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Fungi often adapt to environmental stress by altering their size, shape, or rate of cell division. These morphological changes require reorganization of the cell wall, a structural feature external to the cell membrane composed of highly interconnected polysaccharides and glycoproteins. Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that are typically secreted into the extracellular space to catalyze initial oxidative steps in the degradation of complex biopolymers such as chitin and cellulose. However, their roles in modifying endogenous microbial carbohydrates are poorly characterized. The CEL1 gene in the human fungal pathogen Cryptococcus neoformans (Cn) is predicted by sequence homology to encode an LPMO of the AA9 enzyme family. The CEL1 gene is induced by host physiological pH and temperature, and it is primarily localized to the fungal cell wall. Targeted mutation of the CEL1 gene revealed that it is required for the expression of stress response phenotypes, including thermotolerance, cell wall integrity, and efficient cell cycle progression. Accordingly, a cel1Δ deletion mutant was avirulent in two models of C. neoformans infection. Therefore, in contrast to LPMO activity in other microorganisms that primarily targets exogenous polysaccharides, these data suggest that CnCel1 promotes intrinsic fungal cell wall remodeling events required for efficient adaptation to the host environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Probst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The European hedgehog is in decline, triggering a need to monitor population dynamics to optimise conservation initiatives directed at this species. By counting periosteal growth lines, we determined the age of 388 dead European hedgehogs collected through citizen science in Denmark. The overall mean age was 1.8 years (1.6 years for females and 2.1 years for males), ranging between 0 and 16 years. We constructed life tables showing life expectancies at 2.1 years for females and 2.6 years for males. We discovered that male hedgehogs were more likely to have died in traffic than females, but traffic-related deaths peaked in July for both sexes. A sex difference was detected for non-traffic deaths, as most males died in July, and most females died in September. We created empirical survivorship curves and hazard curves showing that the risk of death for male hedgehogs remains approximately constant with age. In contrast, the risk of death for females increases with age. Most of the collected road-killed individuals died in rural habitats. The degree of inbreeding did not influence longevity. These new insights are important for preparing conservation strategies for the European hedgehog.

Hypothesis: Surface modification of lactic acid bacteria enhances their adsorption and aggregation at air-water interface and enables stabilization of microbubbles that spontaneously transform into water-filled colloidosomes, which can be further modified using LBL formulations., Experiments: The bacterial physicochemical properties were characterized using water contact angle (WCA) measurement, bacterial aggregation assay and zeta potential measurement. Cell viability was enumerated using plate-counting method. The LBL reinforcement of colloidosomes was examined by zeta potential measurement and the formed microstructure was investigated using bright-field microscopy, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Shell permeability of colloidosomes was evaluated using a dye release study., Findings: Bacteria surface-modified using octenyl succinic anhydride (OSA) expressed strong adsorption and aggregation at air-water interface when producing microbubbles. Bacteria with enhanced aggregation ability formed stable shells, enabling complete removal of air and air-water interface without shell disintegration. The formed colloidosomes were studied as they were, or were further reinforced by LBL deposition using polymer or hybrid formulations. Hybrid coating involved assembly of two bacterial species producing colloidosomes with low shell porosity. The findings can be exploited to organize different living bacteria into structured materials and to encapsulate and release substances of diverse sizes and surface properties., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Palm kernel cake (PKC) is an abundant side stream that can only be added to non-ruminant feed in small concentrations due to its content of antinutritional factors, mainly galactomannan, which cannot be digested by non-ruminants. β-mannanases can be added to partially hydrolyze galactomannan to form mannose oligosaccharides, which are known to be prebiotic. We here investigate the action of a β-mannanase from B. subtilis on PKC by colorimetry, NMR and fluorescence microscopy. The amount of mannan oligosaccharides in solution was significantly increased by the β-mannanase and their degree of polymerization (DP) was significantly reduced. There was a dose-response behavior in that larger β-mannanase concentrations increased the amount of soluble mannose oligosaccharides while reducing their average DP. Using confocal immunofluorescence microscopy, solubilization of galactomannan in PKC was clearly visualized. Images show a clear disruption of the cellulose and galactomannan structures of the PKC cell walls. We thus show in this study that using commercial dosages of β-mannanase on PKC can lead to formation of prebiotic compounds. Thus, this study suggests that utilization of PKC in poultry feed formulation might be increased by addition of a β-mannanase and would improve the return on investment.

Fresh roselle are high in moisture and deteriorate easily, which makes drying important for extending shelf-life and increasing availability. This study investigated the influence of different drying methods (oven-drying, freeze-drying, vacuum-drying, and sun-drying) on the quality of roselle calyx expressed as physicochemical properties (moisture content, water activity, soluble solids, color), volatile compounds, and microstructure. Oven-drying and freeze-drying reduced moisture content most while vacuum-drying and sun-drying were not as efficient. All drying methods except sun-drying resulted in water activities low enough to ensure safety and quality. Vacuum-drying had no impact on color of the dry calyx and only small impact on color of water extract of calyx. Drying reduced terpenes, aldehydes, and esters but increased furans. This is expected to reduce fruity, floral, spicy, and green odors and increase caramel-like aroma. Sun-drying produced more ketones, alcohols, and esters. Scanning electron microscopy revealed that freeze-drying preserved the cell structure better, and freeze-dried samples resembled fresh samples most compared to other drying techniques. The study concludes that freeze-drying should be considered as a suitable drying method, especially with respect to preservation of structure.

A Danish strain of the green microalgae Scenedesmus sp. was isolated, identified and characterized with respect to productivity under outdoor cultivation conditions at northern latitudes. The algae were cultivated outdoors in Denmark in closed tubular photobioreactors using only sunlight, simple inorganic nutrients and under ambient temperatures. The biomass composition was evaluated in terms of protein content and quality. The average volumetric and areal biomass productivity obtained for the Scenedesmus sp. isolate during outdoor cultivation was 0.083 g dry matter L -1 and 6.40 g dm m -2  day -1 , respectively. Thus, productivities are comparable to data reported in the literature under similar conditions. A strain-specific nitrogen to protein conversion factor of 5.5 was determined for the Scenedesmus sp. strain enabling more accurate protein estimations from simple nitrogen determination methods like Kjeldahl analysis in the future. The protein content was determined to be 52.4% of dried biomass for this Scenedesmus strain. The sum of essential amino acids was 42% which is high compared to other microalgae. The results are compared and discussed in comparison to other microalgae and soybean as a common plant protein source., (© 2021 Scandinavian Plant Physiology Society.)

