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Many studies show that mixed species stands can have higher gross growth, or so-called overyielding, compared with monocultures. However, much less is known about mortality in mixed stands. Knowledge is lacking, for example, of how much of the gross growth is retained in the standing stock and how much is lost due to mortality. Here, we addressed this knowledge gap of mixed stand dynamics by evaluating 23 middle-aged, unthinned triplets of monospecific and mixed plots of Scots pine (Pinus sylvestris L.) and European beech (Fagus sylvatica L.) repeatedly surveyed over 6–8 years throughout Europe. For explanation of technical terms in this abstract see Box 1. First, mixed stands produced more gross growth (+10%) but less net growth (−28%) compared with the weighted mean growth of monospecific stands. In monospecific stands, 73% of the gross growth was accumulated in the standing stock, whereas only 48% was accumulated in mixed stands. The gross overyielding of pine (2%) was lower than that of beech (18%). However, the net overyielding of beech was still 10%, whereas low growth and dropout of pine caused a substantial reduction from gross to net growth. Second, the mortality rates, the self- and alien-thinning strength, and the stem volume dropout were higher in mixed stands than monospecific stands. The main reason was the lower survival of pine, whereas beech persisted more similarly in mixed compared with monospecific stands.Third, we found a 10% higher stand density in mixed stands compared with monospecific stands at the first survey. This superiority decreased to 5% in the second survey.Fourth, the mixing proportion of Scots pine decreased from 46% to 44% between the first and second survey. The more than doubling of the segregation index (S) calculated by Pielou index (S increased from 0.2 to 0.5), indicated a strong tendency towards demixing due to pine. Fifth, we showed that with increasing water supply the dropout fraction of the gross growth in the mixture slightly decreased for pine, strongly increased for beech, and also increased for the stand as a whole. We discuss how the reduction of inter-specific competition by thinning may enable a continuous benefit of diversity and overyielding of mixed compared with monospecific stands of Scots pine and European beech., The study received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No952314 and under the Marie Skłodowska-Curie grant agreement No 778322. The first author also wishes to thank the German ScienceFoundation (Deutsche Forschungsgemeinschaft) for funding the project “Structure and dynamics of mixed-species stands of Scots pine and European beech compared with monospecific stands; analysis along an ecological gradient through Europe” (# DFG PR 292/15-1). Felipe Bravo is grateful for Funds by the Junta de Castilla y León through the projects “CLU-2019-01 and CL-EI-2021-05 - iuFOR Institute Unit of Excellence” of the University of Valladolid and the co-financing by the European Regional Development Fund (ERDF “Europe drives our growth”). Miren del Río thanks for the support by the Spanish Ministerio de Ciencia e Innovación (# PID2021-126275OB-C21/C22). Dejan Stojanović thanks the Ministry of Education, Science and Technological Development of the Republic of Serbia for funding. Tzvetan Zlatanov thanks the Ministry of Education and Science of the Republic of Bulgaria (# DO1-405/18.12.2020 LTER-BG).

