Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

The Pin Nematode,Paratylenchus neamblycephalus,on Myrobalan Plum and Other Hosts

Elimination of Paratylenchus neoamblvcephalus from soil by fumigation with 1,2-dibromoethane stimulated the growth of Myrobalan seedlings grown in it. Addition of a suspension of P. neoamblycephalus to Myrobalan seedlings inhibited their growth as compared to noninoculated controls. When nematodes were removed from the suspension by settling, and the supernatant liquid was used as inoculum, no stunting occurred. Roots of Myrobalan seedlings inoculated with surface-sterilized P. neoamblycephalus were smaller, darker, and had fewer feeder roots than those of noninoculated controls. Nematodes were observed feeding ectoparasitically, but with heads embedded in roots as deep as the cortex. They were associated with small lesions and dead lateral roots. Clusters of nematodes were common at ruptures in the epidermis, and where lateral roots emerged. Limitation of Myrobalan growth by P. neoamblvcephalus was greater at 20 and 27 C than at 30 C, and was not affected by pH over the range 4.5 to 6.5. Rose, apricot, peach, and all selections and hybrids of Prunus cerasffera tested were hosts for P. neoamblrcephalus. The nematode could not be cultured on various herbaceous plants nor on Myrobalan callus tissue.     

亚硝基化在植物细胞死亡及防御反应中的作用

亚硝基化是近年来新发现的不依赖于环磷酸鸟苷的一氧化氮信号转导途径, 是一氧化氮分子通过共价结合修饰靶蛋白的半胱氨酸残基从而改变其功能的过程。该文重点综述了近年来亚硝基化在细胞死亡和抗病反应这两个紧密关联的生物学过程中的最新研究成果, 总结了亚硝基化通过修饰和调控靶蛋白从而促进或抑制细胞死亡和抗病反应, 并对现有研究结果中某些不一致之处提出自己的观点。最后根据动物学领域的最新研究进展对植物学领域未来亚硝基化的研究方向进行了展望。     

细胞程序死亡与bcl－2基因

细胞程序死亡(PCD),是有别于细胞坏死的另一种重要的衰老、死亡形式,它在胚胎发育、肿瘤发生、免疫系统的克隆选择中起重要作用.bcl-2是调控PCD的基因,但不能抑制所有类型的PCD.最近发现,bcl-X基因编码大小不同的两种蛋白,分别具有刺激和抑制PCD的功能.bcl-2通过抑制PCD可导致细胞癌变,因而bcl-2被看作第三类癌基因.     

细胞程序死亡与bcl－2基因

细胞程序死亡,是有别于细胞坏死的另一种重要的衰老,死亡形式,它在胚胎发育,肿瘤发生,免疫系统的克隆选择中起重要作用,ｂｃｌ－２是调控ＰＣＤ的基因,但不能抑帛有类型的ＰＣＤ,最近发现,ｂｃｌ－Ｘ基因编码大小不同的两种蛋白,分别具有刺激和抑的ＰＤＣＣＤ的功能,ｂｃｌ－２通过抑制ＰＣＤ可导致细胞癌变,因而ｂｃｌ－２被看作第三类癌基因。     

POLYGALACTURONASE INVOLVED IN EXPANSION1 Functions in Cell Elongation and Flower Development in Arabidopsis

Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1’s involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.     

High Ca<Superscript>2+</Superscript> Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death

We investigated the hypothesis that high Ca²⁺ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca²⁺ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca²⁺ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca²⁺ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca²⁺ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.     

JAK2 and SHP2 Reciprocally Regulate Tyrosine Phosphorylation and Stability of Proapoptotic Protein ASK1