Pectin is a major cell wall component that plays important roles in plant development and response to environmental stresses. Arabidopsis thaliana plants expressing a fungal polygalacturonase (PG plants) that degrades homogalacturonan (HG), a major pectin component, as well as loss-of-function mutants for QUASIMODO2 ( QUA2 ), encoding a putative pectin methyltransferase important for HG biosynthesis, show accumulation of reactive oxygen species (ROS), reduced growth and almost complete resistance to the fungal pathogen Botrytis cinerea . Both PG and qua2 plants show increased expression of the class III peroxidase AtPRX71 that contributes to their elevated ROS levels and reduced growth. In this work, we show that leaves of PG and qua2 plants display greatly increased cuticle permeability. Both increased cuticle permeability and resistance to B. cinerea in qua2 are suppressed by loss of AtPRX71 . Increased cuticle permeability in qua2 , rather than on defects in cuticle ultrastructure or cutin composition, appears to be dependent on reduced epidermal cell adhesion, which is exacerbated by AtPRX71 , and is suppressed by the esmeralda1 mutation, which also reverts the adhesion defect and the resistant phenotype. Increased cuticle permeability, accumulation of ROS, and resistance to B. cinerea are also observed in mutants lacking a functional FERONIA, a receptor-like kinase thought to monitor pectin integrity. In contrast, mutants with defects in other structural components of primary cell wall do not have a defective cuticle and are normally susceptible to the fungus. Our results suggest that disrupted cuticle integrity, mediated by peroxidase-dependent ROS accumulation, plays a major role in the robust resistance to B. cinerea of plants with altered HG integrity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lorrai, Francocci, Gully, Martens, De Lorenzo, Nawrath and Ferrari.)

The percentage of respiratory and photorespiratory CO2 refixed in leaves (Pr) represents part of the CO2 used in photosynthesis. The importance of Pr as well as differences between species and functional types are still not well investigated. In this study, we examine how Pr differs between six temperate and boreal woody species: Betula pendula, Quercus robur, Larix decidua, Pinus sylvestris, Picea abies and Vaccinium vitis-idaea. The study covers early and late successional species, deciduous broadleaves, deciduous conifers, evergreen conifers and evergreen broadleaves. We investigated whether some species or functional types had higher refixation percentages than others, whether leaf traits could predict higher Pr and whether these traits and their impact on Pr changed during growing seasons. Photosynthesis CO2 response (A/Ci)-curves, measured early, mid and late season, were used to estimate and compare Pr, mesophyll resistance (rm) and stomatal resistance (rs) to CO2 diffusion. Additionally, light images and transmission electron microscope images were used to approximate the fraction of intercellular airspace and cell wall thickness. We found that evergreens, especially late successional species, refixed a significantly higher amount of CO2 than the other species throughout the entire growing season. In addition, rm, rs and leaf mass per area, traits that typically are higher in evergreen species, were also significantly, positively correlated with Pr. We suggest that this is due to higher rm decreasing diffusion of (photo) respiratory CO2 out of the leaf. Cell wall thickness had a positive effect on Pr and rm, while the fraction of intercellular airspace had no effect. Both were significantly different between evergreen conifers and other types. Our findings suggest that species with a higher rm use a greater fraction of mitochondria-derived CO2, especially when stomatal conductance is low. This should be taken into account when modeling the overall CO2 fertilization effect for terrestrial ecosystems dominated by high rm species., (© The Author(s) 2021. Published by Oxford University Press.)

Global demand for phosphorus (P) requires new agronomic practices to address sustainability challenges while increasing food production. Foliar P fertilization could increase P use efficiency; however, leaf entry pathways for inorganic phosphate ion (Pi) uptake remain unknown, and it is unclear whether foliar P applications can meet plant nutrient demands. We developed two techniques to trace foliar P uptake in P-deficient spring barley ( Hordeum vulgare ) and to monitor the effectiveness of the treatment on restoring P functionality. First, a whole-leaf P status assay was developed using an IMAGING PAM system; nonphotochemical quenching was a proxy for P status, as P-deficient barley developed nonphotochemical quenching at a faster rate than P-sufficient barley. The assay showed restoration of P functionality in P-deficient plants 24 h after foliar P application. Treated leaves reverted to P deficiency after 7 d, while newly emerging leaves exhibited partial restoration compared with untreated P-deficient plants, indicating Pi remobilization. Second, vanadate was tested as a possible foliar Pi tracer using high-resolution laser ablation-inductively coupled plasma-mass spectrometry elemental mapping. The strong colocalization of vanadium and P signal intensities demonstrated that vanadate was a sensitive and useful Pi tracer. Vanadate and Pi uptake predominantly occurred via fiber cells located above leaf veins, with pathways to the vascular tissue possibly facilitated by the bundle sheath extension. Minor indications of stomatal and cuticular Pi uptake were also observed. These techniques provided an approach to understand how Pi crosses the leaf surface and assimilates to meet plant nutrient demands., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

A novel bacterial strain, S40 T , with strong antifungal activity was isolated from the rhizosphere of green potato collected from Zealand, Denmark. Polyphasic analysis with a combined phenotypic, phylogenetic and genomic approach was used to characterize S40 T . Phylogenetic analysis based on the 16S rRNA gene and MLSA (concatenated gyrB , rpoD , infB and atpD sequences) showed that strain S40 T was affiliated with the genus Serratia and with Serratia plymuthica PRI-2C as the closest related strain [average nucleotide identity (ANI), 99.26 %; DNA-DNA hybridization (dDDH), 99.20%]. However, whole genome sequence analyses revealed that S40 T and S. plymuthica PRI-2C genomes displayed lower similarities when compared to all other S. plymuthica strains (ANI ≤94.34 %; dDDH ≤57.6 % relatedness). The DNA G+C content of strain S40 T was determined to be 55.9 mol%. Cells of the strain were Gram-negative, rod-shaped, facultative anaerobic and displayed growth at 10-37 °C (optimum, 25-30 °C) and at pH 6-9 (optimum, pH 6-7). Major fatty acids were C 16 : 0 (27.9 %), summed feature (C 16 : 1  ω 6 c /C 16 : 1  ω7 c ; 18.0 %) and C 17 : 0 cyclo (15.1 %). The respiratory quinone was determined to be Q8 (94 %) and MK8 (95 %) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The results of phenotypic, phylogenetic and genomic analyses support the hypothesis that strain S40 T represents a novel species of the genus Serratia , for which the name Serratia inhibens sp. nov. is proposed. The type strain is S40 T (=LMG 31467 T =NCIMB 15235 T ). In addition, we propose that S. plymuthica PRI-2C is reclassified and transferred to the species S. inhibens as S. inhibens PRI-2C.