Heterogeneity of structure can increase mechanical stability, stress resistance and resilience, biodiversity and many other functions and services of forest stands. That is why many silvicultural measures aim at enhancing structural diversity. However, the effectiveness and potential of structuring may depend on the site conditions. Here, we revealed how the stand structure is determined by site quality and results from site-dependent partitioning of growth and mortality among the trees. We based our study on 90 mature, even-aged, fully stocked monocultures of Scots pine (Pinus sylvestris L.) sampled in 21 countries along a productivity gradient across Europe. A mini-simulation study further analyzed the site-dependency of the interplay between growth and mortality and the resulting stand structure. The overarching hypothesis was that the stand structure changes with site quality and results from the site-dependent asymmetry of competition and mortality. First, we show that Scots pine stands structure across Europe become more homogeneous with increasing site quality. The coefficient of variation and Gini coefficient of stem diameter and tree height continuously decreased, whereas Stand Density Index and stand basal area increased with site index. Second, we reveal a site-dependency of the growth distribution among the trees and the mortality. With increasing site index, the asymmetry of both competition and growth distribution increased and suggested, at first glance, an increase in stand heterogeneity. However, with increasing site index, mortality eliminates mainly small instead of all-sized trees, cancels the size variation and reduces the structural heterogeneity.Third, we modelled the site-dependent interplay between growth partitioning and mortality. By scenario runs for different site conditions, we can show how the site-dependent structure at the stand level emerges from the asymmetric competition and mortality at the tree level and how the interplay changes with increasing site quality across Europe. Our most interesting finding was that the growth partitioning became more asymmetric and structuring with increasing site quality, but that the mortality eliminated predominantly small trees, reduced their size variation and thus reversed the impact of site quality on the structure. Finally, the reverse effects of mode of growth partitioning and mortality on the stand structure resulted in the highest size variation on poor sites and decreased structural heterogeneity with increasing site quality. Since our results indicate where heterogeneous structures need silviculture interventions and where they emerge naturally, we conclude that these findings may improve system understanding and modelling and guide forest management aiming at structurally rich forests., This study was supported by the ERA-Net COFUND programme SUMFOREST, with the national funding organization Federal Ministry of Nutrition and Agriculture (REFORM, grant #2816ERA02S). The project has also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No952314 and under the Marie Skłodowska-Curie grant agreement No 778322, and the 2017 - 2018 Belmont Forum and BiodivERsA joint call for research proposals, under the BiodivScen ERA-Net COFUND programme, with the national funding organization Federal Ministry of Education and Research (grant #16LC1805B). The German Science Foundation funded the project “Structure and growth of mixed Scots pine/European beech stands compared with pure stands analyzed along an ecological gradient through Europe” (grant #PR 292/15-1). H.P. and E.U. were additionally supported by the Bavarian Ministry of Nutrition, Agriculture and Forestry through the projects “Pine-spruce mixed stands in Bavaria” (grant #7831-20339-2012) and “Maintenance of the long term experimental plots in Bavaria (W07, #7831-26625-2017). M.d.R. and R.R.-P. were supported by the projects CLU-2019-01 - iuFOR Institute Unit of Excellence” of the University of Valladolid and VA183P20–SMART, funded by the Junta de Castilla and Leon (Spain) and co-financed by the European Union (ERDF “Europe drives our growth”). J.C. was supported by the National Agency of Agricultural Research (Project No. QK21020307). K.B. was supported by the Polish Government MNiSW 2018–2021 Matching Fund No. 117/H2020/2018. M.M. was supported by the Estonian Research Council grant (PRG1586) and Estonian University of Life Sciences projects number P180024MIME and P200029MIME. R.S. was supported by the Slovak Research and Development Agency project No. APVV- 19-0035.

Tree species mixing has become a crucial tool in European forest management as positive interactions between species have been found to promote the provision of multiple ecosystem services, while at the same time reducing the risks associated with climate change. However, mixing effects have proven to be strongly context-dependant and some species combinations have still not been studied in detail. Here we focus on mixed forests of Scots pine and oak, which are likely to become increasingly popular for balancing wood production and other ecosystem services under climate change. Using 20-year growth data from newly established triplets in Germany and Denmark, this study investigates how mean tree and stand characteristics as well as productivity in mixed Scots pine-oak stands compare with adjacent monocultures and how stable the observed productivity relation is, considering inter-annual variations in local climate. Species mixing on average resulted in 15% higher standing volume and 14% higher volume productivity compared with the weighted mean of the adjacent monocultures. Oak profited most in mixture, showing overyielding of 19%. Overyielding on the stand and species level increased in years with higher water supply. In mixture, standing volume of Scots pine was 25% higher than in monocultures. Both species were found to modify their morphology in mixture. Oak in mixture showed a significantly higher inequality in stem volume compared with monocultures. We hypothesise that the observed overyielding of Scots pine-oak mixtures mainly results from complementary light use, where differences in shade tolerance, crown architecture and leaf phenology may be contributing factors.

