2 to — 2.4 (K_ow is the partition coefficient octanol/water) at pH 8 (pH of sieve tube sap), retained by ion trapping and exhibiting small lateral efflux (q0.7; maleic hydrazide, dalapon); (ii) acidic substances with log K_ow = — 0.7 to — 0.8 at pH 8, retained by ion trapping and subjected to a moderate lateral efflux (0.7>q> 0.5; 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, bromoxynil); (iii) nonionised substances retained by optimum permeability, exhibiting a considerable lateral leakage (q<0.5; glyphosate, amitrole); (iv) substances without basipetal transport in the phloem (atrazine, diuron). Retention of sucrose corresponded quantitatively with that shown in group (i). This classification was also supported by results of uptake and efflux experiments using the isolated conducting tissue. Theoretical translocation profiles were calculated from the determined transport coefficients (q).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - K_ow partition coefficient octanol/water - MCPA 2-methyl-4-chloro-phenoxyacetic acid - q transport coefficient in the vascular bundle - r retention coefficient in the vascular bundle The authors gratefully acknowledge the assistance of H. Fiedler and M. Neugebauer. We are particularly grateful to K. Dutschka, G. Hudepokl, and Dr. J. Knust for producing ¹¹CO₂.     

Climate–ecosystem modelling made easy: The Land Sites Platform

Dynamic Global Vegetation Models (DGVMs) provide a state-of-the-art process-based approach to study the complex interplay between vegetation and its physical environment. For example, they help to predict how terrestrial plants interact with climate, soils, disturbance and competition for resources. We argue that there is untapped potential for the use of DGVMs in ecological and ecophysiological research. One fundamental barrier to realize this potential is that many researchers with relevant expertize (ecology, plant physiology, soil science, etc.) lack access to the technical resources or awareness of the research potential of DGVMs. Here we present the Land Sites Platform (LSP): new software that facilitates single-site simulations with the Functionally Assembled Terrestrial Ecosystem Simulator, an advanced DGVM coupled with the Community Land Model. The LSP includes a Graphical User Interface and an Application Programming Interface, which improve the user experience and lower the technical thresholds for installing these model architectures and setting up model experiments. The software is distributed via version-controlled containers; researchers and students can run simulations directly on their personal computers or servers, with relatively low hardware requirements, and on different operating systems. Version 1.0 of the LSP supports site-level simulations. We provide input data for 20 established geo-ecological observation sites in Norway and workflows to add generic sites from public global datasets. The LSP makes standard model experiments with default data easily achievable (e.g., for educational or introductory purposes) while retaining flexibility for more advanced scientific uses. We further provide tools to visualize the model input and output, including simple examples to relate predictions to local observations. The LSP improves access to land surface and DGVM modelling as a building block of community cyberinfrastructure that may inspire new avenues for mechanistic ecosystem research across disciplines.     

Label-free LC-MSMS analysis of vitreous from autoimmune uveitis reveals a significant decrease in secreted Wnt signalling inhibitors DKK3 and SFRP2

Equine recurrent uveitis is a severe and frequent blinding disease in horses which presents with auto-reactive invading T-cells, resulting in the destruction of the inner eye. Infiltration of inflammatory cells into the retina and vitreous is driven by currently unknown guidance cues, however surgical removal of the vitreous (vitrectomy) has proven therapeutically successful. Therefore, proteomic analyses of vitrectomy samples are likely to result in detection of proteins contributing to disease pathogenesis. Vitreous from healthy and ERU diseased horses were directly compared by quantitative mass spectrometry based on label-free quantification of peak intensities across samples. We found a significant upregulation of complement and coagulation cascades and downregulation of negative paracrine regulators of canonical Wnt signalling including the Wnt signalling inhibitors DKK3 and SFRP2. Based on immunohistochemistry, both proteins are expressed in equine retina and suggest localisation to retinal Müller glial cells (RMG), which may be the source cells for these proteins. Furthermore, retinal expression levels and patterns of DKK3 change in response to ERU. Since many other regulated proteins identified here are associated with RMG cells, these cells qualify as the prime responders to autoimmune triggers.     

Radioimmunoimaging of Liver Metastases with PET Using a 64Cu-Labeled CEA Antibody in Transgenic Mice

Purpose

Colorectal cancer is one of the most common forms of cancer, and the development of novel tools for detection and efficient treatment of metastases is needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (PET) imaging and radioimmunotherapy. Since carcinoembryonic antigen (CEA) is an important target in colorectal cancer, the CEA-specific M5A antibody has been extensively studied in subcutaneous xenograft models; however, the M5A antibody has not yet been tested in advanced models of liver metastases. The aim of this study was to investigate the ⁶⁴Cu-DOTA-labeled M5A antibody using PET in mice bearing CEA-positive liver metastases.

