Pan-neurotrophin receptor p75NTR expression is strongly induced in lesional atopic mast cells.

BACKGROUND: Neurotrophins such as nerve growth factor or brain-derived neurotrophic factor influence neuronal proliferation and differentiation via the low-affinity pan-neurotrophin receptor p75NTR that may play a pivotal role in linking the immune with the nervous system. Because the precise regulation of p75NTR gene transcription in mast cells under states of allergic inflammation has not been investigated in detail so far, the present studies assessed the gene regulation and expression of this receptor. METHODS: Transcriptional expression of p75NTR in human skin was studied in isolated cutaneous cells by means of RT-PCR. In situ lesional mast cell p75NTR expression was analysed by immunohistochemistry. RESULTS: The p75NTR mRNA expression was found in isolated human skin mast cells and keratinocytes. Lower mRNA levels were present in fibroblasts and melanocytes but no transcripts were found in endothelial cells. The p75NTR protein expression was found in situ in lesional and non-lesional mast cells. A significantly increased expression of p75NTR protein was found in atopic dermatitis lesional mast cells when compared with control mast cell expression (P<0.05). CONCLUSION: The demonstration of an increased level of p75NTR gene transcription in lesional mast cells points to an induction of low-affinity neurotrophin receptor sensitivity of mast cells under states of allergic inflammation. Topically administered neurotrophin receptor-modulating compounds may act as anti-inflammatory mediators in cutaneous allergic inflammation.

Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206.

Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.

Mechanism of proline-specific proteinases: (I) Substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney and proline-specific endopeptidase from Flavobacterium meningosepticum, was investigated with a series of N-terminal unprotected (dipeptidyl peptidases IV) and succinylated dipeptidyl-p-nitroanilides (proline-specific endopeptidase). Both enzymes are specific for the S configuration of the amino-acid residue in P1 and P2 position if the penultimate residue is proline. In the case of alanine substrates (Ala in P1, dipeptidyl peptidase IV hydrolyzes such compounds where the configuration of the P2 residue is R. The penultimate residue with dipeptidyl peptidase IV can be, beside proline and alanine, dehydroproline, hydroxyproline and pipecolic acid. Proline substrates (Pro in P1) with an R configuration in P2 are inhibitors of the hydrolysis of proline substrates with an S,S configuration in an uncompetitive (dipeptidyl peptide IV) or mixed inhibition type (proline-specific endopeptidase). Derivatives of Gly-Pro-pNA where the N-terminal amino group is methylated are hydrolyzed by dipeptidyl peptidase IV.

Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.

Although the TGF-beta family of growth factors probably regulates skin and hair follicle development, its exact role is still quite ill-defined. Here, we characterize the correlative expression pattern of the interdependent high affinity receptor proteins for TGF-beta1 and TGF-beta3, TGF-beta receptor type I (TGF-betaRI) and TGF-beta receptor type II (TGF-betaRII), during hair follicle development and cycling in C57BL/6 mice. During neonatal follicle development, TGF-betaRII immunoreactivity is confined to epithelial cells. Focal epidermal TGF-betaRII expression is seen even before actual hair placode formation. In contrast to the TGF-betaRII immunoreactivity in the outer root sheath, precortical hair matrix and inner root sheath cells were TGF-betaRII negative during hair bulb morphogenesis. TGF-betaRI (Alk-5) immunoreactivity largely overlapped the TGF-betaRII expression pattern, but was more widespread. During hair follicle cycling in adolescent mice, TGF-betaRII immunoreactivity was restricted to follicles, and was strikingly hair cycle dependent (maximal immunoreactivity: anagen VI and early catagen). Again, TGF-betaRI (Alk-5) immunoreactivity co-localized with TGF-betaRII immunoreactivity, but was more extensive. Reverse transcriptase polymerase chain reaction analysis of TGF-betaRII mRNA confirmed peak transcript levels in back skin with most hair follicles in the anagen VI-catagen transformation. mRNA levels of TGF-betaRI (Alk-5) did not vary significantly during the hair cycle, whereas those of TGF-betaRI (threonine-serine kinase 7 L) declined during early anagen, and were maximal during the anagen-catagen transition. This provides a basis for defining the choreography of TGF-beta-related signalling during hair follicle morphogenesis and cycling, introduces intraepidermal TGF-betaRII immunoreactivity as a marker for imminent follicle development, and supports the concept that both TGF-betaRII and TGF-betaRI stimulation is involved in, but not restricted to, the control of catagen induction.

