House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.     

Design,synthesis, and binding of homologated truncated 4′-thioadenosine derivatives at the human A3 adenosine receptors

We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH₂) group was inserted in place of the glycosidic bond of a potent and selective A₃ adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A₃ adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.     

Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

A cyclophilin A CPR1 overexpression enhances stress acquisition in Saccharomyces cerevisiae

Cyclophilins are conserved cis-trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. We found the expression of cyclophilin A, Cpr1, changes in response to exposure to yeast Saccharomyces cerevisiae to abiotic stress conditions. The effect of Cpr1 overexpression in stress responses was therefore examined. The CPR1 gene was cloned to the yeast expression vector pVTU260 under regulation of an endogenous alcohol dehydrogenase (ADH) promoter. The overexpression of Cpr1 drastically increased cell viability of yeast in the presence of stress inducers, such as cadmium, cobalt, copper, hydrogen peroxide, tert-butyl hydroperoxide (t-BOOH), and sodium dodecyl sulfate (SDS). The Cpr1 expression also enhanced the cell rescue program resulting in a variety of antioxidanr enzymes including thioredoxin system (particularly, thioredoxin peroxidase), metabolic enzymes (glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase), and molecular chaperones (Hsp104, Hsp90, Hsp60 and Hsp42). Thus, our study illustrates the importance of Cpr1 as a molecular chaperone that improves cellular stress responses through collaborative relationships with other proteins when yeast cells are exposed to adverse conditions, and it also premises the improvement of yeast strains.     

Physiological consequences of programmed necrosis, an alternative form of cell demise

Cell death occurs spontaneously or in response to external stimuli, and can be largely subdivided into apoptosis and necrosis by the distinct morphological and biochemical features. Unlike apoptosis, necrosis was recognized as the passive and unwanted cell demise committed in a non-regulated and disorganized manner. However, under specific conditions such as caspase intervention, necrosis has been proposed to be regulated in a well-orchestrated way as a backup mechanism of apoptosis. The term programmed necrosis has been coined to describe such an alternative cell death. Recently, at least some regulators governing programmed necrosis have been identified and demonstrated to be interconnected via a wide network of signal pathways by further extensive studies. There is growing evidence that programmed necrosis is not only associated with pathophysiological diseases, but also provides innate immune response to viral infection. Here, we will introduce recent updates on the molecular mechanism and physiological significance of programmed necrosis.     

Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca²⁺] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca²⁺]_c in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca²⁺ entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca²⁺]_c increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca²⁺]_c or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca²⁺]_c. Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca²⁺ was measured using Ba²⁺ as a surrogate ion. Similarly, Ba²⁺ influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca²⁺-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca²⁺ release was insignificant in primary B cells and inWEHI-231.     

EVOLUTIONARY ORIGIN OF GLOEOMONAS (VOLVOCALES,CHLOROPHYCEAE), BASED ON ULTRASTRUCTURE OF CHLOROPLASTS AND MOLECULAR PHYLOGENY1

Gloeomonas is a peculiar unicellular volvocalean genus because it lacks pyrenoids in the chloroplasts under the light microscope and has two flagellar bases that are remote from each other. However, ultrastructural features of chloroplasts are very limited, and no molecular phylogenetic analyses have been carried out in Gloeomonas. In this study, we observed ultrastructural features of chloroplasts of three species of Gloeomonas and Chloromonas rubrifilum (Korshikov ex Pascher) Pröschold, B. Marin, U. Schlösser et Melkonian SAG 3.85, and phylogenetic analyses were carried out based on the combined data set from 18S rRNA, ATP synthase beta‐subunit, and P700 chl a–apoprotein A2 gene sequences to deduce the natural phylogenetic positions of the genus Gloeomonas. The present EM demonstrated that the chloroplasts of the three Gloeomonas species and C. rubrifilum SAG 3.85 did not have typical pyrenoids with associated starch grains, but they possessed pyrenoid matrices that protruded interiorly within the stroma regions of the chloroplast. The pyrenoid matrices were large and broad in C. rubrifilum, whereas those of the three Gloeomonas species were recognized in only the small protruded regions of the chloroplast lobes. The present multigene phylogenetic analyses resolved that the three species of Gloeomonas belong to the Chloromonas lineage or Chloromonadinia of the Volvocales, and Chloromonas insignis (Anakhin) Gerloff et H. Ettl NIES‐447 and C. rubrifilum SAG 3.85, both of which have pyrenoids without associated starch grains, were positioned basally to the clade composed of the three species of Gloeomonas. Therefore, Gloeomonas might have evolved from such a Chloromonas species through reduction in pyrenoid matrix size within the chloroplast and by separating their two flagellar bases.     