Procedures

Mice were injected intrasplenically with CEA-positive C15A.3 or CEA-negative MC38 cells and underwent micro-computed tomography (micro-CT) to monitor the development of liver metastases. After metastases were detected, PET/MRI scans were performed with ⁶⁴Cu-DOTA-labeled M5A antibodies. H&E staining, immunohistology, and autoradiography were performed to confirm the micro-CT and PET/MRI findings.

Results

PET/MRI showed that M5A uptake was highest in CEA-positive metastases. The %ID/cm³ (16.5%±6.3%) was significantly increased compared to healthy liver tissue (8.6%±0.9%) and to CEA-negative metastases (5.5%±0.6%). The tumor-to-liver ratio of C15A.3 metastases and healthy liver tissue was 1.9±0.7. Autoradiography and immunostaining confirmed the micro-CT and PET/MRI findings.

Conclusion

We show here that the ⁶⁴Cu-DOTA-labeled M5A antibody imaged by PET can detect CEA positive liver metastases and is therefore a potential tool for staging cancer, stratifying the patients or radioimmunotherapy.     

Diversity of thermophilic and non-thermophilic Crenarchaeota at 80 degrees C

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.     

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.     

The agony of choice: Impact of the host animal species on the enzyme-linked immunosorbent assay performance for host cell protein quantification

Host cell proteins (HCPs) are inevitable process-related impurities in biotherapeutics commonly monitored by enzyme-linked immunosorbent assays (ELISAs). Of particular importance for their reliable detection are the anti-HCP polyclonal antibodies (pAbs), supposed to detect a broad range of HCPs. The present study focuses on the identification of suitable host animal species for the development of high-performance CHO-HCP ELISAs, assuming the generation of pAbs with adequate coverage and specificity. Hence, antibodies derived from immunization of sheep, goats, donkeys, rabbits, and chickens were compared concerning their amount of HCP-specific antibodies, coverage, and performance in a sandwich ELISA. Immunization of sheep, goats, donkeys, and rabbits met all test criteria, whereas the antibodies from chickens cannot be recommended based on the results of this study. Additionally, a mixture of antibodies from the five host species was prepared to assess if coverage and ELISA performance can be improved by a multispecies approach. Comparable results were obtained for the single- and multispecies ELISAs in different in-process samples, indicating no substantial improvement for the latter in ELISA performance while raising ethical and financial concerns.     

Biochemical properties of a novel cell wall protein associated with elongation growth in higher plants

A monoclonal antibody of IgM-type (TIM-11B2) was screened froma hybridoma library. The antibody recognizes a 40 kDa glycoprotein,p40, with high specificity. This protein was detected in allplant species examined so far and was found to be located bothsolubly and ionically-bound within the primary cell wall. The strongest immunobiochemical signals of p40 were found intissues undergoing elongation growth, whereas in other tissuesonly a faint signal could be detected. Those included the non-elongatingparts of different seedlings, such as the apical part of monocotprimary leaves or the leaves of dicots grown in light. Inhibitionof pea epicotyl growth by white light irradiation resulted ina strong decrease of the immunostain signal. On the other hand,induction of rapid coleoptile growth in rice seedlings inducedby submergence resulted in a strong increase of the immunobiochemicalsignal of p40. Time-course studies on the expression of p40during protoplast regeneration revealed that p40 is apparentlynot involved in cell wall formation. The hypothesis that p40is characteristic for tissues with the ability for elongationgrowth is discussed. Comparison of biochemical data and location of p40 with proteinsdescribed up to now indicate that this glycoprotein has notbeen characterized before. Key words: Cell wall protein, elongation growth, monoclonal antibody     

Epidermal RANKL controls regulatory T-cell numbers via activation of dendritic cells

Regulatory CD4(+)CD25(+) T cells are important in suppressing immune responses. The requirements for the maintenance of peripheral CD4(+)CD25(+) T cells remain incompletely understood. Receptor activator of NF-kappaB (RANK) and its ligand (RANKL; also known as CD254, OPGL and TRANCE) are key regulators of bone remodeling, mammary gland formation, lymph node development and T-cell/dendritic cell communication. Here we report that RANKL is expressed in keratinocytes of the inflamed skin. RANKL overexpression in keratinocytes resulted in functional alterations of epidermal dendritic cells and systemic increases of regulatory CD4(+)CD25(+) T cells. Thus, epidermal RANKL expression can change dendritic cell functions to maintain the number of peripheral CD4(+)CD25(+) regulatory T cells. Epidermal RANKL mediated ultraviolet-induced immunosuppression and overexpression of epidermal RANKL suppressed allergic contact hypersensitivity responses and the development of systemic autoimmunity. Therefore, environmental stimuli at the skin can rewire the local and systemic immune system by means of RANKL.