Tree species mixing has been widely promoted as a promising silvicultural tool for reducing drought stress. However, so far only a limited number of species combinations have been studied in detail, revealing inconsistent results. In this study, we analysed the effect of mixing Scots pine and oak (pedunculate oak and sessile oak) trees on their drought response along a comprehensive ecological gradient across Europe. The objective was to improve our knowledge of general drought response patterns of two fundamental European tree species in mixed versus monospecific stands. We focused on three null hypotheses: () tree drought response does not differ between Scots pine and oak, () tree drought response of Scots pine and oak is not affected by stand composition (mixture versus monoculture) and () tree drought response of Scots pine and oak in mixtures and monocultures is not modified by tree size or site conditions. To test the hypotheses, we analysed increment cores of Scots pine and oak, sampled in mixed and monospecific stands, covering a wide range of site conditions. We investigated resistance (the ability to maintain growth levels during drought), recovery (the ability to restore a level of growth after drought) and resilience (the capacity to recover to pre-drought growth levels), involving site-specific drought events that occurred between 1976 and 2015. In monocultures, oak showed a higher resistance and resilience than Scots pine, while recovery was lower. Scots pine in mixed stands exhibited a higher resistance, but also a lower recovery compared with Scots pine in monocultures. Mixing increased the resistance and resilience of oak. Ecological factors such as tree size, site water supply and site fertility were found to have significant effects on the drought response. In the case of Scots pine, resistance was increased by tree size, while recovery was lowered. Resistance of oak increased with site water supply. The observed mixing effect on the tree drought response of Scots pine and oak was in some cases modified by the site conditions studied. Positive mixing effects in terms of resistance and resilience of oak increased with site water supply, while the opposite was found regarding recovery. In contrast, site fertility lessened the positive mixing effect on the resistance of Scots pine. We hypothesise that the observed positive mixing effects under drought mainly result from water- and/or light-related species interactions that improve resource availability and uptake according to temporal and spatial variations in environmental conditions. This work was supported by the European Union as part of the ERA-Net SUMFOREST project REFORM – Mixed species forest management. Lowering risk, increasing resilience (2816ERA02S, PCIN2017-026) and the Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 778322. All contributors thank their national funding institutions for supporting the establishment, mensuration and analysis of the studied triplets. The first author wants to thank the German Federal Ministry of Food and Agriculture (BMEL) for financial support through the Federal Office for Agriculture and Food (BLE) (grant number 2816ERA02S), as well as the Bayerische Staatsforsten (BaySF) and Landesbetrieb Forst Brandenburg for providing suitable research sites. Research on the Lithuanian triplets (LT 1, LT 2) was made possible by the national funding institution Research Council of Lithuania (LMTLT) (agreement number S-SUMFOREST-17-1). The French site FR 1 belongs to the OPTMix experimental site (https://optmix.irstea.fr), which is supported annually by Ecofor, Allenvi, and the French national research infrastructure ANAEE-F. A special thank is due to Peter Biber for supporting the statistical analysis.

Past failures of monocultures, caused by wind-throw or insect damages, and ongoing climate change currently strongly stimulate research into mixed-species stands. So far, the focus has mainly been on combinations of species with obvious complementary functional traits. However, for any generalization, a broad overview of the mixing reactions of functionally different tree species in different mixing proportions, patterns and under different site conditions is needed, including assemblages of species with rather similar demands on resources such as light. Here, we studied the growth of Scots pine and oak in mixed versus monospecific stands on 36 triplets located along a productivity gradient across Europe, reaching from Sweden to Spain and from France to Georgia. The set-up represents a wide variation in precipitation (456–1250 mm year−1), mean annual temperature (6.7–11.5 °C) and drought index by de Martonne (21–63 mm °C−1). Stand inventories and increment cores of trees stemming from 40- to 132-year-old, fully stocked stands on 0.04–0.94-ha-sized plots provided insight into how species mixing modifies stand growth and structure compared with neighbouring monospecific stands. On average, the standing stem volume was 436 and 360 m3 ha−1 in the monocultures of Scots pine and oak, respectively, and 418 m3 ha−1 in the mixed stands. The corresponding periodical annual volume increment amounted to 10.5 and 9.1 m3 ha−1 year−1 in the monocultures and 10.5 m3 ha−1 year−1 in the mixed stands. Scots pine showed a 10% larger quadratic mean diameter (p p p p p p p

The calculation of critical loads and their exceedance is one method to describe the vulnerability of forests to environmental stress caused by anthropogenic impact. Exceedance of critical loads for acidifying inputs and nitrogen was compared to different indicators of the soil and forest conditions in the German part of the extensive forest monitoring (ICP Forests/EU Level I), including more than 1,800 plots. In addition, an empirical relationship between the C/N ratio of the forest floor humus layer (C/NHumus) and the estimated nitrogen output for ten plots of the intensive monitoring (ICP Forests/EU Level II) was established in order to estimate the potential nitrogen output on Level I plots dominated by Norway spruce. Regarding all tree species assessed, the exceedance of critical loads for nitrogen and sulphur is negatively correlated with pH and base saturation up to 30 cm soil depth. The sulphur deposition and the exceedance of critical loads are highly correlated with the sulphur content of leaves and needles, whereas the respective relations for nitrogen were lower. The crown condition was weakly positively related to the sulphur content in tree leaves and needles. For Norway spruce sites, high exceedance of critical loads for nitrogen and nitrogen deposition corresponded well with low C/NHumus. In regions with high nitrogen load and low C/N ratios in the humus layer, the calculated nitrogen output was high. The results support the concept of critical thresholds in that way that their exceedance can impair forest ecosystem functions like nitrogen retention.