The plant hormone auxin serves as central regulator of growth and development. Auxin transporters in the plasma membrane are assumed to define tissue-level patterns of auxin distribution [1, 2]. However, auxin is small enough to diffuse through the plasmodesmata that connect neighboring cells [3], presenting an alternative pathway, whose contribution to auxin transport remained largely unexplored [4]. Here, photoactivation microscopy [5, 6] was used to measure the capacity for small-molecule diffusion in the epidermis of Arabidopsis thaliana leaves. In the elongated epidermis cells covering the midrib and petiole, the plasmodesmata-mediated cell-wall permeability was found to be several times higher in the longitudinal than in the transverse direction. The physiological relevance of this asymmetry was tested through quantification of the shade-avoidance response, which depends on auxin transport from the leaf tip to the petiole in the abaxial side of the leaf [7], with the hypothesis that directionality of diffusion supplements transporter-mediated auxin movement [8]. Triggering the response by auxin application at the tip led to stronger leaf movement in wild-type plants than in gsl8 mutants [9], which lack the callose synthase necessary to establish directionality. The results match the predictions of a mathematical model of auxin transport based on the permeabilities measured in wild-type and mutant plants. It is concluded that plasmodesmata permeability can be selectively modulated within a plant cell and that the conferred directionality in diffusion can influence the tissue-specific distribution patterns of small molecules, like auxin. VIDEO ABSTRACT., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Shell-loosening is of importance in facilitating shrimp peeling. In this study, enzyme and high pressure (HP) improved the shell-loosening at different degrees, which were observed as gaps by microscopy. The shell-loosening gap induced by an endoprotease with broad specificity (Endocut-03L, 53 μm) was much higher than that induced by HP at 100 MPa (HP100, 12 μm), followed by an endoprotease with high specificity (Tail21, 8 μm), and HP at 600 MPa (HP600, 5 μm). The degree of shell-loosening was found to be correlated to the extent of protein changes that were obtained by 2D gel electrophoresis. Shell-loosening due to HP100 and Endocut-03L was mainly caused by physical and enzymatic degradation of high molecular-weight proteins in shell and epidermis and subsequent loss of degradation products, disrupting the structure of muscle-shell connection. However, HP100 was less effective than Endocut-03L due to its stabilizing effect on the shell collagen, lowering its shell-loosening effect., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in severe ER stress. However, the function of these ubiquitous membrane proteins, which belong to the P-type ATPase superfamily, is unknown. We purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and observed that the ATP hydrolytic activity of the protein is stimulated by phosphatidylinositol 4-phosphate (PI4P). Furthermore, SPF1 exhibited negative genetic interactions with SAC1, encoding a PI4P phosphatase, and with OSH1 to OSH6, encoding Osh proteins, which, when energized by a PI4P gradient, drive export of sterols and lipids from the ER. Deletion of SPF1 resulted in increased sensitivity to inhibitors of sterol production, a marked change in the ergosterol/lanosterol ratio, accumulation of sterols in the plasma membrane, and cytosolic accumulation of lipid bodies. We propose that Spf1p maintains cellular sterol homeostasis by influencing the PI4P-induced and Osh-mediated export of sterols from the ER.

The export of photosynthetically produced sugars from leaves depends on plasmodesmatal transport of sugar molecules from mesophyll to phloem. Traditionally, the density of plasmodesmata (PD) along this phloem-loading pathway has been used as a defining feature of different phloem-loading types, with species proposed to have either many or few PD between the phloem and surrounding cells of the leaf. However, quantitative determination of PD density has rarely been performed. Moreover, the structure of PD has not been considered, even though it could impact permeability, and functional data are only available for very few species. Here, a comparison of PD density, structure, and function using data from transmission electron microscopy and live-cell microscopy was conducted for all relevant cell-cell interfaces in leaves of nine species. These species represent the three principal phloem-loading types currently discussed in literature. Results show that relative PD density among the different cell-cell interfaces in one species, but not absolute PD density, is indicative of phloem-loading type. PD density data of single interfaces, even combined with PD diameter and length data, did not correlate with the intercellular diffusion capacity measured by the fluorescence loss in photobleaching method. This means that PD substructure not visible on standard transmission electron micrographs may have a strong influence on permeability. Furthermore, the results support a proposed passive symplasmic loading mechanism in the tree species horse chestnut ( Aesculus hippocastanum ), white birch ( Betula pubescens ), orchard apple ( Malus domestica ), and gray poplar ( Populus x canescens ) as functional cell coupling and PD structure differed from active symplasmic and apoplasmic phloem-loading species., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Sugars produced by photosynthesis in leaves get transported to other organs in the phloem vascular tissue. Three general mechanisms have been proposed for the loading of sugars into the phloem. These differ in the involvement of active transport across the phloem cell's membrane and their capacity for passive intercellular transport through plasmodesmata. This capacity for diffusion from the mesophyll into the phloem cells can be quantified by live-cell microscopy. Instead of sugar molecules, the movement of fluorescent tracers of similar size can be observed. In this chapter, a simple method is described that allows quantification of plasmodesmata-mediated intercellular diffusion across the mesophyll-bundle sheath interface and the bundle sheath-phloem cell interfaces. The fluorescent tracer carboxyfluorescein is loaded into intact leaves and its diffusion monitored with confocal microscopy after photobleaching of a bundle sheath cell.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

A novel bacterial strain, A3 T , was isolated from the intestines of the sea urchin Strongylocentrotus droebachiensis collected in Øresund, Denmark. The strain was Gram-reaction-negative, rod-shaped and facultatively anaerobic, and displayed growth at 5-25 °C (optimum 20 °C), pH 7-9 (optimum at pH 7) and 1-6 % (w/v) NaCl (optimum 3 %). Furthermore, strain A3 T grew on agar, agarose, κ-carrageenan, alginate and laminarin as sole carbon source. Complete liquefaction of agar and κ-carrageenan was observed on solid plate media as a result of enzymatic activities. Major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The respiratory quinones were determined to be ubiquinones Q-8 (92 %) and Q-7 (8 %), and polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 36.9 mol%. Phylogenetical analyses based on the 16S rRNA gene showed that the bacterium was affiliated with the genus Colwellia within the Alteromonadaceae of the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain A3 T and its closest relatives in the genus Colwellia (C. psychrerythraea ATCC 27364 T and C. asteriadis KMD 002 T ) was 97.5 %. The average nucleotide identity between strain A3 T and other members of Colwellia was 78.6-80.5 %, and DNA-DNA hybridization prediction revealed values of less than 23 % relatedness between strain A3 T and other Colwellia species. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain A3 T represents a novel species of the genus Colwellia, for which the name Colwellia echini sp. nov. is proposed. The type strain is A3 T (=LMG 30125 T =NCIMB 15095 T ).

