Differential Expression of Three Members of the 1-Aminocyclopropane-1-Carboxylate Synthase Gene Family in Carnation1

We investigated the expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes in carnation (Dianthus caryophyllus cv White Sim) under conditions previously shown to induce ethylene biosynthesis. These included treatment of flowers with 2,4-dichlorophenoxyacetic acid, ethylene, LiCl, cycloheximide, and natural and pollination-induced flower senescence. Accumulation of ACC synthase transcripts in leaves following mechanical wounding and treatment with 2,4-dichlorophenoxyacetic acid or LiCl was also determined by RNA gel-blot analysis. As in other species, the carnation ACC synthase genes were found to be differentially regulated in a tissue-specific manner. DCACS2 and DCACS3 were preferentially expressed in styles, whereas DCACS1 mRNA was most abundant in petals. Cycloheximide did not induce increased accumulation of ACC synthase transcripts in carnation flowers, whereas the expression of ACC synthase was up-regulated by auxin, ethylene, LiCl, pollination, and senescence in a floral-organ-specific manner. Expression of the three ACC synthases identified in carnation did not correspond to elevated ethylene biosynthesis from wounded or auxin-treated leaves, and there are likely additional members of the carnation ACC synthase gene family responsible for ACC synthase expression in vegetative tissues.

IL-4 determines eicosanoid formation in dendritic cells by down-regulation of 5-lipoxygenase and up-regulation of 15-lipoxygenase 1 expression

Dendritic cell (DC) differentiation from human CD34+ hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34+ HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor α expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase+ (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX+ cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1+ 5-LO-deficient DCs. However, transforming growth factor β1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor β1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX+ cells. In the absence of IL-4, major eicosanoids of CD34+-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B4, whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: Engagement of CD33 induces apoptosis of leukemic cells

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IκBα(S32A/S36A), which is not phosphorylated and prevents NF-κB activation, with LMP1 showed that NF-κB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-κB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-κB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E2 in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT). Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect. The drugs could also induce heterodimerization of dimerization defective mutants. Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs. In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization. This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits.

Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice1

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O2, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.

Integrin engagement mediates tyrosine dephosphorylation on platelet-endothelial cell adhesion molecule 1.

Platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, neutrophils, and monocytes and plays a role during endothelial cell migration. Phosphoamino acid analysis and Western blot analysis with anti-phosphotyrosine antibody show that endothelial PECAM-1 is tyrosine-phosphorylated. Phosphorylation is decreased with endothelial cell migration on fibronectin and collagen and with cell spreading on fibronectin but not on plastic. Cell adhesion on anti-integrin antibodies is also able to specifically induce PECAM-1 dephosphorylation while concurrently inducing pp125 focal adhesion kinase phosphorylation. Inhibition of dephosphorylation with sodium orthovanadate suggests that this effect is at least partially mediated by phosphatase activity. Tyr-663 and Tyr-686 are identified as potential phosphorylation sites and mutated to phenylalanine. When expressed, both mutants show reduced PECAM-1 phosphorylation but Phe-686 mutants also show significant reversal of PECAM-1-mediated inhibition of cell migration and do not localize PECAM-1 to cell borders. Our results suggest that beta 1-integrin engagement can signal to dephosphorylate PECAM-1 and that this signaling pathway may play a role during endothelial cell migration.

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Activation of an interleukin 1 converting enzyme-dependent apoptosis pathway by granzyme B.

Cytotoxic T lymphocytes (CTL) can induce apoptosis through a granzyme B-based killing mechanism. Here we show that in cells undergoing apoptosis by granzyme B, both p45 pro-interleukin 1 beta converting enzyme (ICE) and pro-CPP32 are processed. Using ICE deficient (ICE -/-) mice, embryonic fibroblasts exhibit high levels of resistance to apoptosis by granzyme B or granzyme 3, while B lymphoblasts are granzyme B-resistant, thus identifying an ICE-dependent apoptotic pathway that is activated by CTL granzymes. In contrast, an alternative ICE-independent pathway must also be activated as ICE -/- thymocytes remain susceptible to apoptosis by both granzymes. In ICE -/- B cells or HeLa cells transfected with mutant inactive ICE or Ich-1S that exhibit resistance to granzyme B, CPP32 is processed to p17 and poly(ADP-ribose) polymerase is cleaved indicating that this protease although activated was not associated with an apoptotic nuclear phenotype. Using the peptide inhibitor Ac-DEVD-CHO, apoptosis as well as p45 ICE hydrolysis are suppressed in HeLa cells, suggesting that a CPP32-like protease is upstream of ICE. In contrast, p34cdc2 kinase, which is required for granzyme B-induced apoptosis, remains inactive in ICE -/- B cells indicating it is downstream of ICE. We conclude that granzyme B activates an ICE-dependent cell death pathway in some cell types and requires a CPP32-like Ac-DEVD-CHO inhibitable protease acting upstream to initiate apoptosis.

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.

Brain lipids that induce sleep are novel modulators of 5-hydroxytrypamine receptors.

Amide derivatives of fatty acids were recently isolated from cerebrospinal fluid of sleep-deprived animals and found to induce sleep in rats. To determine which brain receptors might be sensitive to these novel neuromodulators, we tested them on a range of receptors expressed in Xenopus oocytes. cis-9,10-Octadecenamide (ODA) markedly potentiated the action of 5-hydroxytryptamine (5-HT) on 5-HT2A and 5-HT2C receptors, but this action was not shared by related compounds such as oleic acid and trans-9,10-octacenamide. ODA was active at concentrations as low as 1 nM. The saturated analog, octadecanamide, inhibited rather than potentiated 5-HT2C responses. ODA had either no effect or only weak effects on other receptors, including muscarinic cholinergic, metabotropic glutamate, GABA(A), N-methyl-D-asparate, or alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid receptors. Modulation of 5-HT2 receptors by ODA and related lipids may represent a novel mechanism for regulation of receptors that activate G proteins and thereby play a role in alertness, sleep, and mood as well as disturbances of these states.

A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos.

The formation of ventral mesoderm has been traditionally viewed as a result of a lack of dorsal signaling and therefore assumed to be a default state of mesodermal development. The discovery that bone morphogenetic protein 4 (BMP4) can induce ventral mesoderm led to the suggestion that the induction of the ventral mesoderm requires a different signaling pathway than the induction of the dorsal mesoderm. However, the individual components of this pathway remained largely unknown. Here we report the identification of a novel Xenopus homeobox gene PV.1 (posterior-ventral 1) that is capable of mediating induction of ventral mesoderm. This gene is activated in blastula stage Xenopus embryos, its expression peaks during gastrulation and declines rapidly after neurulation is complete. PV.1 is expressed in the ventral marginal zone of blastulae and later in the posterior ventral area of gastrulae and neurulae. PV.1 is inducible in uncommited ectoderm by the ventralizing growth factor BMP4 and counteracts the dorsalizing effects of the dominant negative BMP4 receptor. Overexpression of PV.1 yields ventralized tadpoles and rescues embryos partially dorsalized by LiCl treatment. In animal caps, PV.1 ventralizes induction by activin and inhibits expression of dorsal specific genes. All of these effects mimic those previously reported for BMP4. These observations suggest that PV.1 is a critical component in the formation of ventral mesoderm and possibly mediates the effects of BMP4.