Mast cell and myeloid marker expression during early in vitro mast cell differentiation from human peripheral blood mononuclear cells.

UNLABELLED: In order to characterize the phenotype of human mast cell precursors in the peripheral blood mononuclear fraction and its alterations during in vivo mast cell differentiation, cells were studied before and during culture with stem cell factor or stem cell factor-containing cell supernatants. Prior to culture, 86% of cells were immunoreactive for the monocytic marker CD14, slightly fewer for CD11b and CD64, < 10% expressed FcepsilonRIalpha, rare cells were CD34 + ( < 0,1%), and none stained for CD1, CD33, c-Kit, and tryptase. After 2 wk of culture, there was de novo expression of c-Kit (14% - 43% positive cells), tryptase (26% - 79%), CD33 (57%), and CD64 (64%), an upregulation of FcepsilonRIalpha (23% - 52%), CD11b (93%), and CD68 (95%), but no expression of CD34. Levels of mRNA for FcepsilonRIalpha and c-Kit were detectable prior to culture and increased during culture, together with de novo expression of tryptase. Double staining after 2 wk of culture showed that FcepsilonRIalpha-positive cells were mostly CD14 + (90%), CD64 + (82%), and CD68 + (52%) on flow cytometry. Intracellular tryptase activity was first detectable after 1 wk of culture, increased FcepsilonRIalpha expression was only detectable by week 2. Cultured cells acquired the ability to release histamine during IgE-dependent stimulation, and culture with the c-Kit antibody YB5.B8 resulted in a downregulation of tryptase and FcepsilonRIalpha, but not of c-Kit. These data show that human mast cells develop from c-Kit- and tryptase-negative precursors in the myelomonocytic fraction of peripheral blood and that they upregulate, maintain, and share many phenotypic characteristics of cells from the monocyte/macrophage lineage during early phases of in vitro differentiation. KEYWORDS: c-kit/FcepsilonRI/SCF/tryptase.

Comparative cytokine release from human monocytes, monocyte-derived immature mast cells, and a human mast cell line (HMC-1).

To obtain further information regarding the role of cytokines during mast cell differentiation, we have investigated changes of cytokine secretion in mast cells developing from the human peripheral blood monocytic cell fraction during culture with fibroblast-derived conditioned media. The influence of stem cell factor and an antibody to the respective receptor in our culture system was studied as well. Interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)alpha were spontaneously secreted by cultured cells at day 1 and decreased markedly by day 14. Similar changes occurred also during culture with stem cell factor and were partially abrogated by an anti-receptor antibody. IL-8 was secreted at a high level throughout the culture, whereas no spontaneous secretion of IL-2, IL-3, IL-4, and IL-7 was measured at all. Upon stimulation with phorbol myristate acetate and A23187, cultured cells showed substantially more release of IL-3 and TNF-alpha after 14 d of culture, compared to peripheral blood monocytic cells. Preformed TNF-alpha was found in one of three monocytic cell preparations from peripheral blood, but not in monocytic cell-derived mast cells. During mast cell differentiation, cytokines from monocytic cells are therefore downregulated while the cells assume a pattern typically found in mast cells.

High-dose ultraviolet A1 (UVA1), but not UVA/UVB therapy, decreases IgE-binding cells in lesional skin of patients with atopic eczema.

In order to further elucidate the mechanisms by which high-dose ultraviolet A1 (UVA1) therapy leads to improvement in patients with atopic eczema, we assessed skin sections from patients before and after high-dose UVA1 therapy (n = 5) or conventional UVA/UVB therapy (n = 4) for changes in Langerhans cells and mast cells expressing the high-affinity IgE receptor Fc epsilon RI and in surface-bound IgE by histochemical and immunohistochemical techniques. The two treatment groups exhibited different patterns of changes in the number of Fc epsilon RI+, CD1a+, and mast cells within the dermis: The density of both Langerhans cells and mast cells was decreased after high-dose UVA1 therapy, but not after UVA/UVB therapy. High-dose UVA1 and UVA/UVB therapy significantly increased the number of CD1a+ cells within the epidermis, but only high-dose UVA1 reduced the relative number of IgE+ intraepidermal Langerhans cells typically found in atopic eczema. Reduction of numbers of dermal Langerhans cells and mast cells, as well as relative numbers of intraepidermal IgE+ Langerhans cells, was closely linked to significant clinical improvement by high-dose UVA1, but not UVA/UVB therapy. These studies support the notion that IgE-binding cutaneous cells are involved in the pathogenesis of atopic eczema. We propose that UVA1 radiation exerts its effects in atopic eczema, at least in part, by inhibiting Langerhans cell migration out of the epidermis and, in particular, by reducing the number of IgE-bearing Langerhans cells and mast cells in the dermis.

Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior.

Mastocytosis represents a mast cell proliferative disease that generally runs a benign clinical course, with spontaneous remissions mostly by puberty in childhood-onset disease, although rare forms, particularly in adult-onset disease, can be associated with (pre)malignant hematologic disorders and very rarely present as mast cell leukemia or malignant mastocytosis. Reasons for this divergent clinical behavior of childhood- versus adult-onset disease are unknown. Recently, two activating mutations in the intracellular domain of the proto-oncogene c-kit, which encodes a tyrosine kinase receptor for the mast cell growth factor stem cell factor, have been detected in the human leukemic mast cell line HMC-1. We have therefore studied lesional skin biopsies from patients with adult- and childhood-onset indolent mastocytosis for the presence of these codon 560 and 816 mutations. C-kit coding DNA sequences were amplified and analyzed by mutation-specific restriction analyses, and mutated polymerase chain reaction products were additionally cloned and sequenced. The codon 816 mutation was found in all six samples from adult patients, but not in any of the 11 specimens from children. In addition, the codon 560 mutation could be demonstrated for the first time in indolent mastocytosis, namely in two of four specimens from adult patients, but not in those from two children. These data thus provide a possible explanation for the divergent clinical behavior of adult- versus childhood-onset indolent mastocytosis, with the first being associated with an activating mutation, possibly as part of a neoplastic process, and the latter representing most likely a reactive process of an as yet unknown pathogenesis.

Expression of mast cell growth modulating and chemotactic factors and their receptors in human cutaneous scars.

In order to explore possible mechanisms involved in the previously documented turnover of mast cell subpopulations in human cutaneous scars, we have examined selected factors known to stimulate and/or modulate mast cell hyperplasia (SCF, NGF, TGFbeta1, GM-CSF) and their receptors in human cutaneous scar tissue. On immunohistochemistry, numbers of SCF- and TGFbeta1-positive cells were significantly increased in the epidermis and throughout the dermis in scars (n = 27) of varying ages (4-369 d old), compared with normal skin (n = 12). Furthermore, TRbetaRI, II, and the NGF-p75 receptors were significantly increased in the epidermis, TRbetaRI and NGF-TrkA throughout the dermis, and TRbetaRII, NGF-p75, and GM-CSFR only in the mid- and lower dermis of scars. NGF and GM-CSF expression was in contrast scarce and weak, with no differences between normal skin and scars. In tissue extracts, mRNA levels of SCF, TGFbeta1, TRbetaI and II, and both NGF-receptors, but not GM-CSFR, were significantly increased as well. TRbetaI and II were identified in up to 90% and 83%, respectively, of isolated normal skin mast cells on flow cytometry, and GM-CSFR and NGFR-p75 were identified on 70% and 73%, respectively, of avidin-positive normal mast cells on double immunofluorescence microscopy. As described before for the SCF receptor KIT, GM-CSFR and NGFR-p75 were partly or entirely downregulated on avidin-positive mast cells in scars. The marked upregulation of TGFbeta1, its type I and II receptors, and SCF suggest that these factors play a major role in the orchestration of mast cell increase in human cutaneous scars whereas the role of NGF and GM-CSF is less clear, despite the significant upregulation of their receptors.

Altered expression of mast cell chymase and tryptase and of c-Kit in human cutaneous scar tissue.

In order to explore a possible involvement of mast cells during human wound healing, we studied sections from scars (4-369-d-old) (N = 20) and normal skin (N = 10) for mast-cell-specific tryptase and chymase by enzyme histochemistry, for the stem cell factor receptor c-Kit and the melanosomal marker TA99 by immunohistochemistry, and for simultaneous c-Kit expression and avidin fluorescence by double staining. Enzyme activities and mRNA expression were also studied in tissue extracts. Chymase-reactive mast cell numbers as well as chymase activity and mRNA expression were reduced in all scars, whereas overall numbers of tryptase-reactive cells did not differ from normal skin, although tryptase activity and mRNA expression were increased in scar extracts. In contrast, numbers of c-Kit positive cells were significantly increased in old scars, and in the mid and lower dermis of all scars. A marked reduction of c-Kit reactivity was noted, however, in avidin-positive dermal mast cells and in epidermal basal cells, despite unchanged numbers of melanosome-positive cells, with an associated overall decrease of c-Kit mRNA in scar extracts. These data thus show that numbers of resident mast cells are very low in human cutaneous scars, suggesting massive mediator release from these cells into fresh wounds. Downregulation of stem cell factor receptors may also prevent these cells from increasing in number even in old scars. Instead, scar tissue is populated by a mast cell subpopulation that is chymase-, avidin-, tryptase +, c-Kit +, reflecting most probably an increased immigration and/or proliferation of immature mast cells and their precursors.

