Visualization of cyclooxygenase-2 using a 2,3-diarylsubstituted indole-based inhibitor and confocal laser induced cryofluorescence microscopy at 20 K in melanoma cells in vitro

This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors.COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K.COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1.Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.     

At the edge and on the top: molecular identification and ecology of Daphnia dentifera and D. longispina in high-altitude Asian lakes

The occurrence of members of the highly diverse Daphnia longispina complex in Southern and Central Asian high-mountain lakes has been recognized for more than a century. Until now, however, no molecular data have been available for these populations inhabiting the “Roof of the World.” Here, we present the first identification for D. gr. longispina from that region based on a molecular phylogeny. Our findings show that alpine lakes in the Pamir and Himalaya mountains host populations of widespread species of the complex, for which these are the highest known localities. A spineless morph from the Himalaya region, previously labeled as D. longispina var. aspina, was clustering tightly with D. dentifera, while a population from the Pamir mountain range was grouped with D. longispina. In addition, we analyzed ecological data available for lakes in the Khumbu region (Himalaya) to investigate ecological preferences of non-pigmented D. gr. longispina. The identified factors can at least partly be related to avoidance of high UV conditions by this species. We conclude that the widespread species D. dentifera and D. longispina also colonized the Asian high-mountain lakes, and identify the need for further research to trace the possible effect of rapid environmental changes in this region on the diversity and ecology of high-altitude Daphnia populations.     

SULTR3;1 is a chloroplast‐localized sulfate transporter in Arabidopsis thaliana

Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid‐localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1‐GFP localization, Western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss of SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S‐SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.     

Life-cycle associations involving pairs of holococcolithophorid species: intraspecific variation or cryptic speciation?

New holococcolith-heterococcolith life-cycle associations are documented based on observations of combination coccospheres. Daktylethra pirus is shown to be a life-cycle phase of Syracosphaera pulchra and Syracolithus quadriperforatus a life-cycle phase of Calcidiscus leptoporus. In addition, new observations from cultures confirm the life-cycle associations of Crystallolithus braarudii with Coccolithus pelagicus and of Zygosphaera hellenica with Coronosphaera mediterranea. In all four cases previous work has shown that the heterococcolithophorid species is associated with another holococcolithophorid. Two other examples of a heterococcolithophorid being associated with two holococcolithophorids have previously been identified, so this seems to be a common phenomenon. The six examples are reviewed to determine whether a single underlying mechanism is likely to be responsible for all cases. It is concluded that there is no single mechanism but rather that the six examples fall into three categories: (a) in two cases the holococcolith types are probably simply ecophenotypic morphotypes; (b) in two other cases the holococcolith types are discrete and are paralleled by morphometric differences in the heterococcolith types; (c) in the final two cases the holococcolith types are discrete but are not paralleled by any obvious morphological variation in the heterococcolith morphology. We infer that cryptic speciation may be widespread in heterococcolithophorid phases and that study of holococcolithophorid phases can provide key data to elucidate this phenomenon.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

Metabolites from intestinal microbes shape Treg

Intestinal bacterial metabolites are an important communication tool between the host immune system and the commensal microbiota to establish mutualism. In a recent paper published in Science, Wendy Garrett and her colleagues report an exciting role of the three most abundant microbial-derived short-chain fatty acids (SCFA), acetic acid, propionic acid and butyric acid, in colonic regulatory T cell (cTreg) homeostasis.A number of studies have shown that increased cTreg numbers and their immunoregulatory function are promoted by the presence of commensal intestinal microbes (either individual species such as Bacteroides fragilis¹, defined benign consortia of bacteria such as the altered Schaedler flora² or groups of Clostridia³). In a recent paper in Science, Garrett and colleagues report how these effects are generated through molecular exchanges between the host and the enormous load of microbes carried in the lower intestine⁴.Smith et al.⁴ investigated the role of SCFA, which are bacterial fermentation products produced by a wide variety of bacteria through anaerobic acidogenic pathways. SCFA released by colonic bacteria have long been known to be important as a carbon source for colonic epithelial cells⁵. From this new work we can now see that signaling effects of SCFA also regulate cTreg homeostasis.Microbiota-derived SCFA were found to increase total (thymic-derived) cTreg numbers. The homing characteristics to the colon and the regulatory functions of these cells (such as IL-10 production) were also enhanced through SCFA treatment.These effects are mediated by the G-protein-coupled free fatty acid receptor 43 (GPR43). Using mice that are genetically deficient in this receptor, Smith et al. showed that this signaling pathway is responsible for the increased cTreg numbers in vivo and that signaling by SCFA reduces the susceptibility to chronic intestinal inflammation. As they found GPR43 expression on cTreg (compared with lower GPR43 expression on Treg from other sites) this may be a direct effect, e.g. alterations in histone deacetylation. However, other cell types in the GI tract also express GPR43, including enteroendocrine cells and other leukocytes, therefore indirect effects are not yet excluded. In fact, Atarashi and colleagues have recently published their studies of how Clostridial species induce cTreg⁶. They found that bacterial-derived SCFA stimulate epithelial cells to produce TGFβ, contributing to Treg differentiation and expansion.Whereas other species-specific bacterial molecules, such as B. fragilis-derived PSA, have previously been demonstrated to have immunomodulatory functions², the report by Smith et al. is an elegant demonstration of the ubiquitous and pervasive bacterial metabolites that impact on the mucosal immune system. There is really a rather promiscuous exchange of metabolites between the microbiota and the host, with metabolic pathways that require components of both eukaryotic and prokaryotic cells. Bile acids are a great example of such a mixed pathway, where a dysbiosis caused by obesity promotes liver cancer through alterations in the microbial bile acid metabolism⁷. Although Smith et al. do not see any SCFA-mediated effects on central Treg compartments (outside the colon), other bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed increased incidence of intestinal inflammation and systemic immune-mediated disorders such as autoimmune or allergic diseases (Figure 1), which are often linked to changes within the microbiota due to diet or antibiotic use⁸.Open in a separate windowFigure 1Bacterial metabolites that reach systemic sites likely modulate adaptive or innate immune cell function at systemic sites. This may eventually rationalize the observed correlation of microbiota composition and susceptibility to systemic immune-mediated disorders such as autoimmune or allergic diseases.A clinical situation in which the colon faces a deficiency of SCFA happens after surgery that diverts the fecal stream into a stoma bag, leaving the distal colon without its normal contents. This operation may be carried out to protect a low surgical anastomosis after removal of a tumor. The result is that the defunctioned colon frequently becomes inflamed, a condition recognized as ''diversion colitis''. In some cases, treatment with SCFA has been able to treat the condition successfully⁹. The lack of SCFA as a carbon source for colonocytes was previously considered as a key factor in the aetiopathogenesis of the condition, although this will need to be reviewed in the light of the new data on the effects of SFCA on colonic Treg numbers and function.Our colonic health depends on our intestinal microbiota and what we feed them. Changes in Western dietary patterns, e.g., due to reduced intake of plant fibers, might drastically impact the production of SCFA within the intestine. Furthermore, Smith et al. demonstrate a direct effect of antibiotic (vancomycin) treatment on SCFA levels, which in turn affects intestinal immune regulation by reducing the number of cTreg.Taken together, this draws a picture of a superorganism composed of the host (us) and our microbiota, with the metabolic interface as an important communication tool. This allows the host and the microbiota to adapt to and communicate with each other. Originally, germ-free animals were derived to challenge the notion that the existence of higher organisms was irrevocably linked to their associated microbiotas¹⁰. Although the germ-free program succeeded¹¹, it has provided us with powerful tools to show that the original notion was justified: pervasive metabolic interactions and signaling make us the sum of our prokaryotic and eukaryotic cellular components.     

Zinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin

Metastasis is the major cause of breast cancer mortality. The strength of cell adhesion to extracellular matrix is critical to cancer cell migration. Integrins, the primary mediators of cell to extra-cellular matrix adhesion, contain distinct divalent cation-binding sites. Binding of manganese and magnesium is vital to integrin-mediated cancer cell adhesion and migration. We hypothesized that zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. MDA-MB-231 cells were cultured in a zinc-depleted medium supplemented with 0 (control), 2.5, 5, 10, 25 and 50 μM of zinc to mimic severe zinc-deficiency, moderate zinc-deficiency, adequate zinc and three levels of zinc-supplementation: low-, moderate- and high-levels of zinc-supplementation, respectively. Zinc treatments had no effect on cellular zinc concentration, cell number and cell viability. Zinc at 5–50 μM reduced migration distance of MDA-MB-231 cells on fibronectin by 43–86% and migration rate on fibronectin by 72–90%. Zinc induced a dose-dependent inhibition of cell adhesion to fibronectin (R²=?0.98). Zinc at 10–50 μM reduced magnesium-facilitated cell adhesion to fibronectin in a dose-dependent manner (R²=?0.90). However, zinc had no effect on manganese-facilitated cell adhesion to fibronectin. Zinc at 5–50 μM caused rounding of the normally elongated, irregular-shaped MDA-MB-231 cells and disappearance of F-actin. Anti-integrin α5- and β1-subunit blocking antibodies inhibited magnesium-facilitated cell adhesion to fibronectin by 95 and 99%, respectively. In summary, zinc inhibited MDA-MB-231 cell migration on fibronectin by interfering with magnesium-dependent integrin-, likely integrin α5/β1-, mediated adhesion.     

Influence of extracellular pH on the cytotoxicity, cellular accumulation, and DNA interaction of novel pH-sensitive 2-aminoalcoholatoplatinum(II) complexes

Extracellular acidity is a frequent pathophysiological condition of solid tumors offering possibilities for improving the tumor selectivity of molecular therapy. This might be accomplished by prodrugs with low systemic toxicity, attaining their full antitumor potency only under acidic conditions, such as bis(2-aminoalcoholato-κ²N,O)platinum(II) complexes that are activated by protonation of alcoholato oxygen, resulting in cleavage of platinum–oxygen bonds. In this work, we examined whether the pH dependency of such compounds is reflected in differential biological activity in vitro. In particular, the pH dependence of cytotoxicity, cellular accumulation, DNA platination, GMP binding, effects on DNA secondary structure, cell cycle alterations, and induction of apoptosis was investigated. Enhanced cytotoxicity of five of these complexes in non-small-cell lung cancer (A549) and colon carcinoma (HT-29) cells at pH 6.0 in comparison with pH 7.4 was confirmed: 50 % growth inhibition concentrations ranged from 42 to 214 μM in A549 cells and from 35 to 87 μM in HT-29 cells at pH 7.4 and decreased at pH 6.0 to 11–50 and 7.3–25 μM, respectively. The effects induced by all five pH-sensitive compounds involve increased 5′-GMP binding, cellular accumulation, and DNA platination as well as stronger effects on DNA secondary structure at pH 6.0 than at pH 7.4. As exemplified by treatment of A549 cells with a 2-amino-4-methyl-1-pentanolato complex, induction of apoptosis is enhanced at pH 6.5. These results confirm the increased reactivity and in vitro activity of these compounds under slightly acidic conditions, encouraging further evaluation of ring-closed aminoalcoholatoplatinum(II) derivatives in solid tumors in vivo.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.