A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

A DNA vaccine against cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) prevents tumor growth

Co-stimulatory signaling pathway triggered by the binding of B7.1/B7.2 (CD80/86) of antigen-presenting cells (APCs) to CD28 of T cells is required for optimal T-cell activation. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding with a greater affinity. Ipilimumab, a monoclonal antibody against CTLA-4, has shown positive efficacy in a pivotal clinical trial for the treatment of metastatic melanoma and was approved by FDA. However, the cost of monoclonal antibody-based therapeutics might limit the number of patients treated. To develop a novel therapeutics specifically targeting CTLA-4, we constructed a DNA vaccine by cloning the sequence of CTLA-4 fused with a transmembrane domain sequence of placental alkaline phosphatase (PLAP) into a mammalian expression plasmid, pVAC-1. Immunization with the resulting construct, pVAC-1-hCTLA-4, elicited antibody specific to human CTLA-4 with cross reactivity to murine CTLA-4, which was sufficient for inhibiting B16F10 tumor growth in c57BL/6 mice in the absence of measurable toxicity. Coupling liposome with pVAC-1-mCTLA-4 could break tolerance to self-antigen in BALB/c mice and induce potent immunity against murine CTLA-4, and suppress growth of subcutaneous renal cell carcinoma (Renca).     

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting. In a previous study on interleukin (IL)-2 receptor β (IL-2Rβ) and IL-4 receptor α (IL-4Rα), which are constitutively internalized without ligand stimulation, we revealed that Hrs bound to IL-2Rβ and IL-4Rα in a ubiquitin-independent manner, and identified a hydrophobic amino acid cluster in the cytoplasmic region of IL-2Rβ and IL-4Rα as the Hrs-interacting domain. However, a chimeric receptor containing the hydrophobic amino acid cluster inserted into the C-terminal of IL-2Rα was not delivered to late endosomes, but recycled back to the plasma membrane. In the present study, we explored the functional domain related to endosomal sorting in IL-2Rβ together with the hydrophobic amino acid cluster, and discovered the importance of an approximately 30-amino acid stretch following the C-terminus of the hydrophobic amino acid cluster in IL-2Rβ. Even though the amino acid stretch following the hydrophobic amino acid cluster was composed of arbitrary amino acids, such a stretch was also permissive for the sorting ability, suggesting that the hydrophobic amino acid cluster functions as an endosomal sorting signal. These findings clarify part of the molecular mechanism underlying the ubiquitin-independent endosomal sorting of cytokine receptors that are constitutively internalized without ligand stimulation.     

The role of SDF-1-CXCR4/CXCR7 axis in biological behaviors of adipose tissue-derived mesenchymal stem cells in vitro

Numerous studies have reported that CXCR4 and CXCR7 play an essential, but differential role in stromal cell-derived factor-1 (SDF-1)-inducing cell chemotaxis, viability and paracrine actions of BMSCs. Adipose tissue-derived mesenchymal stem cells (ADSCs) have been suggested to be potential seed cells for clinical application instead of bone marrow derived stroma cell (BMSCs). However, the function of SDF-1/CXCR4 and SDF-1/CXCR7 in ADSCs is not well understood. This study was designed to analyze the effect of SDF-1/CXCR4 and SDF-1/CXCR7 axis on ADSCs biological behaviors in vitro. Using Flow cytometry and Western blot methods, we found for the first time that CXCR4/CXCR7 expression was increased after treatment with SDF-1 in ADSCs. SDF-1 promoted ADSCs paracrine, proliferation and migration abilities. CXCR4 or CXCR7 antibody suppressed ADSCs paracrine action induced by SDF-1. The migration of ADSCs can be abolished by CXCR4 antibody, while the proliferation of ADSCs was only downregulated by CXCR7 antibody. Our study indicated that the angiogenesis of ADSCs is, at least partly, mediated by SDF-1/CXCR4 and SDF-1/CXCR7 axis. However, only binding of SDF-1/CXCR7 was required for proliferation of ADSCs, and CXCR7 was required for migration of ADSCs induced by SDF-1. Our studies provide evidence that the activation of either axis may be helpful to improve the effectiveness of ADSCs-based stem cell therapy.     

Relevance of signaling molecules for apoptosis induction on influenza A virus replication

Apoptosis is an important mechanism to maintain homeostasis in mammals, and disruption of the apoptosis regulation mechanism triggers a range of diseases, such as cancer, autoimmune diseases, and developmental disorders. The severity of influenza A virus (IAV) infection is also closely related to dysfunction of apoptosis regulation. In the virus infected cells, the functions of various host cellular molecules involved in regulation of induction of apoptosis are modulated by IAV proteins to enable effective virus replication. The modulation of the intracellular signaling pathway inducing apoptosis by the IAV infection also affects extracellular mechanisms controlling apoptosis, and triggers abnormal host responses related to the disease severity of IAV infections. This review focuses on apoptosis related molecules involved in IAV replication and pathogenicity, the strategy of the virus propagation through the regulation of apoptosis is also discussed.     

Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4B^C74Ain vitro and in vivo, we show that the effects of ATG4B^C74A are cell type, treatment, and context-dependent. ATG4B^C74A expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.     

Nuclear-encoded chloroplast RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes in a red alga, Cyanidioschyzon merolae

The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon merolae.     

Inhibiting neuropeptide Y Y1 receptor modulates melanocortin receptor- and NF-κB-mediated feeding behavior in phenylpropanolamine-treated rats

Neuropeptide Y (NPY) and nuclear factor-kappa B (NF-κB) are involved in regulating anorexia elicited by phenylpropanolamine (PPA), a sympathomimetic drug. This study explored whether NPY Y1 receptor (Y1R) is involved in this process, and a potential role for the proopiomelanocortin system was identified. Rats were given PPA once a day for 4 days. Changes in the hypothalamic expression of the NPY, Y1R, NF-κB, and melanocortin receptor 4 (MC4R) levels were assessed and compared. The results indicated that food intake and NPY expression decreased, with the largest reductions observed on Day 2 (approximately 50% and 45%, respectively), whereas NF-κB, MC4R, and Y1R increased, achieving maximums on Day 2 (160%, 200%, and 280%, respectively). To determine the role of Y1R, rats were pretreated with Y1R antisense or a Y1R antagonist via intracerebroventricular injection 1 h before the daily PPA dose. Y1R knockdown and inhibition reduced PPA anorexia and partially restored the normal expression of NPY, MC4R, and NF-κB. The data suggest that hypothalamic Y1R participates in the appetite-suppression from PPA by regulating MC4R and NF-κB. The results of this study increase our understanding of the molecular mechanisms in PPA-induced anorexia.