Release of stem cell factor from a human keratinocyte line, HaCaT, is increased in differentiating versus proliferating cells.

Stem cell factor, a recently discovered growth factor for hematopoietic stem cells, mast cells, and melanocytes, was initially reported to be produced by fibroblasts. In this study, we investigated the secretion of this factor from human HaCaT cells during in vitro culture and compared it to synthesis by cells in the skin. Release of stem cell factor from freshly cultured keratinocytes was comparable to that of HaCaT cells and was nearly half that produced by fibroblasts and umbilical vein endothelial cells. No stem cell factor was detectable in culture supernatants of melanocytes. HaCaT cells underwent spontaneous differentiation after a period of proliferation until confluency. Depending on duration of culture, they released increasing amounts of stem cell factor (approximately 150 pg/10(6) cells on day 3 (proliferating cells) vs approximately 450 pg/10(6) cells on day 14 (differentiating cells) measured by enzyme-linked immunosorbent assay. Stimulation for 24 h with the calcium ionophore A 23187 (10(-6) to 10(-8) M) further enhanced release. Western blot analysis of HaCaT cell lysates with a stem cell factor antibody revealed two proteins with the known molecular weights of membrane-bound and soluble stem cell factor. By semiquantitative reverse transcriptase polymerase chain reaction, full-length as well as spliced type stem cell factor mRNA was found to be increased in differentiating versus proliferating HaCaT cells. Keratinocytes are thus potentially important sources of stem cell factor in human skin, and HaCaT cells provide a useful model for further studies of stem cell factor from keratinocytes.

Intercellular adhesion molecule-1 and hair follicle regression.

Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology.

Induction of MHC class II antigen expression on human HMC-1 mast cells.

Mast cells are not only the primary effector cells of immediate type immune reactions, but they have recently also been considered to contribute to the induction of an immune response. Data on the ability of the cells to express major histocompatibility complex (MHC) antigens and the mechanisms involved are however controversial or unclear. We have therefore studied the expression of MHCI and the induction of MHCII molecules on leukemic HMC-1 mast cells by immunohistochemistry. Cells were incubated for up to 72 h in the presence of IFN gamma, TNF alpha and IL-4, and immunohistochemical staining was done with monoclonal antibodies with specificity for HLA class I heavy chain, HLA-DQ (Tü22), HLA-DR (Tü36) and HLA-DQ, -R and -P (Tü35). All unstimulated mast cells expressed MHC class I, but almost no class II antigens. Incubation with IFN gamma caused a rapid, dose-dependent induction of MHC class II molecules, with Tü35 staining maximally one third of the cells within 24 h at the highest dose tested (100 IU/ml), with decline on extended culture. TNF alpha (2 ng/ml) was less effective but caused more persistent induction with time. IL-4 (200 ng/ml) had hardly any effects at all. Staining with Tü22 and Tü36 was always lower than with Tü35, and additive or even synergistic results were obtained when cells were stimulated with a combination of IFN gamma and TNF alpha. These findings support the concept that mast cells can facultatively participate in immune recognition processes depending on the type of pathological conditions in their microenvironment which allow expression of MHC class II molecules.

Fibroblast-Derived Factors Induce Different Mast Cell Characteristics in Human Myeloid Cell Lines and Peripheral Monocytes.

Mast cell differentiation markers were studied by culturing monocytic (U937, THP1) a promyelotic (HL60) and a human mast cell line (HMC1) and peripheral human blood monocytes with fibroblast supernatants and feeder layers. Mast cell differentiation occurred on the basis of metachromasia, chloroacetate esterase, expression of mast cell-specific antigens (APAAP) and production of tryptase in all cell types, but to varying degrees and not at all with a FcεRI marker in monocytic lines. Direct cellular contact is thus not necessary for induction of mast cell differentiation by fibroblasts.

Stem cell factor, a novel cutaneous growth factor for mast cells and melanocytes.