Element concentrations in tree rings can be used to monitor changes in environmental quality. With regard to the detection of incipient soil acidification, the manganese concentration in soils and plants is a significant marker for the switch of acid buffering in soils mainly with the exchange of base cations or with the dissolution of aluminium oxides. This is a site-specific non-linear event, indicating the onset of Al3+ dominance in the soil solution, were damages to vegetation due to acid stress become possible. This turning point is also a marker for the attainment of pH 4.2 in soils, the critical threshold used for critical load calculations. On a plot of the German environmental monitoring in forests the element concentrations in tree rings of 60-year-old spruces reveal a distinct decline in the Mn concentration, beginning in the late 1960s ending in the late 1970s. With this information it was possible to assume a base saturation in the soil of about 15–20% in the late 1960s, and to model the development of the base saturation of the site. A decline from 17.5 to 6% within one decade could be related to the deposition. This is in accordance with the base saturation of 6.5%, measured in 1993 for this site, but also for adjacent spruce sites on the same geological substrate. The knowledge of the time span were this site-specific non-linear event occurred is essential for the reconstruction of the soil chemistry of a site. Moreover, it enables the assignment of observations like ‘forest damages’ to the onset of changes in environmental quality.

Based on the results of the atmospheric depo- sition classification of the year 1989, a methodical ap- proach should be introduced, which—based on the modelled total deposition rates—enables us to cha- racterise the input situation of forest monitoring plots and to delimit load areas in Germany. In 1989, the deposition situation in nearly 1,800 forest monitoring sites (BZE/extensive Soil Condition Inventory) in Ger- many could be explained by four factors (or three, excluding sea salt impact) with the help of a factor analysis. The factor values were grouped into six depo- sition types with typical compounds and regional pat- terns. The classified input rates of the soil inventory plots adequately represent the stress situation and deposition changes in Germany. The application of the statistical approach on the level of Brandenburg clarifies the special local input situation. Due to the special combination of deposed elements, the sources of emis- sions can be characterised as well. When the soil inventory is repeated, a project planned for 2006, this approach can be used in order to determine homogenous areas for stratified data evaluation.

In the 1970s unexpected forest damages, called “new type of forest damage” or “forest decline”, were observed in Germany and other European countries. The Federal Republic of Germany and the German Federal States implemented a forest monitoring system in the early 1980s, in order to monitor and assess the forest condition. Due to the growing public awareness of possible adverse effects of air pollution on forests, in 1985 the ICP Forests was launched under the convention on long-range transboundary air pollution (CLRTAP) of the United Nations Economic Commission for Europe (UN-ECE). The German experience in forest monitoring was a base for the implementation of the European monitoring system. In 2001 the interdisciplinary case study “concept and feasibility study for the integrated evaluation of environmental monitoring data in forests”, funded by the German Federal Ministry of Education and Research, concentrated on in-depths evaluations of the German data of forest monitoring. The objectives of the study were: (a) a reliable assessment of the vitality and functioning of forest ecosystems, (b) the identification and quantification of factors influencing forest vitality, and (c) the clarification of cause-effect-relationships leading to leaf/needle loss. For these purposes additional data from external sources were acquired: climate and deposition, for selected level I plots tree growth data, as well as data on groundwater quality. The results show that in particular time series analysis (crown condition, tree growth, and tree ring analysis), in combination with climate and deposition are valuable and informative, as well as integrated evaluation of soil, tree nutrition and crown condition data. Methods to combine information from the extensive and the intensive monitoring, and to transfer process information to the large scale should be elaborated in future.

The 17-alpha-substituted triterpene 1 [(17alpha)-23-(E)-dammara-20,23-diene-3beta,25-diol] showed promising activity in animal models of immunosuppression and inflammation. Using a mouse model for inflammatory skin diseases (oxazolone-induced allergic contact dermatitis, ACD) as the directing in vivo test system, Structure-activity-relationship studies with the aim to understand the necessary structural requirements for the biological activity of 1 were conducted. Furthermore, we anticipated to identify biologically active compounds with the 17beta configuration, which are thermodynamically more stable and much easier to synthesize. This was achieved by identifying the 17-beta substituted dammarane 5B and its analogues.

We report the synthesis and results from the in vitro evaluation of 6-(adamantan-2-ylidene-hydroxybenzoxazole)-O-sulfamate 1 as an irreversible inhibitor of human steroid sulfatase (STS). Highly straightforward, condensation of 2-methyl-6-hydroxybenzoxazole with 2-adamantanone, subsequent elimination of water and sulfamoylation provide the title compound in 45% overall yield from the inexpensive 2,4-dihydroxyacetophenone. 1 was found to be a potent irreversible inhibitor of purified human steroid sulfatase (STS) and specific for this enzyme relative to human arylsulfatases A and B. In cellular assays with human keratinocytes, sebocytes and fibroblasts, 1 blocked STS activity with IC(50) values in the range of 0.15-0.8 nM, and in MCF-7 breast cancer cells with IC(50)=2.3 nM, while it did not bind to estrogen receptors alpha and beta. Thus, 1 is a candidate for further investigation of its potential as a drug to be used in androgen- and estrogen-dependent diseases.

