Structural basis of blocking integrin activation and deactivation for anti-inflammation

Integrins mediate leukocyte accumulation to the sites of inflammation, thereby enhancing their potential as an important therapeutic target for inflammatory disorders. Integrin activation triggered by inflammatory mediators or signaling pathway is a key step to initiate leukocyte migration to inflamed tissues; however, an appropriately regulated integrin deactivation is indispensable for maintaining productive leukocyte migration. While typical integrin antagonists that block integrin activation target the initiation of leukocyte migration, a novel class of experimental compounds has been designed to block integrin deactivation, thereby perturbing the progression of cell migration. Current review discusses the mechanisms by which integrin is activated and subsequently deactivated by focusing on its structure-function relationship.     

Antiangiogenic and antitumor activities of IL-27

IL-27 is a novel IL-6/IL-12 family cytokine playing an important role in the early regulation of Th1 responses. We have recently demonstrated that IL-27 has potent antitumor activity, which is mainly mediated through CD8(+) T cells, against highly immunogenic murine colon carcinoma. In this study, we further evaluated the antitumor and antiangiogenic activities of IL-27, using poorly immunogenic murine melanoma B16F10 tumors, which were engineered to overexpress single-chain IL-27 (B16F10 + IL-27). B16F10 + IL-27 cells exerted antitumor activity against not only s.c. tumor but also experimental pulmonary metastasis. Similar antitumor and antimetastatic activities of IL-27 were also observed in IFN-gamma knockout mice. In NOD-SCID mice, these activities were decreased, but were still fairly well-retained, suggesting that different mechanisms other than the immune response are also involved in the exertion of these activities. Immunohistochemical analyses with Abs against vascular endothelial growth factor and CD31 revealed that B16F10 + IL-27 cells markedly suppressed tumor-induced neovascularization in lung metastases. Moreover, B16F10 + IL-27 cells clearly inhibited angiogenesis by dorsal air sac method, and IL-27 exhibited dose-dependent inhibition of angiogenesis on chick embryo chorioallantoic membrane. IL-27 was revealed to directly act on HUVECs and induce production of the antiangiogenic chemokines, IFN-gamma-inducible protein (IP-10) and monokine induced by IFN-gamma. Finally, augmented mRNA expression of IP-10 and monokine induced by IFN-gamma was detected at the s.c. B16F10 + IL-27 tumor site, and antitumor activity of IL-27 was partially inhibited by the administration of anti-IP-10. These results suggest that IL-27 possesses potent antiangiogenic activity, which plays an important role in its antitumor and antimetastatic activities.     

Involvement of a class III peroxidase and the mitochondrial protein TSPO in oxidative burst upon treatment of moss plants with a fungal elicitor

Production of apoplastic reactive oxygen species (ROS), or oxidative burst, is among the first responses of plants upon recognition of microorganisms. It requires peroxidase or NADPH oxidase (NOX) activity and factors maintaining cellular redox homeostasis. Here, PpTSPO1 involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance was tested for its role in biotic stress in Physcomitrella patens, a nonvascular plant (moss). The fungal elicitor chitin caused an immediate oxidative burst in wild-type P. patens but not in the previously described ΔPrx34 mutants lacking the chitin-responsive secreted class III peroxidase (Prx34). Oxidative burst in P. patens was associated with induction of the oxidative stress-related genes AOX, LOX7, and NOX, and also PpTSPO1. The available ΔPpTSPO1 knockout mutants overexpressed AOX and LOX7 constitutively, produced 2.6-fold more ROS than wild-type P. patens, and exhibited increased sensitivity to a fungal necrotrophic pathogen and a saprophyte. These results indicate that Prx34, which is pivotal for antifungal resistance, catalyzes ROS production in P. patens, while PpTSPO1 controls redox homeostasis. The capacity of TSPO to bind harmful free heme and porphyrins and scavenge them through autophagy, as shown in Arabidopsis under abiotic stress, seems important to maintenance of the homeostasis required for efficient pathogen defense.     

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N-acetyl-L-Met, N-acetyl-L-Gln and N-acetyl-L-Leu, but had a lower hydrolytic activity towards N-acetyl-L-Phe. The kinetic parameters K(m) and k(cat) were determined to be 24.6 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn(2+), Mn(2+) and Ni(2+)), and Mn(2+) addition caused an alteration in substrate specificity. Site-directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal-binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.     