Previously we have shown that tyrosine 718 of ASK1 when phosphorylated is critical for SOCS1 binding and SOCS1-mediated degradation of ASK1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are unknown. In this study, we identified JAK2 and SHP2 as a Tyr-718-specific kinase and phosphatase, respectively. Interferon-γ (IFN-γ) induced degradation of ASK1 in normal but not in SOCS1-KO endothelial cells (EC). IFN-γ-induced tyrosine phosphorylation of ASK1 at Tyr-718 was blocked by a JAK2-specific inhibitor. IFN-γ enhanced the association between JAK2 and ASK1, and the ASK1-JAK2 complex was labile and was stabilized by the proteasomal inhibitor MG132. Furthermore, JAK2, but not JAK1, directly bound to and phosphorylated ASK1 at Tyr-718, leading to an enhanced association of ASK1 with SOCS1 and subsequent ASK1 degradation. Next, we showed that overexpression of the SH2-containing protein-tyrosine phosphatase-2 (SHP2) augmented, whereas a phosphatase-inactive mutant of SHP2 inhibited, TNF-induced ASK1 dephosphorylation. SHP2 associated with ASK1 in response to tumor necrosis factor in EC. An SHP-2 substrate-trapping mutant formed a complex with tyrosine-phosphorylated ASK1, suggesting that ASK1 is a direct SHP2 substrate. Moreover, SHP2 wild type, but not a catalytically inactive mutant, dissociated SOCS1 from ASK1. IFN-γ-induced ASK1 Tyr(P)-718 was enhanced in mouse EC deficient in SHP2 (SHP2-KO). In contrast, tumor necrosis factor-induced dephosphorylation of ASK1 at Tyr(P)-718 and activation of ASK1-JNK signaling, as well as EC apoptosis, are significantly reduced in SHP2-KO EC. Our data suggest that JAK2-SOCS1 and SHP2 reciprocally regulate ASK1 phosphorylation and stability in response to cytokines.Myocardial infarction due to atherosclerosis of coronary arteries remains the leading cause of death in the United States. It has become clear that increases in inflammatory mediators represent a common pathogenic mechanism for atherosclerosis (1). The vascular cell that normally limits the inflammatory and atherosclerotic process is the EC.³ Proinflammatory stimuli induce EC dysfunction, which is characterized by an enhanced sensitivity of vascular cells to proinflammatory and proapoptotic stimuli. Studies from our laboratory and others have demonstrated that ASK1 (apoptosis signal-regulating kinase-1), a member of MAP3K family (2, 3), is an effector of inflammation in EC (4–8). Almost all inflammatory stimuli such as tumor necrosis factor-α (TNF), interleukin-1 (IL-1), and reactive oxygen species activate ASK1. Activated ASK1 subsequently recruits and activates its downstream target MAP2Ks (MKK3/7 and MKK4/7), which in turn activate MAPKs (JNK and p38). Studies from ASK1-deficient mice have also linked ASK1 to cardiovascular pathogenesis. ASK1 deletion in mice attenuated angiotensin II-induced cardiac hypertrophy and remodeling. Neointimal formation due to proliferation of smooth muscle cells in a cuff injury model is also attenuated by ASK1 deletion in mice (9, 10).Although the linkage of ASK1 to inflammation is very strong, the mechanism by which inflammatory stimuli, including TNF, activate ASK1 is not fully understood. The identification of proteins associated with ASK1 and their regulation on ASK1 have provided some insights into the mechanism for ASK1 activation. ASK1 is a 170-kDa protein that is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. One important regulatory mechanism of ASK1 activity is its Ser/Thr phosphorylation and dephosphorylation by kinases and phosphatases. ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site and inhibits ASK1 activity (11, 12). TNF activates ASK1 in part by dissociating these cellular inhibitors from ASK1 (4, 7). Recently, we have identified PP2A as a phosphatase in TNF-induced dephosphorylation of ASK1 Ser(P)-967 (13). In addition to the 14-3-3-binding site, Ser(P)-967, ASK1 is phosphorylated at Ser-83 by Akt, leading to inhibition of ASK1 activity. In contrast, autophosphorylation of ASK1 at Thr-838 leads to oligomerization and activation (14). Phosphorylation of Thr-845 can be negatively regulated by the phosphatase PP5 (15). Similarly, we found that the ASK1 autophosphorylation at Thr-813 and Thr-842 also positively regulates ASK1 signaling (16).In contrast to Ser/Thr phosphorylation, regulation of ASK1 by tyrosine phosphorylation is less well understood. We have recently shown that ASK1 is phosphorylated at Tyr-718, and this phosphorylation is critical for the binding to suppressor of cytokine signaling-1 (SOCS1), a subunit of ubiquitin ligase responsible for ASK1 degradation (17). Tyrosine phosphorylation of ASK1 is up-regulated in response to growth factors and cytokines such as IFN-γ, whereas this phosphorylation can be down-regulated by TNF treatment, resulting in ASK1 dissociation from SOCS1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are not known.The cytoplasmic tyrosine kinase, JAK2, autophosphorylates in response to growth factors and cytokines, including IFN-γ. JAK2 then activates cytokine receptors and other cytoplasmic proteins such as the STATs by phosphorylating their key tyrosine residue. The JAK/STAT pathway can be regulated by SH2-containing protein-tyrosine phosphatases such as SHP2 (18–20). SHP2 is ubiquitously expressed and composed of two SH2 domains on the N-terminal and C-terminal protein-tyrosine phosphatase (PTP) domain. The SH2 domain of SHP2 mediates the association with phosphotyrosine-containing proteins present on activated receptors as well as on activated JAKs and STATs; this association triggers activation of the tyrosine phosphatase domain and subsequent dephosphorylation of substrates. SHP2 signals downstream of receptor tyrosine kinases and cytokine receptors, and in most cases it serves to positively transduce signals from these receptors. In other instances SHP2 has been shown to exhibit inhibitory signaling properties by negatively regulating the JAK-STAT pathway (19).In this study, we demonstrate that the IFN-γ-activated kinase JAK2 and TNF-activated SHP2 are the tyrosine kinase and phosphatase for Tyr-718 on ASK1, respectively. The actions of both JAK2 and SHP2 affect protein turnover of ASK1 and thus regulate ASK1/JNK-dependent proinflammatory and proapoptotic pathways in EC.     