Metabolic highways may be orchestrated by the assembly of sequential enzymes into protein complexes, or metabolons, to facilitate efficient channeling of intermediates and to prevent undesired metabolic cross-talk while maintaining metabolic flexibility. Here we report the isolation of the dynamic metabolon that catalyzes the formation of the cyanogenic glucoside dhurrin, a defense compound produced in sorghum plants. The metabolon was reconstituted in liposomes, which demonstrated the importance of membrane surface charge and the presence of the glucosyltransferase for metabolic channeling. We used in planta fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to study functional and structural characteristics of the metabolon. Understanding the regulation of biosynthetic metabolons offers opportunities to optimize synthetic biology approaches for efficient production of high-value products in heterologous hosts., (Copyright © 2016, American Association for the Advancement of Science.)

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Immunosenescence, characterized by complex modifications of immunity with age, could be related to frailty syndrome in elderly individuals, leading to an inadequate response to minimal aggression. Functional decline (i.e., the loss of ability to perform activities of daily living) is related to frailty and decreased physiological reserves and is a frequent outcome of hospitalization in older patients. Links between immunosenescence and frailty have been explored and 20 immunological parameters, including insulin-like growth factor-1 (IGF-1), thymopoeisis, and telomere length, were shown to be affected in elderly patients with functional decline. A strong relationship between IGF-1 and thymic ouput was evidenced. IGF-1, a mediator of growth hormone (GH), was subsequently shown to induce interleukin-7 secretion in cultured primary human thymic epithelial cells. We are exploring the stress hypothesis in which an acute stressor is used as the discriminator of frailty susceptibility. GH can counteract the deleterious immunosuppressive effects of stress-induced steroids. Under nonstress conditions, the immunosenescent system preserves physiological responses, while under stress conditions, the combination of immunosenescence and a defect in the somatotrope axis might lead to functional decline., (© 2015 New York Academy of Sciences.)

The epidermis on leaves protects plants from pathogen invasion and provides a waterproof barrier. It consists of a layer of cells that is surrounded by thick cell walls, which are partially impregnated by highly hydrophobic cuticular components. We show that the Arabidopsis T-DNA insertion mutants of REDUCED WALL ACETYLATION 2 (rwa2), previously identified as having reduced O-acetylation of both pectins and hemicelluloses, exhibit pleiotrophic phenotype on the leaf surface. The cuticle layer appeared diffused and was significantly thicker and underneath cell wall layer was interspersed with electron-dense deposits. A large number of trichomes were collapsed and surface permeability of the leaves was enhanced in rwa2 as compared to the wild type. A massive reprogramming of the transcriptome was observed in rwa2 as compared to the wild type, including a coordinated up-regulation of genes involved in responses to abiotic stress, particularly detoxification of reactive oxygen species and defense against microbial pathogens (e.g., lipid transfer proteins, peroxidases). In accordance, peroxidase activities were found to be elevated in rwa2 as compared to the wild type. These results indicate that cell wall acetylation is essential for maintaining the structural integrity of leaf epidermis, and that reduction of cell wall acetylation leads to global stress responses in Arabidopsis.

Exposing a plant to stress situations, such as grafting, generally triggers antioxidant defense systems. In fruit tree grafting, quince (Cydonia oblonga) is widely used as a rootstock for pear (Pyrus communis L.), but several economically important pear cultivars are incompatible with available quince rootstocks. In this study, grafts were established using an in vitro callus graft system mimicking the events taking place in fruit trees. In vitro grown callus from pear [P. communis L. cv. 'Conference' (Co) and cv. 'William' (Wi)] and quince (C. oblonga Mill. clone 'BA29') was used to establish the compatible homografts 'Co/Co', 'Wi/Wi' and 'BA29/BA29', the compatible heterograft 'Co/BA29' and the incompatible heterograft 'Wi/BA29'. The main objective was to determine whether specific isoforms of genes involved in oxidative stress [superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT)] are differentially expressed at the graft interface from compatible and incompatible unions throughout 3 weeks after grafting. Reactive oxygen species (ROS) levels and programmed cell death were also evaluated in the course of graft development. Genes differentially expressed between compatible and incompatible heterografts were identified. Transcript levels of six antioxidant genes (SOD1, SOD3, APX3, APX6, CAT1 and CAT3) were down-regulated 10 days after grafting (DAG) in the incompatible heterograft in comparison to the compatible one. Likewise, SOD enzymatic activities were significantly higher at 1 and 10 days after wounding in the compatible cultivar 'Co' than in the incompatible one 'Wi'. These findings, together with live cell imaging of ROS-specific probes, ultrastructural mitochondrial changes and DNA fragmentation related to apoptotic processes, give indications that within incompatible rootstock/scion interfaces, either the level of ROS is increased or there is a less efficient detoxification system., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Cereal grain germination is central for plant early development, and efficient germination has a major role in crop propagation and malting. Endosperm starch is the prime energy reserve in germination and seedling establishment. In this study, it was hypothesized that optimized starch granule structure, and not only the endosperm starch content per se, is important for germination and seedling establishment. For that purpose, wild-type (WT), and specifically engineered degradable hyperphosphorylated (HP) starch and more resistant amylose-only (AO) starch barley lines were used. The transgenics showed no severe phenotypes and the WT and HP lines degraded the starch similarly, having 30% residual starch after 12 d of germination. However, the AO line showed significant resistance to degradation, having 57% residual starch. Interestingly, protein and β-glucan (BG) degradation was stimulated for both HP and AO lines as compared with the WT. At late seedling establishment stages, specific sugars were rapidly consumed in the AO line. α-Amylase activity was distinctly suppressed in both the HP and the AO lines. Pre-germination β-amylase deposition was low in the AO grains and β-amylase was generally suppressed in both HP and AO lines throughout germination. As further supported by scanning electron microscopy and histochemical analyses on grain and seedlings, it was concluded that inadequate starch granule deposition in combination with the suppressed hydrolase activity leads to temporal and compensating re-direction of starch, sugar, and protein catabolism important to maintain metabolic dynamics during grain germination and seedling establishment., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

SUPPRESSOR OF VARIEGATION 4 (SVR4, also called MRL7) and its homolog SVR4-like (also called MRL7-Like) were originally identified as important proteins for proper function of the chloroplast in Arabidopsis. Both are nuclear-encoded chloroplast-located proteins, and knockout mutants of either gene result in seedling lethality. Transmission electron microscopy analysis revealed that chloroplast development is arrested at an early developmental stage in both mutants. Accordingly, in the mutant plants severely decreased levels of photosynthetic pigments as well as subunits of the photosynthetic complexes could be detected. In absence of either of the two proteins chloroplast DNA organization was clearly affected. Immunological analysis revealed that SVR4 is a component of the transcriptionally active chromosome (TAC) from barley chloroplasts. Analyses of gene expression indicate that SVR4 and SVR4-like are required for proper function of the plastid transcriptional machinery. We propose that SVR4 and SVR4-like function as molecular chaperones ensuring proper organization of the nucleoids in chloroplasts., (© 2013 Scandinavian Plant Physiology Society.)

Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Aims: We address the question of the expression and the role of the growth hormone/insulin-like growth factor (GH/IGF) axis in the thymus., Methods: Using RT-qPCR, the expression profile of various components of the somatotrope GH/IGF axis was measured in different thymic cell types and during thymus embryogenesis in Balb/c mice. The effect of GH on T cell differentiation was explored via thymic organotypic culture., Results: Transcription of Gh, Igf1, Igf2 and their related receptors predominantly occurred in thymic epithelial cells (TEC), while a low level of Gh and Igf1r transcription was also evidenced in thymic T cells (thymocytes). Gh, Ghr, Ins2, Igf1, Igf2, and Igfr1 displayed distinct expression profiles depending on the developmental stage. The protein concentrations of IGF-1 and IGF-2 were in accordance with the profile of their gene expression. In fetal thymus organ cultures (FTOC) derived from Balb/c mice, treatment with exogenous GH resulted in a significant increase of double negative CD4-CD8- T cells and CD4+ T cells, together with a decrease in double positive CD4+CD8+ T cells. These changes were inhibited by concomitant treatment with GH and the GH receptor (GHR) antagonist pegvisomant. However, GH treatment also induced a significant decrease in FTOC Gh, Ghr and Igf1 expression., Conclusion: These data show that the thymotropic properties of the somatotrope GH/IGF-1 axis involve an interaction between exogenous GH and GHR expressed by TEC. Since thymic IGF-1 is not increased by GH treatment, the effects of GH upon T cell differentiation could implicate a different local growth factor or cytokine., (Copyright © 2012 S. Karger AG, Basel.)

The abundance of a selection of transcript species involved in inflammation, immunosenescence and stress response was compared between PBMC of 35 geriatric patients with hip fracture in acute phase (days 2-4 after hospitalization) or convalescence phase (days 7-10) and 28 healthy aged controls. Twenty-nine differentially abundant transcripts were identified in acute phase versus healthy ageing. Twelve of these transcripts remained differentially abundant in convalescence phase, and 22 were similarly differentially abundant in acute phase of geriatric infectious diseases. Seven of these 22 transcripts were previously identified as differentially abundant in PBMC of healthy aged versus healthy young controls, with further alteration for CD28, CD69, LCK, CTSD, HMOX1, and TNFRSF1A in acute phase after geriatric hip fracture and infectious diseases. The next question is whether these alterations are common to other geriatric diseases and/or preexist before the clinical onset of the diseases., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background and Aims: Although inflammatory and hormonal markers have been associated with further functional adverse outcomes in community- dwelling seniors, these markers have not been studied from this perspective in acutely ill older patients. This prospective study was designed to determine whether biological markers can improve the predictive value of a clinical screening tool to assess the risk of functional decline in hospitalized older patients., Methods: Patients aged 75 years and over admitted for hip fracture, acute heart failure or infection (n=118) were recruited. The clinical screening tool SHERPA was filled in on admission, with concomitant measurement of interleukin-6 (IL-6), insulin-like growth factor 1 (IGF-1), C-reactive protein (CRP), white blood cells, urea, albumin, pre-albumin and total cholesterol. Functional decline was defined as a decrease of one point in the activities of daily living scale between pre-admission and 3-month post-discharge status. We compared the discrimination calibration of SHERPA vs SHERPA+, a logistic regression model including SHERPA and selected biomarkers., Results: Three months after discharge, functional decline had occurred in 46 patients. IL-6 and IGF-1 were selected, since their levels were significantly different between decliners and non-decliners, and were included in the new logistic regression model SHERPA+. Areas under the ROC curve [95% CI] for functional decline prediction were 0.73 [0.63-0.81] for SHERPA vs 0.79 [0.69-0.86] for SHERPA+ (p=0.14). However, SHERPA+ was better calibrated, as the average predicted risk of functional decline within subgroups matched the proportion which actually underwent functional decline (Brier score=0.185). Since functional decline was higher in patients with hip fracture, the SHERPA+ model was challenged by including the diagnosis. Only SHERPA, IGF-1 and diagnosis were significantly associated with functional decline., Conclusions: Selected biological markers may marginally improve the clinical prediction of post-discharge functional decline in hospitalized patients, and may allow to stratify them appropriately. The predictive value of these biomarkers is not fully independent of disease status. Further studies are needed to confirm these results in a cohort representative of older patients admitted through the emergency department.

The abundance of a preselection of transcripts involved in inflammation, immunosenescence and stress response was compared between PBMC of healthy aged donors and aged patients in acute phase of heart failure and at recovery. This study identified 22 transcripts differentially abundant in acute phase of heart failure versus healthy aged subjects. Transcripts involved in inflammation and oxidative stress were more abundant. Those associated with T-cell functions were less abundant. The results were compared to two other major acute geriatric issues: infectious diseases and hip fracture. In acute phase, compared to healthy aged subjects, the abundance of 15/22 transcripts was also altered in both geriatric infectious diseases and hip fracture. Many variations had not vanished at the recovery phase. The abundance of CD28, CD69, LCK, HMOX1, TNFRSF1A transcripts, known to be altered in healthy aged versus healthy young subjects, was further affected in acute phase of the three geriatric diseases considered. The transcript levels of BCL2, CASP8, CCL5, DDIT3, EGR3, IL10RB, IL1R2, SERPINB2 and TIMP1 were affected in all three pathological conditions compared to healthy aged, but not versus healthy young subjects. In conclusion, this work provides critical targets for therapeutic research on geriatric heart failure, infectious diseases and hip fracture., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Despite more than 130 years of research, phloem loading is far from being understood in gymnosperms. In part this is due to the special architecture of their leaves. They differ from angiosperm leaves among others by having a transfusion tissue between bundle sheath and the axial vascular elements. This article reviews the somewhat inaccessible and/or neglected literature and identifies the key points for pre-phloem transport and loading of photoassimilates. The pre-phloem pathway of assimilates is structurally characterized by a high number of plasmodesmata between all cell types starting in the mesophyll and continuing via bundle sheath, transfusion parenchyma, Strasburger cells up to the sieve elements. Occurrence of median cavities and branching indicates that primary plasmodesmata get secondarily modified and multiplied during expansion growth. Only functional tests can elucidate whether this symplasmic pathway is indeed continuous for assimilates, and if phloem loading in gymnosperms is comparable with the symplasmic loading mode in many angiosperm trees. In contrast to angiosperms, the bundle sheath has properties of an endodermis and is equipped with Casparian strips or other wall modifications that form a domain border for any apoplasmic transport. It constitutes a key point of control for nutrient transport, where the opposing flow of mineral nutrients and photoassimilates has to be accommodated in each single cell, bringing to mind the principle of a revolving door. The review lists a number of experiments needed to elucidate the mode of phloem loading in gymnosperms.

