A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants

Members of the plant NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family display protein sequence homology with the SLC15/PepT/PTR/POT family of peptide transporters in animals. In comparison to their animal and bacterial counterparts, these plant proteins transport a wide variety of substrates: nitrate, peptides, amino acids, dicarboxylates, glucosinolates, IAA, and ABA. The phylogenetic relationship of the members of the NRT1/PTR family in 31 fully sequenced plant genomes allowed the identification of unambiguous clades, defining eight subfamilies. The phylogenetic tree was used to determine a unified nomenclature of this family named NPF, for NRT1/PTR FAMILY. We propose that the members should be named accordingly: NPFX.Y, where X denotes the subfamily and Y the individual member within the species

Integration of physiological and developmental responses of the plant to nitrate

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Mutation of a nitrate transporter, AtNRT1 : 4, results in a reduced petiole nitrate content and altered leaf development

Unlike nitrate uptake of plant roots, less is known at the molecular level about how nitrate is distributed in various plant tissues. In the present study, characterization of the nitrate transporter, AtNRT1:4, revealed a special role of petiole in nitrate homeostasis. Electrophysiological studies using Xenopus oocytes showed that AtNRT1:4 was a low-affinity nitrate transporter. Whole-mount in situ hybridization and RT-PCR demonstrated that AtNRT1:4 was expressed in the leaf petiole. In the wild type, the leaf petiole had low nitrate reductase activity, but a high nitrate content, indicating that it is the storage site for nitrate, whereas, in the atnrt1:4 mutant, the petiole nitrate content was reduced to 50-64% of the wild-type level. Moreover, atnrt1:4 mutant leaves were wider than wild-type leaves. This study revealed a critical role of AtNRT1:4 in regulating leaf nitrate homeostasis, and the deficiency of AtNRT1: 4 can alter leaf development

The functional haplotype of peptidylarginine deiminase IV (S55G, A82V and A112G) associated with susceptibility to rheumatoid arthritis dominates apoptosis of acute T leukemia Jurkat cells

Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previously, our study showed that inducible PADI4 causes haematopoietic cell death. Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn't induce apoptosis however it promoted inducible PADI4-decreased cell viability and -enhanced apoptosis. Through in vitro and in vivo PADI enzyme activity assay, we demonstrated that PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis were compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c was released from mitochondria to cytoplasm in significant amounts. Western blotting data showed the increase in apoptosomal caspase activation during programmed cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 increasing their enzyme activity could enhance apoptosis through the mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs

Ornithine decarboxylase attenuates leukemic chemotherapy drugs-induced cell apoptosis and arrest in human promyelocytic HL-60 cells

Ornithine decarboxylase (ODC), the rate-limiting enzyme of the polyamine biosynthetic pathway, plays an important role in cell cycle, tumor promotion and anti-apoptosis. In our previous studies, overexpression of ODC prevented apoptosis induced by tumor necrosis factor-alpha and methotrexate. We further investigated the apoptotic mechanisms of the cancer chemotherapeutic drugs, including etoposide (VP-16), paclitaxel (TAX) and cisplatin (CDDP), and the influences of ODC on apoptosis and cell cycle. Our results showed that the investigated drugs induced caspase-dependent apoptosis, the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (Delta psi(m)) in HL-60 cells, all of which were reversed by putrescine, glutathione or N-acetyl-L-Cysteine. Overexpression of ODC prevented the cancer chemotherapeutic drugs-induced apoptosis, ROS generation and the disruption of Delta psi(m). After drug administrations, the decline of Bcl-2, cytochrome c release and caspases' activation were inhibited by ODC overexpression. In cell cycle, ODC overexpressed cells seemed to overcome the G1 arrest and G2/M arrest, caused by VP-16 and TAX, respectively, and kept on the cell cycle rolling. Overexpression of ODC increased the expression of Cyclin A, D, E and Cdk4 and the enzyme activity of Cdk1 and Cdk2 after the treatment of VP-16 and TAX, respectively. In conclusions, the cancer chemotherapeutic drugs-induced apoptosis is through ROS-related, mitochondria-mediated and caspase-dependent pathways. With higher ODC activity, cells are resistant to the cancer chemotherapeutic drugs-induced apoptosis and keep on the cell cycle rolling with the significant interference in G1/S arrest caused by VP-16 and G2/M arrest by TAX. (c) 2008 Elsevier Ltd. All rights reserved

Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells

Curcumin, a well-known dietary pigment derived from the food flavoring turmeric (Curcuma longa) exhibits anti-proliferative, anti-inflammatory, and anti-oxidative activities. Recently, studies have shown that a chemopreventive effect of curcumin could be due to the hyperproduction of reactive oxygen species (ROS) inducing apoptosis in tumor cells. In our previous studies, ornithine decarboxylase (ODC) overexpression prevented tumor necrosis factor alpha (TNF-alpha)- and methotrexate-induced apoptosis via reduction of ROS. Furthermore, ODC is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. In this study, we found that enzyme activity and protein expression of ODC were reduced during curcumin treatment. Overexpression of ODC in human promyelocytic leukemia HL-60 parental cells could reduce curcumin-induced apoptosis, which leads to loss of mitochondrial membrane potential (Delta psi(m)), through reducing intracellular ROS. Moreover, ODC overexpression prevented cytochrome c release and the activation of caspase-9 and caspase-3 following curcumin treatment. These results demonstrate that curcumin-induced apoptosis occurs through a mechanism of down-regulating ODC and along a ROS-dependent mitochondria-mediated pathway. (C) 2007 Elsevier Inc. All rights reserved

The PKC delta inhibitor, rottlerin, induces apoptosis of haematopoietic cell lines through mitochondrial membrane depolarization and caspases' cascade

Rottlerin is a widely selective protein kinase C delta (PKC delta) inhibitor isolated from Mallotus philippinensis. It shown to be effective against several human tumor cell lines and in potentiating chemotherapy-induced cytotoxcicity. Using the trypan blue exclusion assay, we demonstrated that rottlerin reduced the viability in a dose- and time-dependent manner of human leukemia HL60 cells, human acute T cell leukemia Jurkat cells and mouse macrophage RAW 264.7 cells. Rottlerin caused apoptosis and the apaptotic processing was inhibited by a caspase inhibitor, z-VAD-fmk, in these haematopoietic cells. The apoptosis-inducing activities were determined by nuclear condensation, sub-G, appearance, DNA fragmentation, loss of mitochondrial membrane potential release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Expression of PKC delta and Bcl-2 protein inhibited Delta psi(m) change and repressed cell death. These studies suggest that the cytotoxic effects of rottlerin through inhibition Of PKC delta cause mitochondrial dysfunction, cytochrome c release from mitochondria into cytoplasm and the activation of caspases' cascade. (c) 2005 Elsevier Inc. All rights reserved

Ornithine decarboxylase interferes with macrophage-like differentiation and matrix metalloproteinase-9 expression by tumor necrosis factor alpha via NF-kappa B

Ornithine decarboxylase (ODC), a tumor promoter, provokes cell proliferation, and inhibits cell death; but the mechanism involved in cell differentiation remains unknown. Herein, we examine whether it functions during macrophage-like differentiation. Previous studies reveal that ODC. a rate-limiting enzyme of polyamine biosynthesis, and polyamines are involved in restraining immune response in activated macrophage. By using 12-O-tetradecanoylphorbol-13-acetate (TPA)-differentiated human promyelocytic HL-60 and promonocytic U-937 cells, we discover that polyamines block the expression, secretion and activation of MMP-9. Meanwhile conventional expression of ODC represses tumor necrosis factor-alpha (TNF-alpha) expression and nuclear factor-kappaB (NF-kappa B) activation as well as MMP-9 enzyme activity. Following stimulation by TNF-alpha, the secretion of MMP-9 is restored in ODC-overexpressed cells. In addition, the NF-kappa B inhibitors (pyrrolidinedithiocarbamate, BAY-11-7082 and lactacystin) suppress the TPA-induced MMP-9 enzyme activity. Concurrently, both the irreversible inhibitor of ODC, alpha-difluoromethytomithine, and TNF-a could not recover MMP-9 activation following NF-kappa B inhibitor treatment in parental cells. Furthermore, ODC could directly inhibit and attenuate NF-kappa B DNA binding and transcriptional activation. Therefore, we suggest that ODC inhibits the TNF-alpha-elevated MMP-9 activation via NF-kappa B as TPA-induced macrophage-like differentiation and this interrupting mechanism may provide a new conceivable resolution why leukemia is poorly differentiated besides atypical growth. (c) 2007 Elsevier Ltd. All rights reserved

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-alpha) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNIF-alpha-induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential (Delta psi(m)) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained Delta psi(m) and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of Delta psi(m) and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt Delta psi(m) or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of Delta psi(m) and apoptosis when cells were treated with TNF-alpha. ODC overexpression avoided the decline of BcI-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of BcI-2 maintained Delta psi(m) and prevented apoptosis, but could not reduce ROS until four hours after TNF-alpha treatment. According to these data, we suggest that TNF-alpha induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF-alpha-induced apoptosis by decreasing intracellular ROS to avoid BcI-2 decline, maintain Delta psi(m), prevent cytochrome c release and deactivate the caspase cascade pathway

Overexpression of peptidylarginine deiminase IV features in apoptosis of haematopoietic cells

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (Delta psi(m)), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy Delta psi(m), release cytochrome c to cytoplasm and activate the caspase cascade