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.

Netherton syndrome (NS; OMIM 256500) is a genetic skin disease resulting from defects in the serine protease inhibitor Kazal-type 5 (SPINK5) gene, which encodes the protease inhibitor lympho-epithelial Kazal type inhibitor (LEKTI). We established a SPINK5 knockdown skin model by transfecting SPINK5 small interfering RNA (siRNA) into normal human epidermal keratinocytes, which were used together with fibroblast-populated collagen gels to generate organotypic skin cultures. This model recapitulates some of the NS skin morphology: thicker, parakeratotic stratum corneum frequently detached from the underlying epidermis and loss of corneodesmosomes. As enhanced serine protease activity has been implicated in the disease pathogenesis, we investigated the impact of the kallikreins KLK5 [stratum corneum trypsin-like enzyme (SCTE)] and KLK7 [stratum corneum chymotrypsin-like enzyme (SCCE)] on the SPINK5 knockdown phenotype by generating double knockdowns in the organotypic model. Knockdown of KLK5 or KLK7 partially ameliorated the epidermal architecture: increased epidermal thickness and expression of desmocollin 1 (DSC1), desmoglein 1 (DSG1) and (pro)filaggrin. Thus, inhibition of serine proteases KLK5 and KLK7 could be therapeutically beneficial in NS.

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC.The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC “memory” of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

We analyzed the effect of isoprenoid depletion by fluvastatin on bradykinin (BK)- and epidermal growth factor (EGF)-mediated Ca2+ mobilization and prostaglandin E2 production, in the human keratinocyte cell line HaCaT. BK and EGF stimulated Ca2+ mobilization in an agonist-dependent manner. The synthesis of prostaglandin E2 paralleled the level of Ca2+ mobilization induced by BK and EGF. Treatment with fluvastatin increased the EGF-promoted but not the BK-promoted Ca2+ mobilization and prostaglandin E2 production in Ca(2+)-containing medium. In the absence of extracellular Ca2+, fluvastatin treatment led to an increase in intracellular Ca2+ release by both agonists. This effect was abolished by depleting the intracellular pool of Ca2+ with thapsigargin. Our findings showed that the intracellular Ca2+ release was dependent on the metabolism of mevalonate and that the Ca2+ mobilization modulated prostaglandin E2 synthesis in human keratinocytes.

As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the leptomycin family, the known leptomycin A (1), leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev, which is required for the cytoplasmic expression of unspliced and incompletely spliced viral mRNAs, is located predominantly in the nucleolus. In this study, we show that Rev translocates from the nucleolus to the cytoplasm in HeLa and COS cells transfected with Rev under conditions where rRNA synthesis is inhibited (e.g., with actinomycin D). Dominant-negative mutants with mutations in the activation domain of Rev, which are known to inhibit wild-type Rev function in trans, are unable to leave the nucleus upon actinomycin D treatment. More importantly, when present in excess, these mutants inhibit the translocation of wild-type Rev. This correlation of inhibitory activities suggests that Rev function depends on its transport to and presence (at least transient) in the cytoplasm. In this context, we discuss the possibility that Rev is actively involved in the transport of HIV-1-specific mRNAs containing the Rev response element (a highly structured RNA sequence, which is specifically recognized by the Rev trans-activator). We also discuss the potential of nucleocytoplasmic export of Rev as a target for anti-HIV chemotherapy.