Background: Infectious diseases are significant causes of morbidity and mortality among elderly populations. However, the relationship between oxidative stress, immune function and inflammatory response in acute phase of the infectious disease is poorly understood., Results: Herein the abundance of a selection of 148 transcripts involved in immunosenescence and stress response was compared in total RNA of PBMC of 28 healthy aged probands and 39 aged patients in acute phase of infectious disease (day 2-4 after hospitalization) or in convalescence phase (day 7-10). This study provides a list of 24 differentially abundant transcript species in the acute phase versus healthy aged. For instance, transcripts associated with inflammatory and anti-inflammatory reactions (TNFRSF1A, IL1R1, IL1R2, IL10RB) and with oxidative stress (HMOX1, GPX1, SOD2, PRDX6) were more abundant while those associated with T-cell functions (CD28, CD69, LCK) were less abundant in acute phase. The abundance of seven of these transcripts (CD28, CD69, LCK, CTSD, HMOX1, TNFRSF1A and PRDX6) was already known to be altered in healthy aged probands compared to healthy young ones and was further affected in aged patients in acute phase, compromising an efficient response., Conclusion: This work provides insights of the state of acute phase response to infections in elderly patients and could explain further the lack of appropriate response in the elderly compared to younger persons.

Age-related changes of gene expression contribute to the physiological alteration observed with human ageing. Herein, the abundance of a selection of 148 transcripts involved in immunosenescence and stress response was compared in total RNA of PBMC of healthy young to middle-age probands (35.0 +/- 6.5 year old) and healthy old probands (82.5 +/- 6.8 year old). This study provides a list of 16 differentially abundant transcripts species in the healthy old probands. Thus, these changes of abundance can be considered as easily accessible biomarkers of ageing. Some of these differential abundances like CD28, CD69, LCK (decreased abundance in old subjects), CD86, Cathepsin D, H and S (increased abundance in old subjects) might explain biochemical and cytochemical changes observed at the protein level in the immune system and thus might correspond to regulatory processes affecting the ageing process. Indeed these changes reflect the low-grade pro-inflammatory status observed in old persons and suggest a hypo-responsiveness of T-cells together with an increase in antigen presentation potential. In addition, among the differentially abundant transcripts were transcripts involved in the oxidative stress response HMOX1 and HSPA6 mRNAs were found as more abundant in PBMC from elderly subjects., (Copyright 2009 Elsevier Inc. All rights reserved.)

One factor that contributes to a successful fruit tree grafting is the establishment of symplasmic contacts in the graft interface to facilitate the transfer of compounds between scion and stock. Using novel experimental and theoretical approaches we investigated whether the localized incompatibility, experienced in some Prunus grafts, could be related to insufficient plasmodesmal coupling at an early stage of development within one of the partners. Dye-coupling analysis using fluorescent tracers combined with confocal laser scanning microscopy were performed in cultured callus from either the plum rootstock (Prunus cerasifera Ehrh. x Prunus munsoniana W. Wight et Hedr.) cv. 'Marianna 2624' or from the apricot (Prunus armeniaca L.) cv. 'Moniqui' growing in vitro. Fluorescein was loaded into callus cells in a caged form. Following photoactivation of fluorescence within single cells, the uncaged fluorescein could be traced as it was spreading cell-to-cell revealing the existence of functional plasmodesmata. This set of experiments was performed within the 'stock' partner in callus fusions ('callus grafts') as well as in ungrafted callus. The results indicated species-related as well as developmental-related differences in plasmodesmal conductivity. The results further pointed to a novel control factor of connectivity that reaches the graft partner and changes its innate rate of communication: when combining the poorly transporting apricot cultivar with the well-transporting plum cultivar, communication between plum callus cells was much reduced, compared to that in plum homografts. For further support of the hypothesis, we carried out a quantitative analysis in which fluorescein was esterloaded into the callus. Fluorescence redistribution after photobleaching of fluorescein in individual cells gave a measure for the plasmodesmal contact between the cells. We found significant differences between the species with regard to mobile fraction and halftime of redistribution, which confirmed that callus cells are not interconnected to the same extent in Marianna 2624 and Moniqui.

Background: Despite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse age-related changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels., Methodology/principal Findings: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/beta TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p<0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p<0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/beta TREC ratio (p<0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/beta TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal., Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults., Trial Registration: ClinicalTrials.gov NTC00601419.

Transgenic tobacco (Nicotiana tabacum) was studied to localize the activity of phloem loading during development and to establish whether the endoplasmic reticulum (ER) of the companion cell (CC) and the sieve element (SE) reticulum is continuous by using a SUC2 promoter-green fluorescent protein (GFP) construct targeted to the CC-ER. Expression of GFP marked the collection phloem in source leaves and cotyledons as expected, but also the transport phloem in stems, petioles, midveins of sink leaves, nonphotosynthetic flower parts, roots, and newly germinated seedlings, suggesting that sucrose retrieval along the pathway is an integral component of phloem function. GFP fluorescence was limited to CCs where it was visualized as a well-developed ER network in close proximity to the plasma membrane. ER coupling between CC and SEs was tested in wild-type tobacco using an ER-specific fluorochrome and fluorescence redistribution after photobleaching (FRAP), and showed that the ER is continuous via pore-plasmodesma units. ER coupling between CC and SE was quantified by determining the mobile fraction and half-life of fluorescence redistribution and compared with that of other cell types. In all tissues, fluorescence recovered slowly when it was rate limited by plasmodesmata, contrasting with fast intracellular FRAP. FRAP was unaffected by treatment with cytochalasin D. The highest degree of ER coupling was measured between CC and SE. Intimate ER coupling is consistent with a possible role for ER in membrane protein and signal exchange between CC and SE. However, a complete lack of GFP transfer between CC and SE indicated that the intraluminal pore-plasmodesma contact has a size exclusion limit below 27 kD.

