Tumor necrosis factor-alpha-induced caspase-1 gene expression. Role of p73

Tumour necrosis factor-alpha (TNF-alpha) is a cytokine that is involved in many functions, including the inflammatory response, immunity and apoptosis. Some of the responses of TNF-alpha are mediated by caspase-1, which is involved in the production of the pro-inflammatory cytokines interleukin-1beta, interleukin-18 and interleukin-33. The molecular mechanisms involved in TNF-alpha-induced caspase-1 gene expression remain poorly defined, despite the fact that signaling by TNF-alpha has been well studied. The present study was undertaken to investigate the mechanisms involved in the induction of caspase-1 gene expression by TNF-alpha. Treatment of A549 cells with TNF-alpha resulted in an increase in caspase-1 mRNA and protein expression, which was preceded by an increase in interferon regulatory factor-1 and p73 protein levels. Caspase-1 promoter reporter was activated by the treatment of cells with TNF-alpha. Mutation of the interferon regulatory factor-1 binding site resulted in the almost complete loss of basal as well as of TNF-alpha-induced caspase-1 promoter activity. Mutation of the p53/p73 responsive site resulted in reduced TNF-alpha-induced promoter activity. Blocking of p73 function by a dominant negative mutant or by a p73-directed small hairpin RNA reduced basal as well as TNF-alpha-induced caspase-1 promoter activity. TNF-alpha-induced caspase-1 mRNA and protein levels were reduced when p73 mRNA was down-regulated by small hairpin RNA. Caspase-5 gene expression was induced by TNF-alpha, which was inhibited by the small hairpin RNA-mediated down-regulation of p73. Our results show that TNF-alpha induces p73 gene expression, which, together with interferon regulatory factor-1, plays an important role in mediating caspase-1 promoter activation by TNF-alpha.     

The Nitrogen Metabolism of Groundnut Plants: the Role of {gamma}-methyleneglutamine and {gamma}-methyleneglutamic Acid

The changes occurring in the nitrogenous compounds during thegrowth of groundnut seedlings in the dark and light were compared,particular attention being centred on the levels of -methyleneglutamine,the principal amide of these plants, and -methyleneglutamicacid. The distribution of amino acids and amides in the mainorgans of normal young and mature plants was also examined.Suggestions are made concerning the possible pathways of synthesisand the functions of -methyleneglutamic acid and -methyleneglutaminein groundnut plants.     

Id2 reverses cell cycle arrest induced by {gamma}-irradiation in human HaCaT keratinocytes

Id2 plays a key role in epithelial cells, regulating differentiation, the cell cycle, and proliferation. Because human skin constantly renews itself and is the first target of irradiation, it is of primary interest to evaluate whether such a gene may be regulated in keratinocytes exposed to ionizing radiation. We show here that Id2 is induced in response to gamma-irradiation and have investigated the consequence of this regulation on cell fate. Using RNA interference, we observed that Id2 extinction significantly reduces cell growth in human keratinocytes through the control of the G(1)-S transition of the cell cycle. We have investigated whether the impact of Id2 on the cell cycle may have a physiological role on the cell's ability to cope with radiative stress. Indeed, when Id2 is down-regulated through interfering RNA, cells are more sensitive to irradiation. Conversely, when Id2 is overexpressed, this somehow protects the cell. We propose that Id2 favors reentering the cell cycle after radiation-induced cell cycle arrest to permit the recovery of keratinocytes exposed to ionizing radiation.     

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.     

The Role of Glutamate Decarboxylase and {gamma}-Aminobutyric Acid in Germinating Barley

The variations in glutamate decarboxylase activity and in glutamicacid and -ABA concentration have been measured in barley embryosduring the uptake of water and in the roots and shoots for upto 6 days of growth. Glutamate decarboxylase activity was relativelysteady in the embryos during soaking but rose rapidly once growthbegan. This development paralleled an increase in the concentrationof glutamic acid in both roots and shoots at a time when theconcentration of -ABA was falling. During soaking in aeratedwater, the -ABA content of the embryos rose for 36 h, at whichpoint it accounted for 35 per cent of the soluble amino acids.-ABA was found to be a major free amino acid in roots but notin shoots. Experiments in vivo involving ¹⁴C-labelled glutamicacid and -ABA indicated that carbon from -ABA passed very rapidlyinto the citric-acid cycle intermediates and also that, throughoutthe period studied, -ABA was formed from glutamic acid despitethe alterations in relative concentrations of these amino acidsin the growing tissue.     

Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2-α1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1-β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1 β2 receptors can dimerize via K24'C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.     

The Enzymic Decarboxylation of {gamma}-methyleneglutamic Acid by Plant Extracts

-Methyleneglutamic acid, an acidic amino-acid isolated fromgroundnut plants, was decarboxylated by enzymes present in extractsof Capsicum fruits, barley roots, and tulip leaves, and alsoby intact cells of Clostridium welchii S.R. I2. The amino-acidwas attacked in a similar manner to, but in all cases at a slowerrate than, l-glutamic acid. The nature of the enzyme responsiblefor the decarboxylation of -methyleneglutamic acid was furtherinvestigated using preparations from barley roots (which donot contain the amino-acid) and from tulip leaves (in whichthe amino-acid is normally present, together with larger amountsof its amide form, -methyleneglutamine). The effects of pH,inhibitors, and partial heat denaturation upon the enzyme systemspresent in the barley and tulip extracts indicated that a singleenzyme was responsible for the decarboxylation of both l-glutamicacid and -methyleneglutamic acid. Although the Cl. welchii rapidlydeamidated and then decarboxylated l-glutamine, -methyleneglutaminewas not attacked by the organism.     

Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR

Colorectal cancer is a leading cause of cancer-related morbidity and mortality in the United States. Curcumin, the yellow pigment in turmeric, possesses inhibitory effects on growth of a variety of tumor cells by reducing cell proliferation and inducing apoptosis. Effects of the peroxisome proliferator-activated receptor-gamma (PPARgamma) on stimulating cell differentiation and on inducing cell cycle arrest have attracted attention from the perspective of treatment and prevention of cancer. The aim of this study was to elucidate the mechanisms by which curcumin inhibits colon cancer cell growth. In the present report, we observed that curcumin, in a dose-dependent manner, inhibited the growth of Moser cells, a human colon cancer-derived cell line, and stimulated the trans-activating activity of PPARgamma. Further studies demonstrated that activation of PPARgamma was required for curcumin to inhibit Moser cell growth. Activation of PPARgamma mediated curcumin suppression of the expression of cyclin D1, a critical protein in the cell cycle, in Moser cells. In addition, curcumin blocked EGF signaling by inhibiting EGF receptor (EGFR) tyrosine phosphorylation and suppressing the gene expression of EGFR mediated by activation of PPARgamma. In addition to curcumin reduction of the level of phosphorylated PPARgamma, inhibition of cyclin D1 expression played a major and significant role in curcumin stimulation of PPARgamma activity in Moser cells. Taken together, our results demonstrated for the first time that curcumin activation of PPARgamma inhibited Moser cell growth and mediated the suppression of the gene expression of cyclin D1 and EGFR. These results provided a novel insight into the roles and mechanisms of curcumin in inhibition of colon cancer cell growth and potential therapeutic strategies for treatment of colon cancer.