Under equilibrium conditions, the affinities of five anti-IgG2a mAb isolated from virus-infected mice were comparable to other high-affinity auto-antibodies. Similar to rheumatoid factors, these anti-IgG2a auto-antibodies bound to aggregated or complexed IgG2a with 50 to 1500-fold higher avidity than their monomeric counterparts. Despite their high functional affinity to IgG2a, flow cytometric analysis revealed no binding or marginal mAb binding to four distinct lines of B cells expressing different densities of membrane-anchored IgG2a. If, however, surface IgG2a was cross-linked by polyclonal light chain-specific antibodies, IgM and IgA mAb binding resulted, and was detected as an increase in mean fluorescence intensity compared with isotype-matched control antibodies. The binding of one IgM mAb to cross-linked IgG2a patches of the cell surface was also visualized by confocal microscopy. Pretreatment of cells with aggregated IgG2a caused increased fluorescence intensity, demonstrating that the IgM and IgA mAb were also able to interact with IgG2a aggregates bound on the B cell surface via Fc gamma RIIB. It also permitted efficient co-ligation of the aggregated B cell receptors (BCR) with Fc gamma RIIB-fixed immune complexes known to deliver a negative signal in B cell activation. Cross-linking of IgG2a complexes bound to Fc gamma RI on macrophages or dendritic cells with antigen-specific BCR and/or T cells via their Fc gamma RIIB may accelerate the physical contact of cells involved in the antigen-specific response.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal cell carcinoma (BCC) is a distinctive manifestation in nevoid basal cell carcinoma syndrome (NBCCS) patients. Both inherited and acquired mutations of patched 1 (PTCH1), a tumor-suppressor gene controlling the activity of Smoothened (SMO), are the primary cause of the constitutive activation of the Hedgehog (HH) pathway, leading to the emergence of BCCs in NBCCS. LDE225, a distinct, selective antagonist of SMO, showed potent inhibition of basaloid tumor nest formation and mediated regression of preformed basaloid tumors in organ cultures of skin derived from Ptch1 heterozygous knockout mice. In a double-blind, randomized, vehicle-controlled, intraindividual study, a total of 8 NBCCS patients presenting 27 BCCs were treated twice daily with 0.75% LDE225 cream or vehicle for 4 weeks. Application of 0.75% LDE225 cream was well tolerated and showed no skin irritation. Of 13 LDE225-treated BCCs, 3 showed a complete, 9 a partial, and only 1 no clinical response. Except for one partial response, the vehicle produced no clinical response in any of the 14 treated BCCs. Treatment with 0.75% LDE225 cream in NBCCS patients was very well tolerated and caused BCC regression, thus potentially offering an attractive therapeutic alternative to currently available therapies for this indication. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub

As one of the regulatory gene products in the HIV-1 genome, Rev protein must be translocated from the nucleus to the cytoplasm to exert its function. Therefore, inhibition of Rev protein translocation could be a useful target for HIV therapy. An extract from the Streptomyces strain A92-308902 with very potent inhibitory activity was found in the course of a high throughput screening with a Rev translocation assay (RTA). Bioassay-guided fractionation with gel filtration, normal-phase and reversed-phase chromatography yielded six RTA-active metabolites belonging to the leptomycin family, the known leptomycin A (1), leptomycin B (2), kazusamycin B (3), and kazusamycin A (4). and the hitherto unknown dilactonmycin (5) and delactonmycin (6), together with an inactive cyclic hexadepsipeptide L-156,620 (7). The structures were established mainly by spectroscopic methods (UV, FT-IR, FAB-MS, 1H-NMR, 13C-NMR(JMOD), DQ-COSY, ROESY, HSQC, and HMBC). The configuration of all CC bonds of 1–6 was unambiguously established by analysis of coupling constants and ROESY spectra. All isolated leptomycins 1–6 inhibit Rev translocation at nanomolar concentrations. Six derivatives (2a–c and 4a–c) of leptomycin B (2) and kazusamycin A (4) were also prepared and tested in the RTA for preliminary investigations on structure-activity relationships.

European Journal of Forest Research, 128 (6), ISSN:1612-4677, ISSN:1612-4669

Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjected into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigen-antibody complex strongly suggest protein unfolding is not required for nuclear protein transport.

PKC isoforms tau, alpha, and beta play fundamental roles in the activation of T cells and other immune cell functions. Here we show that the PKC inhibitor AEB071 both abolishes the production of several cytokines by activated human T cells, keratinocytes, and macrophages in vitro and inhibits an acute allergic contact dermatitis response in rats. To translate these findings into humans, single and multiple ascending oral doses of AEB071 were administered to healthy volunteers and patients with psoriasis, respectively. AEB071 was well tolerated with no clinically relevant laboratory abnormalities. Ex vivo stimulation of lymphocytes from subjects exposed to single doses of AEB071 resulted in a dose-dependent inhibition of both lymphocyte proliferation and IL2 mRNA expression. Clinical severity of psoriasis was reduced up to 69% compared with baseline after 2 weeks of treatment, as measured by the Psoriasis Area Severity Index (PASI) score. The improvement in psoriasis patients was accompanied by histological improvement of skin lesions and may be partially explained by a substantial reduction of p40+ dermal cells, which are known to mediate psoriasis. These data suggest that AEB071 could be an effective novel treatment regimen for psoriasis and other autoimmune diseases, and that AEB071 warrants long-term studies to establish safety and efficacy.

The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds.

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl–arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest Ki = 76 nM), they showed only weak inhibition of STS activity in cells (lowest IC50 around 2 μM). Extended structure–activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC50 = 270 nM).