Ontogenesis of oxytocin (OT) and vasopressin (VP) gene expression and function were investigated in murine thymus. OT and VP transcripts were detected in the thymus on embryonic days 13 and 15, respectively. Corresponding messenger RNAs were evidenced in thymic epithelial cells by in situ hybridization with a neurophysin probe. From all OT and VP receptors, only OTR was expressed by all T-cell subsets, while V1bR was found in double positive and single positive CD8 cells. In fetal thymic organ cultures, OTR antagonist d[D-Tyr(Et)2, Thr4]OVT increased early apoptosis of CD8 cells, while V1bR antagonist (Sanofi SSR149415) inhibited T-cell differentiation, and favored CD8 T-cell commitment.

Caged probes offer a novel approach to study plant cell-to-cell communication. Instead of introducing fluorescent molecules into cells by microinjection, their caged counterparts can be preloaded into the tissue by diffusion. Following spatially controlled photoactivation, movement of the uncaged fluorochrome can be followed in time and direction by confocal laser scanning microscopy. In the onion bulb scale epidermis used as a model system, symplasmic transport of the tracer out of a target cell was followed. Transport via the symplasmic pathway was challenged by plasmolysing the tissue. The experiments confirmed the symplasmic nature of tracer transport.

Transgenic tobacco (Nicotiana tabacum) plants expressing green fluorescent protein (GFP) from the AtSUC2 promoter were used to study the function of different vein classes in developing leaves. In sink leaves, unloading capacity occurred acropetally, with the class I (midrib) and class II veins becoming functional in phloem unloading before the maturation of the class III veinal network. In contrast, in developing cotyledons and source leaves, loading capacity occurred in a basipetal direction. There was a strong correlation between loading capacity, as assessed by (14)C Suc uptake and companion cell expression of AtSUC2-GFP. Developing cotyledons were shown to utilize all available vein classes for loading. A second line of transgenic plants was produced in which GFP, expressed from the AtSUC2 promoter, was targeted to the endoplasmic reticulum instead of the cytoplasm. In these AtSUC2-GFP-ER plants, GFP was unable to traffic into the sieve element and was restricted solely to the companion cells of source leaf tissues. Partial shading of leaves undergoing the sink-source transition demonstrated that the activation of the AtSUC2 promoter in tobacco was influenced by light. Functional and structural maturation of the minor veins required light or a product of light. The activation of the AtSUC2 promoter within major veins appears to be regulated differently from that in the minor veins. The relationship between AtSUC2 activation and the activity of endogenous tobacco Suc transporters is discussed.

The research field on focal adhesion-related kinases started a decade ago, but the term focal adhesion was introduced for the first time nearly 20 yr before. Since its identification, many studies have enlightened the role of the first intermediate of focal adhesion-related signals in a large number of biologic and physiologic processes. In this review, we try to integrate the most recent data about the known focal adhesion-related kinases, and we focus on three topics in which they deserve great interest: neuroendocrine-immune interactions, developmental biology, and proliferative diseases.

Sequential immunoscintigrams were used to describe the relative distribution and kinetics of 8 mg 131I-labeled human IgM monoclonal antibody 16.88 in 20 patients with colorectal cancer. The results show that the initial activity was higher and the clearance rate was faster (P < 0.05) from the left ventricle and liver than from most organs. In bone marrow the reverse was observed (P < 0.05). The biological half-life of 131I(-16.88) in tumor tissue (range 35.4-47.5 h) was longer (P < 0.01) than that in normal tissue (30.2-41.9 h). The image contrast ratio between liver metastases and background increased from 0.8 to 1.3 and for lesions outside the liver from 1.1 to 1.6. The estimated effective dose equivalent was 0.12 mSv/MBq. A second infusion 2 weeks after the first with the addition of unlabeled 16.88 up to 1000 mg for improvement of tumor tissue uptake was not of clinical relevance.

Promising clinical results have been obtained with radiolabeled antibodies in lymphoma patients. The higher uptake by lymphomas of 67Gallium (67Ga) compared with monoclonal antibodies makes selective radiotherapy by the widely available 67Ga appealing. However, the gamma radiation of 67Ga used in scintigraphy is considered to be almost non-toxic to lymphoma cells. However, in addition to photon radiation 67Ga emits low energy Auger electrons and 80-90 keV conversion electrons which could be cytotoxic. The objective of the present study was the assessment of radiotoxicity of 67Ga on a lymphoid cell line: U937. Proliferation (MTT-assay) and clonogenic capacity (CFU-assay) were measured after 3 and 6 days incubation with 10, 20 and 40 microCi ml-1 67Ga. Growth inhibition was 36% after 3 days incubation and 63% after 6 days incubation with 40 microCi 67Ga ml-1. Clonogenic capacity was reduced by 51% after 3 days and 72% after 6 days incubation with 40 microCi ml-1 67Ga. A survival curve showed an initial shoulder and became steeper beyond 200-250 pCi cell-1 (low linear energy transfer type). Iso-effect doses of 67Ga and 90Yttrium (90Y) were determined. The iso-effect dose of 40 microCi 67Ga ml-1 (cumulative dose of conversion electrons 306 cGy) was 2.5 microCi 90Y ml-1 (cumulative dose 494 cGy) and the iso-effect dose of 80 microCi 67Ga ml-1 was 5.0 microCi 90Y/ml. The main cytotoxic effect of 67Ga seems to be induced by the 80 keV conversion electrons. We conclude that the conversion electrons of 67Ga have a cytotoxic effect on U937 cells and that in our experiments a 16-fold higher microCi-dose of 67Ga than of 90Y was needed for the same cytotoxic effect. We believe that 67Ga holds promise for therapeutic use.