Tissue-Specific Differences in PD-1 and PD-L1 Expression during Chronic Viral Infection: Implications for CD8 T-Cell Exhaustion

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8⁺ T cells. We examined the expression of PD-1 and its ligands, PD-L1 and PD-L2, in multiple tissues during the course of chronic viral infection and determined how the amount of PD-1 expressed, as well as the anatomical location, influenced the function of exhausted CD8 T cells. The amount of PD-1 on exhausted CD8 T cells from different anatomical locations did not always correlate with infectious virus but did reflect viral antigen in some tissues. Moreover, lower expression of PD-L1 in some locations, such as the bone marrow, favored the survival of PD-1^Hi exhausted CD8 T cells, suggesting that some anatomical sites might provide a survival niche for subpopulations of exhausted CD8 T cells. Tissue-specific differences in the function of exhausted CD8 T cells were also observed. However, while cytokine production did not strictly correlate with the amount of PD-1 expressed by exhausted CD8 T cells from different tissues, the ability to degranulate and kill were tightly linked to PD-1 expression regardless of the anatomical location. These observations have implications for human chronic infections and for therapeutic interventions based on blockade of the PD-1 pathway.Chronic viral infections are often associated with CD8⁺ T-cell dysfunction (30). This dysfunction, termed exhaustion, includes defects in the ability to produce antiviral cytokines, poor cytotoxicity, a loss of antigen-independent self-renewal, and the inability to vigorously re-expand following antigen exposure (30). These functional deficiencies contrast with the highly functional memory CD8⁺ T cells that are generated after acute infection and maintained via interleukin-7 (IL-7)- and IL-15-mediated homeostatic proliferation (30). During chronic viral infections, T-cell exhaustion often correlates with poor control of viral replication (3, 8, 38, 39). Thus, there is considerable interest in developing strategies to reverse exhaustion and restore function in virus-specific CD8⁺ T cells during chronic infections.Recent studies have revealed an important role for the negative regulatory molecule PD-1 in CD8 T-cell exhaustion during chronic viral infections (29). PD-1, a member of the CD28/CTLA-4 family of costimulatory/coinhibitory receptors, contains both ITIM and ITSM motifs in the intracellular tail and can deliver negative signals, at least partly via recruitment of the phosphatase Shp-2 (29). A role for PD-1 in regulating T-cell responses to chronic viral infections was first observed using lymphocytic choriomeningitis virus (LCMV) infection of mice, where PD-1 was found to be highly expressed on exhausted CD8⁺ T cells from chronically infected animals but not on functional memory CD8⁺ T cells from mice that had cleared an acute strain of the virus (3). In vivo blockade of the PD-1 pathway led to a dramatic increase in the number of virus-specific CD8⁺ T cells, improved functionality of these cells, and enhanced control of viral replication (3). These observations were extended to human chronic viral infections, and a series of studies have demonstrated that human immunodeficiency virus (HIV)-, hepatitis C virus (HCV)-, and HBV-specific CD8⁺ T cells upregulate PD-1 in humans compared to CD8⁺ T cells specific for nonpersisting viruses such as influenza virus or vaccinia virus (6-8, 24, 26, 32, 33, 42). Increasing PD-1 expression also correlates with disease status during HIV infection (8, 42). In vitro blockade of PD-1-PD-L interactions can reinvigorate exhausted virus-specific T-cell responses in humans and appears to have a prominent impact on proliferative expansion and/or prevention of apoptosis in these cases (9, 24, 32). Finally, recent results from in vivo blockade in the macaque simian immunodeficiency virus (SIV) infection model demonstrated the effectiveness of blocking PD-1 in primates during chronic viral infection (36). In these studies, PD-1 blockade enhanced virus-specific T and B-cell responses, lowered viral load, and improved the survival of chronically infected animals. Thus, PD-1 has emerged as not only a major regulator of T-cell exhaustion and viral control during chronic infection but also as an important potential therapeutic target.Despite these important studies and the clear impact of PD-1 blockade on the reversal of T-cell exhaustion, important questions remain. For example, previous work has demonstrated that PD-1 expression is not uniform on subsets of exhausted CD8 T cells (4). However, the expression of PD-1 on exhausted CD8 T cells in multiple tissues, and the relationship between PD-1 expression in these tissues to viral load, the PD-1 ligands and function has not been examined. Given the nonlymphoid accumulation of virus-specific CD8 T cells during chronic viral infections (11, 39) and the predilection of many important chronic infections for replicating in anatomically restricted locations (e.g., HCV and the liver, HIV and mucosal tissues, etc.), the dynamics of PD-1 expression by exhausted CD8 T cells outside the blood and spleen could have important therapeutic implications.In the present study we examined these issues using the mouse model of LCMV infection. Our results demonstrate that exhausted CD8 T cells have a wide range of PD-1 expression in different tissues of chronically infected mice. Virus-specific CD8 T cells in some anatomical locations such as the liver, brain, and bone marrow (BM) expressed high PD-1 for substantially longer than virus-specific CD8⁺ T cells from the spleens or blood of the same mice. Although PD-1 expression in the spleen correlated well with reduced gamma interferon (IFN-γ) and tumor necrosis factor (TNF) production, the PD-1^Hi virus-specific CD8⁺ T cells from the BM remained capable of producing antiviral cytokines ex vivo. In contrast, a strong negative correlation between PD-1 expression and cytotoxicity existed for exhausted CD8 T cells from all tissues tested. PD-L1 expression was high in the spleen, whereas in the BM antigen-presenting cell (APC) populations expressed lower amounts of PD-L1. Survival of PD-1^Hi CD8⁺ T cells from the BM was decreased in the presence of splenic APCs, suggesting that different tissue microenvironments in vivo could selectively support the persistence of PD-1^Hi exhausted CD8 T cells. Since PD-1 expression differs by anatomical location, these observations suggest that PD-1 blockade in vivo will have varying impacts on exhausted CD8 T cells from different tissues or anatomical locations. These observations have implications for human chronic infections such as HBV, HCV, and HIV.     

Enhanced Anti-tumor Activity by the Combination of the Natural Compounds (?)-Epigallocatechin-3-gallate and Luteolin: POTENTIAL ROLE OF p53*

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3–5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser¹⁵ phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.