Steroid sulfatase (STS) is an attractive target for the potential therapy of a number of estrogen- and androgen-dependent disorders. Most potent STS inhibitors known so far act as irreversible enzyme blockers and feature an aryl sulfamate moiety; even minor modifications at the sulfamate group result in drastically decreased activity. On the basis of a recently reported subclass of highly potent STS inhibitors, i.e., chromenone sulfamates, we now extended the investigation of structure-activity relationships to hitherto unstudied sulfamate replacements. Thereby, we discovered 2-(1-adamantyl)-4-(thio)chromenone-6-carboxylic acids (5d and 5j) as potent, reversible inhibitors of STS. In a cell-free system using purified human STS, both new inhibitors show similar Ki values (0.50 microM and 0.53 microM, respectively). However, the thio analogue 5j is superior to 5d (IC50 = 0.18 microM versus 9.4 microM) in a cellular assay system using CHO cells overexpressing STS. Compound 5j is an example of a reversible STS inhibitor with potent activity toward the target enzyme in a cellular test system. Moreover, 5d,j are stable and have no estrogenic potential.

We report the synthesis and results from the in vitro evaluation of 6-(adamantan-2-ylidene-hydroxybenzoxazole)-O-sulfamate 1 as an irreversible inhibitor of human steroid sulfatase (STS). Highly straightforward, condensation of 2-methyl-6-hydroxybenzoxazole with 2-adamantanone, subsequent elimination of water and sulfamoylation provide the title compound in 45% overall yield from the inexpensive 2,4-dihydroxyacetophenone. 1 was found to be a potent irreversible inhibitor of purified human steroid sulfatase (STS) and specific for this enzyme relative to human arylsulfatases A and B. In cellular assays with human keratinocytes, sebocytes and fibroblasts, 1 blocked STS activity with IC(50) values in the range of 0.15-0.8 nM, and in MCF-7 breast cancer cells with IC(50)=2.3 nM, while it did not bind to estrogen receptors alpha and beta. Thus, 1 is a candidate for further investigation of its potential as a drug to be used in androgen- and estrogen-dependent diseases.

The stereochemical course of the oxidation of chiral oxazolidinone-substituted enecarbamates has been studied for singlet oxygen ((1)O(2)), dimethyldioxirane (DMD), and m-chloroperbenzoic acid (mCPBA) by examining of the special structural and stereoelectronic features of the enecarbamates. Valuable mechanistic insight into these selective oxidations is gained. Whereas the R(1) substituent on the chiral auxiliary is responsible for the steric shielding of the double bond and determines the sense of the pi-facial diastereoselectivity, structural characteristic such as the Z/E configuration and the nature of the R(2) group on the double bond are responsible for the extent of the diastereoselectivity. Stereoelectronic steering by the vinylic nitrogen functionality controls the mode selectivity (ene reaction vs [2+2] cycloaddition) in the case of (1)O(2).

Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases. Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput screening. A series of analogues was prepared in order to explore the essential structural elements for STS inhibition, and first structure-activity relationships were established. Mechanistic investigations revealed that the compounds are reversible, competitive inhibitors of STS.

Chiral oxazolidinone-substituted enecarbamates 1 are epoxidized in a diastereoselectivity up to 93:7 for both DMD and mCPBA. The diastereofacial differentiation depends on the steric interaction between the R1 substituent on the oxazolidinone ring and the incoming electrophile. The stereochemical course of epoxidation was assessed by chemical correlation with the known optically active diols.

[structure: see text] Chiral oxazolidinone-substituted enecarbamates 1 are epoxidized in a diastereoselectivity up to 93:7 for both DMD and mCPBA. The diastereofacial differentiation depends on the steric interaction between the R(1) substituent on the oxazolidinone ring and the incoming electrophile. The stereochemical course of epoxidation was assessed by chemical correlation with the known optically active diols.

The cellular target of leptomycin B (LMB), a nuclear export inhibitor, has been identified as CRM1 (exportin 1), an evolutionarily conserved receptor for the nuclear export signal of proteins. However, the mechanism by which LMB inhibits CRM1 still remains unclear. CRM1 in a Schizosaccharomyces pombe mutant showing extremely high resistance to LMB had a single amino acid replacement at Cys-529 with Ser. The mutant gene, named crm1-K1, conferred LMB resistance on wild-type S. pombe , and Crm1-K1 no longer bound biotinylated LMB. 1 H NMR analysis showed that LMB bound N -acetyl- l -cysteine methyl ester through a Michael-type addition, consistent with the idea that LMB binds covalently via its α,β-unsaturated δ-lactone to the sulfhydryl group of Cys-529. When HeLa cells were cultured with biotinylated LMB, the only cellular protein bound covalently was CRM1. Inhibition by N -ethylmaleimide (NEM), an alkylating agent, of CRM1-mediated nuclear export probably was caused by covalent binding of the electrophilic structure in NEM to the sulfhydryl group of Cys-529, because the crm1-K1 mutant showed the normal rate for the export of Rev nuclear export signal-bearing proteins in the presence of not only LMB but also NEM. These results show that the single cysteine residue determines LMB sensitivity and is selectively alkylated by LMB, leading to CRM1 inactivation.

