The Cytotoxic and Proapoptotic Activities of Hypnophilin are Associated with Calcium Signaling in UACC‐62 Cells

Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC‐62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC‐62 cells, which was blocked in cells stimulated in Ca²⁺‐free media. HNP treatment with BAPTA‐AM, an intracellular Ca²⁺ chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC‐62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC‐62 cells, after treatment with HNP, is associated with Ca²⁺ influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:479‐485, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21507     

Periostin Mediates the Increased Pro‐Angiogenic Activity of Gastric Cancer Cells under Hypoxic Conditions

This study was conducted to investigate the biological role of periostin in gastric cancer (GC) under hypoxia. Western blot analysis revealed that along with an upregulation of hypoxia‐inducible factor‐1alpha, there was a time‐dependent induction of periostin in MKN‐45 cells under hypoxia (2% O₂), increasing by eightfold as compared to normoxic cells. Pretreatment with 30 µM PD98059, an inhibitor of ERK1/2, significantly reduced hypoxia‐stimulated periostin expression (P < 0.01). Periostin knockdown in MKN‐45 cells was achieved by specific small interfering RNA (siRNA). The conditioned medium from periostin siRNA‐transfected MKN‐45 cells induced significantly less (P < 0.01) endothelial tube formation than control siRNA‐transfected cells. Additionally, periostin silencing markedly decreased the mRNA expression and secretion of vascular endothelial growth factor (VEGF) in hypoxic MKN‐45 cells. Thus, our data suggest that periostin is a hypoxia‐response gene and mediates a cross talk between GC and endothelial cells under hypoxia, partially through regulation of the VEGF expression. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:364‐369, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21498     

Synthesis and alkylation of aza‐glycinyl dipeptide building blocks

Aza‐glycinyl dipeptides are useful building blocks for the synthesis of a diverse array of azapeptides. The construction of the aza‐glycine residue is however challenging, because of the potential for side reactions, such as those leading to formation of oxadiazalone, hydantoin and symmetric urea by‐products. Employing N,N′‐disuccinimidyl carbonate to activate benzophenone hydrazone, we have developed a more efficient approach for the synthesis of aza‐glycinyl dipeptides. Alkylation of the semicarbazone of the resulting protected aza‐glycinyl dipeptides using tetraethylammonium hydroxide and propargyl bromide provided an efficient entry into the aza‐propargylglycinyl peptide building blocks, which have served previously in various reactions including Sonogashira cross‐couplings, dipolar cycloadditions and intramolecular exo‐dig cycloadditions to furnish a variety of azapeptide building blocks. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug‐resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α‐helical AMPs surprisingly have a helix‐breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane‐active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Introduction of histidine units using benzotriazolide activation

N^α‐Boc‐N^im‐(4‐toluenesulfonyl‐l ‐histidylbenzotriazole) enables convenient acylation of N‐, O‐, S‐, and C‐nucleophiles with no detectable racemization. We report efficient syntheses of novel histidine‐containing di‐, tri‐, and tetra‐peptides and models for the preparation of potentially biologically active histidine N‐, O‐, S‐, and C‐conjugates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

MFG-E8 and HMGB1 Are Involved in the Mechanism Underlying Alcohol-Induced Impairment of Macrophage Efferocytosis

Efferocytosis is a unique phagocytic process for macrophages to remove apoptotic cells in inflammatory loci. This event is maintained by milk fat globule-EGF factor 8 (MFG-E8), but attenuated by high mobility group box 1 (HMGB1). Alcohol abuse causes injury and inflammation in multiple tissues. It alters efferocytosis, but precise molecular mechanisms for this effect remain largely unknown. Here, we showed that acute exposure of macrophages to alcohol (25 mmol/L) inhibited MFG-E8 gene expression and impaired efferocytosis. The effect was mimicked by hydrogen peroxide. Moreover, N-acetylcysteine (NAC), a potent antioxidant, blocked acute alcohol effect on inhibition of macrophage MFG-E8 gene expression and efferocytosis. In addition, recombinant MFG-E8 rescued the activity of alcohol-treated macrophages in efferocytosis. Together, the data suggest that acute alcohol exposure impairs macrophage efferocytosis via inhibition of MFG-E8 gene expression through a reactive oxygen species dependent mechanism. Alcohol has been found to suppress or exacerbate immune cell activities depending on the length of alcohol exposure. Thus, we further examined the role of chronic alcohol exposure on macrophage efferocytosis. Interestingly, treatment of macrophages with alcohol for seven days in vitro enhanced MFG-E8 gene expression and efferocytosis. However, chronic feeding of mice with alcohol caused increase in HMGB1 levels in serum. Furthermore, HMGB1 diminished efferocytosis by macrophages that were treated chronically with alcohol, suggesting that HMGB1 might attenuate the direct effect of chronic alcohol on macrophage efferocytosis in vivo. Therefore, we speculated that the balance between MFG-E8 and HMGB1 levels determines pathophysiological effects of chronic alcohol exposure on macrophage efferocytosis in vivo.     

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells,the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α‐helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus‐induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose‐dependent and time‐dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Turbellarian assemblages in freshwater lagoons in southern Brazil

Turbellarians, which typically feed on bacteria, algae, rotifers, oligochaetes, dipteran larvae, microcrustaceans, and other organisms, are abundant in diverse types of wetlands. Despite their importance, abundance, and species richness in freshwater environments, turbellarians are seldom considered in studies on biodiversity. We analyzed the structure of turbellarian communities in shore areas of three categories of permanent wetlands classified according to their perimeter as small, intermediate, and large during an annual cycle. In total, 1847 turbellarians were collected representing 42 species and 15 genera, from the orders Catenulida, Macrostomida, Lecithoepitheliata, Rhabdocoela, and Tricladida. Sixteen species were common to the three categories of wetlands, whereas nineteen species were unique to a particular category. Species composition varied among wetlands of different sizes; small, intermediate, and large wetlands had different dominant species. We found seasonal differences in community composition over the year, but no significant differences in mean values of observed species richness among wetlands with different sizes and among seasonal samples. The estimated species richness was, however, higher in the small wetlands, followed by the large and intermediate wetlands. In the summer, abundance was significantly lower in the small water bodies than in the intermediate and large bodies of water. Our results reinforce the need for conservation of wetlands of different sizes.     

A conceptual model of the Amblyomma americanum life cycle in northeast Missouri

The geographic distribution of Amblyomma americanum (the lone star tick) has increased as has its role as a pathogen vector. The objectives of this study were to determine seasonal activity patterns of each life stage of A. americanum in the northwestern part of the species range and the relationship of these activity patterns among life stages and degree days. Tick activity was monitored over four years since 2007 in a forest and old field habitat located in northeast Missouri. Every other week from February to December, ticks were collected using bait and drag methods. Autocorrelations demonstrated yearly seasonal patterns in each life stage and cross‐correlations between life stages depicted a relationship between activity at a life stage and the previous stage's activity. Cross‐correlations indicated that degree days were related to activity. These data indicated that A. americanum generally complete their life cycle in a minimum of two years in northeast Missouri, with overwintering occurring predominantly in the nymphal and adult stages. These data provide a baseline to compare the life cycle of A. americanum in northeast Missouri to populations in different parts of the species range or at different times in the region.