The diagnostic value of 99mTc-labeled monoclonal antibody E48 F(ab')2 (750 MBq, 1 mg) was evaluated in 10 patients with a histologically proven squamous cell carcinoma of the head and neck and with clinical evidence of cervical lymph node involvement. Preoperative findings on lymph node status obtained by radioimmunoscintigraphy (RIS), computerized tomography, magnetic resonance imaging, and palpation were defined per side (left and/or right side of the neck) as well as per lymph node level (I through V) and compared with the histopathological outcome of the neck dissection specimen. In 10 patients, all 8 known tumors at the primary site were detected by RIS. Furthermore, RIS was correct in 13 of 13 tumor involved neck sides and in 17 of 20 tumor involved lymph node levels. False-negative observations comprised 3 levels containing tumor deposits smaller than 1 cm in diameter, 2 of which were not detected by any other diagnostic modality. Palpation, computerized tomography, and magnetic resonance imaging were correct in, respectively, 13, 15, and 15 of the 20 tumor involved levels. There were 2 false-positive observations with monoclonal antibody E48 and 3 with palpation. No false-positive detections occurred with computerized tomography or magnetic resonance imaging. In two of the patients, RIS provided clinically important information which was not provided by any other diagnostic method. In one patient, recurrence of laryngeal carcinoma was established at the primary site after previous radiotherapy. In another patient, bilateral instead of unilateral lymph node involvement became apparent. These preliminary data indicate that radioimmunoscintigraphy with monoclonal antibody E48 may be helpful in the diagnosis of metastatic and recurrent head and neck cancer.

An assay for the determination of 4-aminopyridine in serum has been developed using 3,4-diaminopyridine as internal standard and reversed-phase high-performance liquid chromatography with detection at 244 nm. A mobile phase of acetonitrile-methanol-ethanol-1% ammonium carbonate (75:10:10:5) provided excellent separation of both compounds. Samples were extracted on solid-phase columns. The linearity, precision, recovery and the limit of detection were all sufficient for the routine use of this assay in clinical studies of patients treated with 4-aminopyridine.

Line-source experiments were conducted to assess the performance of a gamma-camera equipped with a specially designed 511-keV collimator for the planar imaging of positron emitters. The results were compared with the camera performance with routinely used collimators and radionuclides (thallium-201, technetium-99m and gallium-67). With positron emitters, scatter contributed less to the widening of the line spread function than with radionuclides emitting lower photon energies. These observations can be explained by the relative deterioration in the discrimination power of the gamma-camera to reject scattered radiation at low energies. Planar 511-keV imaging may provide relevant clinical information, as we showed by fluorodeoxyglucose studies in a patient with a myocardial infarction and in a patient with a malignant lymphoma. It is concluded that positron emitters can be effectively applied for planar imaging with the generally available gamma-cameras. This study implies that radiotracers developed for positron emission tomography may find a place in the practice of conventional nuclear medicine.

Twenty colorectal cancer patients were given an intravenous injection of human IgM monoclonal antibody (MAb) 16.88 (8 mg) conjugated to 131I for tumor localization. After a 2-week interval, a second injection with 200, 500, or 1000 mg of unlabeled antibody added was given to groups of five patients each. at the end of the 2-hour infusion, 66% of the radioactivity remained in the circulation. Blood clearance of the 131I-labeled MAb 16.88 was biphasic with a mean half-life (T1/2 alpha) of 12 hours and T1/2 beta of 45 hours. Clearance rate was 0.09 L/hour. More than 90% of the 131I in serum was protein bound, with an immunoreactive fraction of 80% in the first 48 hours. Size exclusion chromatography indicated no degradation products other than 131I in serum and urine. The urinary excretion rate of 131I increased to 1.5% of the dose per hour at 24 hours, with 50% of the dose excreted in 34 hours. The pharmacokinetic profile of 131I-labeled MAb 16.88 was neither influenced by the total protein dose of antibody administered nor affected by specific uptake in tumor tissue in individual patients, as determined on early immunoscintigrams. The larger antibody doses showed a slightly slower excretion of 131I. The assays applied to determine immunogenicity were enzyme-linked immunosorbent assay, radioimmunoassay, and the dot-blot assay. They had sensitivities ranging from 5 ng/mL to 0.5 micrograms/mL for goat or rabbit antihuman IgM. The assays did not reveal antihuman antibody responses.

The kinetics of systemically and locally administered cefuroxime or ceftazidime in wound fluid were investigated in the period after vascular prosthetic implantation. Cefuroxime or ceftazidime was administered intravenously in patients. Simultaneously 250 mg ceftazidime or cefuroxime was added to preclotted blood. Locally administered antibiotics could not be detected in our samples. In groin fluid samples 24-48 h after the operation the average concentration of cefuroxime was 8.3 and of ceftazidime 5.0 mg/l. The decline of the concentration of cefuroxime or ceftazidime in groin fluid seems to be much slower than one would expect from the half-lives of the antibiotics. We conclude that cefuroxime and to a lesser extent ceftazidime are suitable as prophylactic agents in arterial reconstruction.

Human IgM monoclonal antibody 16.88 recognised an intracellular antigen strongly expressed in colorectal cancer tissue in 51% of our patients. Tumour localisation was carried out with 185 MBq 131I-16.88 (8 mg) in 20 of these patients with advanced disease. In 16 patients (80%) immunoscintigraphy was positive in at least one organ site with disease. Of all sites, 55% could be visualized. In general, lesions less than 3 cm could not be detected. Sequential immunoscintigrams of liver metastases showed variable patterns. Initial "cold" lesions corresponded to liver metastases with poor blood supply as indicated by 99mTc-sulphur-colloid and 99mTc-HMPAO scintigraphy, respectively. The mean (S.D.) biological half-life (whole body clearance of radioactivity) was 37.6 (5.0) h. A second infusion of 131I-16.88 with the addition of high doses of unlabelled 16.88 could be done safely, but did not result in better visualisation of tumour lesions or affect radioactivity clearance from the body.

Human IgM monoclonal antibody (MAb) 16.88 recognizes an antigen strongly expressed by colon cancer tissue. We used 131I-labelled 16.88 for biodistribution and pharmacokinetic studies in nude mice bearing WiDr or NIH:OVCAR-3 xenografts. Serum half-life was 8 h. Maximum tumour uptake was between 1 and 8 h after administration and amounted to, respectively, 3% and 1% of the injected dose g-1 for WiDr and NIH:OVCAR-3 tumours. Half-lives in these tumours were approximately 24 h. Tumour to normal colon uptake ratios increased from 2.3 at 24 h to 17 at 5 days after injection. Simultaneously, pharmacokinetic studies were performed in patients with advanced colon cancer reactive with 16.88. They were injected with 5 mCi 131I-16.88 by intravenous infusion over 2 h. Serum half-life was 20 h with greater than 90% of the 131I bound to 16.88. Within 40 h 50% of the injected dose was excreted as free 131I in the urine. In one patient an accelerated clearance was found, possibly caused by pre-existing antibodies reacting with 16.88. None of the patients showed an immune response against 16.88 antibody. Immunoscintigraphy showed positive tumour localization in the majority of the patients, best visualized at later days. We conclude that 16.88 has tumour localization properties while its human origin accounts for the lack of immunogenicity.