Photo-response of the brown marmorated stink bug, <Emphasis Type="Italic">Halyomorpha halys</Emphasis> (Stål) (Heteroptera: Pentatomidae), and its role in the hiding behavior

As part of a study aimed at determining the factors affecting overwintering habitat choice by Halyomorpha halys, we examined the behavioral response to light of diapausing adults (which have no reproductive activity) and non-diapausing adults. Bugs were tested individually in a choice chamber (14, 18 and 24°C) containing a dark refuge with opaque (black) sides and roof. Each bug’s position in the choice chamber was recorded at regular intervals. The results indicated that diapausing bugs prefer to settle in a dark place (p < 0.05). This preference appeared to weaken at 24°C; however, this apparent trend was probably due to the greater overall activity of the bugs under warmer conditions. In tests of non-diapausing bugs, lower proportions of the bugs were observed inside the dark refuge at higher temperatures; however, the preference for dark places was exhibited under all temperature conditions. Based on these results, we infer that H. halys adults have a fundamental preference for dark places whether or not they are in diapauses, which plays a main role in hiding behavior. This preference becomes most obvious in the winter, with the decrease in behavioral activity resulting from lower temperatures. We propose that this common preference for location is one of the factors responsible for the bugs’ convergence on specific microhabitats and the overwintering aggregate formation of H. halys.     

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca²⁺]_i in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca²⁺]_i in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca²⁺]_i in response to MG-132. MDL-28170 induced neither an increase in [Ca²⁺]_i in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.     

15-Deoxy-Δ-prostaglandin J2 enhanced the anti-tumor activity of camptothecin against renal cell carcinoma independently of topoisomerase-II and PPARγ pathways

Renal cell carcinoma (RCC) is chemoresistant cancer. Although several clinical trials were conducted to explore effective medications, the chemoresistance of RCC has not yet been conquered. An endogenous ligand for peroxisome proliferator-activated receptor-γ (PPARγ), 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), induces apoptosis in RCC. Here, we examined synergistic effects of several carcinostatics on the anti-tumor activity of 15d-PGJ₂ in Caki-2 cell line by MTT assay. A topoisomerase-I inhibitor, camptothecin (CPT), exhibited synergistically toxicity with 15d-PGJ₂, but neither 5-fluorouracil nor cisplatin did. The combination of 15d-PGJ₂ and a topoisomerase-II inhibitor, doxorubicine, did not cause synergistic cell growth inhibition. The synergistic effect of topoisomerase-I and II inhibitors was not also detected. A PPARγ antagonist, GW9662, did not prevent Caki-2 from undergoing 15d-PGJ₂-induced cytotoxicity. The treatment of CPT combined with 15d-PGJ₂ activated caspase-3 more than the separate treatment. These results suggest that 15d-PGJ₂ exhibited the anti-tumor activity synergistically with CPT independent of topoisomerase-II and PPARγ.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy.The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase NuSAP-chromatin interaction suggests additional functions for NuSAP, as recently identified for other nuclear spindle assembly factors with a role in gene expression or DNA damage response.     

ER stress-induced protein, VIGG, disturbs plant cation homeostasis, which is correlated with growth retardation and robustness to ER stress

VIGG is a putative endoplasmic reticulum (ER) resident protein induced by virus infection and ER stress, and is correlated with fruit quality in grapevine. The present study was undertaken to determine the biological function of VIGG in grapevine. Experiments using fluorescent protein-VIGG fusion protein demonstrated that VIGG is localized in ER and the ER targeting sequence is in the N-terminus. The overexpression of VIGG in Arabidopsis plant led to growth retardation. The rosette leaves of VIGG-overexpressing plants were smaller than those of the control plants and rolled at 42 days after seeding. VIGG-overexpressing plants revealed robustness to ER stress as well as the low expression of ER stress marker proteins, such as the luminal binding proteins. These characteristics of VIGG-overexpressing plants were supported by a microarray experiment that demonstrated the disruption of genes related to ER stress response and flowering, as well as cation mobility, in the plants. Finally, cation homeostasis in the plants was disturbed by the overexpression of VIGG. Taken together, these results suggest that VIGG may disturb cation homeostasis in plant, which is correlated with the robustness to ER stress and growth retardation.     

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.     

15-Deoxy-Δ12,14-prostaglandin J2 enhanced the anti-tumor activity of camptothecin against renal cell carcinoma independently of topoisomerase-II and PPARγ pathways

Renal cell carcinoma (RCC) is chemoresistant cancer. Although several clinical trials were conducted to explore effective medications, the chemoresistance of RCC has not yet been conquered. An endogenous ligand for peroxisome proliferator-activated receptor-γ (PPARγ), 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), induces apoptosis in RCC. Here, we examined synergistic effects of several carcinostatics on the anti-tumor activity of 15d-PGJ(2) in Caki-2 cell line by MTT assay. A topoisomerase-I inhibitor, camptothecin (CPT), exhibited synergistically toxicity with 15d-PGJ(2), but neither 5-fluorouracil nor cisplatin did. The combination of 15d-PGJ(2) and a topoisomerase-II inhibitor, doxorubicine, did not cause synergistic cell growth inhibition. The synergistic effect of topoisomerase-I and II inhibitors was not also detected. A PPARγ antagonist, GW9662, did not prevent Caki-2 from undergoing 15d-PGJ(2)-induced cytotoxicity. The treatment of CPT combined with 15d-PGJ(2) activated caspase-3 more than the separate treatment. These results suggest that 15d-PGJ(2) exhibited the anti-tumor activity synergistically with CPT independent of topoisomerase-II and PPARγ.     

Bis-THF motif of acetogenin binds to the third matrix-side loop of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreductase

Natural acetogenins are among the most potent inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I). Our photoaffinity labeling study suggested that the hydroxylated bis-THF ring moiety of acetogenins binds at "site A" in the third matrix-side loop connecting the fifth and sixth transmembrane helices in the ND1 subunit [Kakutani et al. (2010) Biochemistry 49, 4794-4803]. Nevertheless, since this proposition was led using a photoreactive Δlac-acetogenin derivative, it needs to be directly verified using a natural acetogenin-type probe. We therefore conducted photoaffinity labeling using a photoreactive natural acetogenin mimic ([(125)I]diazinylated natural acetogenin, [(125)I]DANA), which has a small photolabile diazirine group, in place of a hydroxy group, attached to the bis-THF ring moiety. Analysis of the photocross-linked protein in bovine heart submitochondrial particles unambiguously revealed that [(125)I]DANA binds to the membrane subunit ND1 with high specificity. The photocross-linking was completely blocked in the presence of just a 5-fold excess of bullatacin, indicating that [(125)I]DANA is an excellent mimic of natural acetogenins and hence binds to the site that accommodates natural products. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by different proteases and their combinations indicated that the cross-linked residue is predominantly located at the supposed site A in the third matrix-side loop.     

The reconstituted 'humanized liver' in TK-NOG mice is mature and functional

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA’s proteolytic activity for mushroom defense against pathogens is discussed.