Collaborative environmental DNA sampling from petal surfaces of flowering cherry <Emphasis Type="Italic">Cerasus</Emphasis> × <Emphasis Type="Italic">yedoensis</Emphasis> ‘Somei-yoshino’ across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the “Ohanami Project”, to obtain environmental DNA samples from petal surfaces of Cerasus?×?yedoensis ‘Somei-yoshino’ across the Japanese archipelago during spring 2015. C. × yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained from a wide distribution; this DNA likely originated from the pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.     

Antitumor activity exhibited by Fas ligand (CD95L) overexpressed on lymphoid cells against Fas+ tumor cells

Lymph node (LN) cells of Fas-mutant mice lpr/lpr (lpr) and lpr ^cg /lpr ^cg (lpr ^cg ) express an increased level of Fas ligand (FasL) (CD95L). We examined the antitumor potential of cell-bound FasL on these LN cells against Fas⁺ tumor cells. Fas⁺ F6b and Fas⁻ N1d cells were produced from murine hepatoma MH134 (Fas⁻) by gene transfection. lpr and lpr ^cg LN cells inhibited growth of F6b but not N1d cells in vitro. Neither gld/gld lpr/lpr (gld/lpr) LN cells, which lack both FasL and Fas, nor wild-type LN cells showed growth-inhibitory activities against F6b and N1d cells. The effector cells and molecule were CD4⁻CD8⁻ T cells and FasL, respectively. The tumor neutralization test and adoptive transfer demonstrated that lpr and lpr ^cg , but not gld/lpr, LN cells retarded the growth of F6b cells. Although anti-Fas antibody and FasL cause severe liver failure, wild-type mice injected with lpr LN cells appeared clinically normal. Adoptive transfer of lpr LN cells to F6b-bearing mice exerted the same antitumor activity in wild-type and gld/lpr recipient mice, indicating the applicability of cell-bound FasL for Fas-mediated target therapy of cancer. These results suggest that antitumor activity was dependent on the Fas-FasL system and that lymphoid cells overexpressing FasL can be powerful antitumor effector cells against Fas⁺ tumor cells. Received: 16 March 1998 / Accepted: 28 July 1998     

A method for the synthesis of an oseltamivir PET tracer

A protocol applicable for the synthesis of an oseltamivir positron emission tomography (PET) tracer was developed. Acetylation of amine 3 with CH(3)COCl, followed by deprotection and aqueous workup, produced oseltamivir 4 from 3 within 10 min. The obtained 4 was sufficiently pure for PET studies. This method can be extended to PET tracer synthesis using CH(3)(11)COCl.     

Functionally undefined gene, yggE, alleviates oxidative stress generated by monoamine oxidase in recombinant Escherichia coli

Real-time PCR analysis showed that yggE gene was about two and three times up-regulated in Escherichia coli cells exposed to UVA irradiation and thermal elevation, respectively, suggesting that this gene is responsive to physiological stress. The yggE gene was introduced into E. coli BL21 cells, together with a monoamine oxidase (MAO) gene as a model source for oxidative stress generation. The distribution of independently isolated transformants (two dozen isolates) was examined in terms of MAO activity and cell vitality. In the case of control strain expressing MAO alone, the largest number of transformants existed in the low range of MAO activity less than 2 units mg⁻¹ and the number significantly decreased at increased MAO activity. On the other hand, the distribution of MAO/YggE-coexpressing transformants shifted to higher MAO activity with frequent appearance in the activity range of 4–8 units mg⁻¹. The yggE gene product therefore has a possible function for alleviating the stress generated in the cells.     

Thymus, innate immunity and autoimmune arthritis: interplay of gene and environment

A hypomorphic mutation of the gene encoding zeta-associated protein-70 (ZAP-70), a signaling molecule in T cells, produces autoimmune arthritis in mice under a microbially conventional condition but not in a clean environment. The genetic anomaly alters thymic selection of self-reactive T cells as well as natural regulatory T cells and their respective functions. Highly self-reactive polyclonal T cells, including arthritogenic ones, thus produced by the thymus strongly recognize self-antigens presented by antigen-presenting cells, stimulate them to up-regulate co-stimulatory molecules and secrete cytokines that drive na?ve self-reactive T cells to differentiate into autoimmune effector Th17 cells. Administration of microbial products and activation of complement can facilitate the differentiation, evoking clinically overt arthritis in a microbially clean environment. Furthermore, mutation-dependent graded attenuation of T cell receptor signaling alters disease phenotypes and the dependency of disease occurrence on the environment. These findings provide a model of how genetic and environmental factors, in association, cause autoimmune diseases such as rheumatoid arthritis.