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Lung Epithelial Cells Exposed to High Energy Protons

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.     

The Boron Requirement and Cell Wall Properties of Growing and Stationary Suspension-Cultured Chenopodium album L. Cells

Suspension-cultured Chenopodium album L. cells are capable of continuous, long-term growth on a boron-deficient medium. Compared with cultures grown with boron, these cultures contained more enlarged and detached cells, had increased turbidity due to the rupture of a small number of cells, and contained cells with an increased cell wall pore size. These characteristics were reversed by the addition of boric acid (≥7 μm) to the boron-deficient cells. C. album cells grown in the presence of 100 μm boric acid entered the stationary phase when they were not subcultured, and remained viable for at least 3 weeks. The transition from the growth phase to the stationary phase was accompanied by a decrease in the wall pore size. Cells grown without boric acid or with 7 μm boric acid were not able to reduce their wall pore size at the transition to the stationary phase. These cells could not be kept viable in the stationary phase, because they continued to expand and died as a result of wall rupture. The addition of 100 μm boric acid prevented wall rupture and the wall pore size was reduced to normal values. We conclude that boron is required to maintain the normal pore structure of the wall matrix and to mechanically stabilize the wall at growth termination.The ultrastructure and physical properties of plant cell walls are known to be affected by boron deficiency (Kouchi and Kumazawa, 1976; Hirsch and Torrey, 1980; Fischer and Hecht-Buchholz, 1985; Matoh et al., 1992; Hu and Brown, 1994; Findeklee and Goldbach, 1996). Moreover, boron is predominantly localized in the cell wall when plants are grown with suboptimal boron (Loomis and Durst, 1991; Matoh et al., 1992; Hu and Brown, 1994; Hu et al., 1996). In radish, >80% of the cell wall boron is present in the pectic polysaccharide RG-II (Matoh et al., 1993; Kobayashi et al., 1996), which is now known to exist as a dimer that is cross-linked by a borate ester between two apiosyl residues (Kobayashi et al., 1996; O''Neill et al., 1996). Dimeric RG-II is unusually stable at low pH and is present in a large number of plant species (Ishii and Matsunaga, 1996; Kobayashi et al., 1996, 1997; Matoh et al., 1996; O''Neill et al., 1996; Pellerin et al., 1996; Kaneko et al., 1997). The widespread occurrence and conserved structure of RG-II (Darvill et al., 1978; O''Neill et al., 1990) have led to the suggestion that borate ester cross-linked RG-II is required for the development of a normal cell wall (O''Neill et al., 1996; Matoh, 1997).One approach for determining the function of boron in plant cell walls is to compare the responses to boron deficiency of growing plant cells that are dividing and synthesizing primary cell walls with those of growth-limited plant cells in which the synthesis of primary cell walls is negligible. Suspension-cultured cells are well suited for this purpose because they may be reversibly transferred from a growth phase to a stationary phase. Continuous cell growth phase is maintained by frequent transfer of the cells into new growth medium (King, 1981; Kandarakov et al., 1994), whereas a stationary cell population is obtained by feeding the cells with Suc and by not subculturing them. Cells in the stationary phase are characterized by mechanically stabilized primary walls and reduced biosynthetic activity. Here we describe the responses of suspension-cultured Chenopodium album L. cells in the growth and stationary phases to boron deficiency. These cells have a high specific-growth rate, no significant lag phase, and reproducible changes in their wall pore size during the transition from the growth phase to the stationary phase (Titel et al., 1997).