Mechanisms affecting mast cell and melanocyte growth and function are still poorly understood. This report summarizes the current state of knowledge on a recently described growth factor for both these cell types and for primitive haematopoietic stem cells. Stem cell factor (SCF), also named mast cell growth factor or kit-ligand, has only recently been cloned and has been shown to be encoded on human chromosome 12. It may be of specific importance in cutaneous physiology and pathology since it is produced by several cell types in the skin (e.g. fibroblasts, keratinocytes, endothelial cells) and since it affects melanocyte and mast cell growth, survival, secretion and adhesion as well as migration into tissues. Defects in the genes encoding for the SCF receptor (c-kit-protein) have been shown to be responsible for human piebaldism. A pathogenetic role in mastocytosis has recently been proposed, but remains to be proven. SCF receptor expression is decreased on cells of some malignant cell lines compared to their physiological counterparts, making it unlikely that SCF is a key factor in malignant transformation and cellular hyperproliferation. In haematopoiesis, SCF acts primarily in concert with other growth factors, and we show here that alone in serum-free culture it has no effect on mast cell growth. Furthermore, there is evidence that besides SCF, additional mast cell growth factors are secreted by fibroblasts and keratinocytes, suggesting a complex orchestration of several growth factors in the regulation of cutaneous growth and differentiation in which SCF plays only one part.

GM-CSF downmodulates c-kit, Fc(epsilon)RI(alpha) and GM-CSF receptor expression as well as histamine and tryptase levels in cultured human mast cells.

GM-CSF is known primarily as a hematopoietic growth factor, but it has also been shown to inhibit mast cell differentiation in vitro. In order elucidate the mechanisms involved, we investigated the effects of GM-CSF in vitro on the differentiation of human leukemic mast cells (HMC-1 cells) and normal cord blood-derived mast cells (CBMC) under the influence of SCF, NGF, and fibroblast supernatant (FS). Under all culture conditions, GM-CSF induced a dose- and time-dependent reduction in intracellular histamine levels, tryptase activity, and numbers of cells immunoreactive for c-Kit and FcepsilonRIalpha. This effect leveled off between 10-100 ng/ml and after 4 days of culture. There was an associated decrease in mRNA expression for c-kit, FcepsilonRIalpha and tryptase. In contrast, no significant changes in the expression of the NGF receptor TrkA were noted under the same conditions. The GM-CSF receptor was found in HMC-1 cells and CBMC at both the mRNA and protein levels, but its expression decreased during culture with FS, and even more markedly during culture with GM-CSF. GM-CSF thus selectively inhibits in vitro induction and/or upregulation of all major mast cell characteristics in HMC-1 cells and CBMC irrespective of the growth factors present, and a concomitant downregulation of GM-CSF receptors can counteract these effects. GM-CSF may therefore function as a regulatory factor in mast cell growth and differentiation under normal and pathological conditions.

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells.

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the Fc epsilon RI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.

Similarities of the substrate cleavage catalyzed by proline specific endopeptidase and dipeptidyl peptidase IV.

16 substrates of the types succinyl-alanyl-alanine-pX-anilide, and succinyl-alanyl-proline-pX anilide having different substituents in para-position of the aryl residue were synthesized and characterized. The influence of electronic as well as hydrophobic substituent constants, sigma and pi, on the hydrolysis of the substrates catalyzed by the enzyme Proline Specific Endopeptidase (PSE) has been investigated. In the Hansch approach, the catalytic constants lg kcat and lg (kcat/KM) of succinyl-alanyl-alanine-pX-anilides hydrolyzed by PSE correlate significantly with electronic substituent constants sigma, whereas no correlation in the case of succinyl-alanyl-proline-pX-anilides. The intercorrelation of the constants of the former substrates with corresponding data from Dipeptidyl Peptidase IV (DP IV) catalyzed hydrolysis of alanyl-alanine-pX-anilides suggest that both enzymes act by similar catalytic mechanism.

Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues.

The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein-ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled µ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.

Predicting the segregation profile of the Pt25Rh75(100) surface from first-principles.

The segregation profile of the Pt(25)Rh(75)(100) surface is studied by the combination of density functional theory calculations with the cluster-expansion method and Monte Carlo simulations. We construct the stability diagram for the surface layers, which allows the prediction of the most stable atomic configuration for a given average concentration in those layers. On this basis, we apply the cluster-expansion Hamiltonian in grand-canonical Monte Carlo simulations for the prediction of the temperature-dependent concentration profile. The experimentally found enrichment of Pt in the top layer and depletion in the second layer is nicely confirmed by the calculations.