Leptomycin B (LMB) is a Streptomyces metabolite that inhibits nuclear export of the human immunodeficiency virus type 1 regulatory protein Rev at low nanomolar concentrations. Recently, LMB was shown to inhibit the function of CRM1, a receptor for the nuclear export signal (NES). Here we show evidence that LMB binds directly to CRM1 and that CRM1 is essential for NES-dependent nuclear export of proteins in both yeast and mammalian cells. Binding experiments with a biotinylated derivative of LMB and a HeLa cell extract led to identifying CRM1 as a major protein that bound to the LMB derivative. Microinjection of a purified anti-human CRM1 antibody into the mammalian nucleus specifically inhibited nuclear export of NES-containing proteins, as did LMB. Consistent with this, CRM1 was found to interact with NES, when assayed with immobilized NES and HeLa cell extracts. This association was disrupted by adding LMB or purified anti-human CRM1 antibody. The inhibition of CRM1 by LMB was also observed in fission yeast. The fission yeast crm1 mutant was defective in the nuclear export of NES-fused proteins, but not in the import of nuclear localization signal (NLS)-fused proteins. Interestingly, a protein containing both NES and NLS, which is expected to shuttle between nucleus and cytoplasm, was highly accumulated in the nucleus of the crm1 mutant cells or of cells treated with LMB. These results strongly suggest that CRM1 is the target of LMB and is an essential factor for nuclear export of proteins in eukaryotes.

Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of >5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.

Background: The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. Results: The Streptomyces metabolite, leptomycin B, and other antibiotics of the leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, leptomycin B suppresses HIV-1 replication. Conclusions: Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.

In order to monitor the effects of drugs on interleukin-8 (IL-8) production by cells, a microtiter plate assay that determines four parameters simultaneously was established (i) levels of secreted IL-8 (supernatant ELISA), (ii) levels of intracellular IL-8 (cell ELISA), (iii) intracellular localization (fluorescence microscopy), and (iv) the amount of cellular protein (colorimetric assay). The quantitative and qualitative determination of intracellular IL-8 was achieved by immunofluorescence using the ELF-detection system (Molecular Probes, Eugene, OR), which is approximately 10 times more sensitive than conventional immunofluorescence detection systems. Thus, a 32× objective magnification (without immersion oil) is sufficient to precisely assess the subcellular localization of IL-8. Experiments were carried out with human umbilical vein endothelial cells. Drugs interfering with transcription or translation and inhibitors of protein kinase C inhibited both production and secretion of IL-8. Brefeldin A (BFA), colchicine, and the HMGCoA-reductase inhibitor fluvastatin disrupted the characteristic Golgi localization of IL-8, but only BFA inhibited its secretion. This assay can therefore be used to distinguish drugs that inhibit both IL-8 synthesis and secretion from those that inhibit IL-8 secretion only. It can easily be adapted to other cellular proteins for which a sensitive detection method is required.

Chemolrines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not yet clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilised on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1α on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.

Laser-scanning confocal microscopy was used in conjunction with a highly fluorescent Ca 2+ indicator fluo-3 to visualize real-time alterations in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) in individual living Jurkat T-cells during the first minutes of activation by phytohaemagglutinin (PHA) at the physiological temperature (37°C). With a delay of 30–120 s, PHA induced a strong [Ca 2+ ] i peak in the micromolar range (1–3 μM). The rise in [Ca 2+ ] i lasted for 1–2 minutes, and was followed by a sustained plateau of elevated [Ca 2+ ] i in the 0.2–0.5 μM range. Some cells (10–20%) responded with additional asynchronous 0.5–1.5 μM peaks during the plateau phase. These oscillations continued for 10–20 minutes. The spans of the peaks ranged from 30 to 100 s, intervals between peaks varied from 60 to 300 s. It was shown that the initial [Ca 2+ ] i peak was associated with Ca 2+ mobilisation from internal sources, whereas the plateau was maintained by an influx of Ca 2+ from external medium. In K + -rich medium or in the presence of quinine, a K + channel blocker, no secondary response to PHA-activation characterised by an elevated plateau was observed. The data suggest that the Ca 2+ influx was dependent on the membrane potential and/or the extracellular K + -concentration. Optical sectioning showed that the intracellular Ca 2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including those exhibiting Ca 2+ oscillations. This suggests that Ca 2+ signals are localized not only in cytoplasm at the cell plasma membrane but can be also transferred directly into the nucleus.