The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Trehalose mediated growth inhibition of Arabidopsis seedlings is due to trehalose-6-phosphate accumulation

Trehalose-6-phosphate (T6P) is required for carbon utilization during Arabidopsis development, and its absence is embryo lethal. Here we show that T6P accumulation inhibits seedling growth. Wild-type seedlings grown on 100 mm trehalose rapidly accumulate T6P and stop growing, but seedlings expressing Escherichia coli trehalose phosphate hydrolase develop normally on such medium. T6P accumulation likely results from much-reduced T6P dephosphorylation when trehalose levels are high. Metabolizable sugars added to trehalose medium rescue T6P inhibition of growth. In addition, Suc feeding leads to a progressive increase in T6P concentrations, suggesting that T6P control over carbon utilization is related to available carbon for growth. Expression analysis of genes from the Arabidopsis trehalose metabolism further supports this: Suc rapidly induces expression of trehalose phosphate synthase homolog AtTPS5 to high levels. In contrast, T6P accumulation after feeding trehalose in the absence of available carbon induces repression of genes encoding T6P synthases and expression of T6P phosphatases. To identify processes controlled by T6P, we clustered expression profile data from seedlings with altered T6P content. T6P levels correlate with expression of a specific set of genes, including the S6 ribosomal kinase ATPK19, independently of carbon status. Interestingly, Suc addition represses 15 of these genes, one of which is AtKIN11, encoding a Sucrose Non Fermenting 1 (SNF1)-related kinase known to play a role in Suc utilization.     

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.     

肉桂醛抗人宫颈癌相关机制的研究

目的探讨不同浓度的肉桂醛对HeLa细胞P21、CDK4蛋白表达的影响及意义。方法不同浓度的经过纯度鉴定的肉桂醛处理体外培养的HeLa细胞,培养24 h后免疫组织化学和Western blotting法检测HeLa细胞P21、CDK4蛋白表达的变化。结果肉桂醛纯度〉96.24%;肉桂醛能显著增高P21和降低CDK4蛋白在HeLa细胞中的表达,各浓度肉桂醛处理组的P21、CDK4蛋白表达与溶剂对照组相比差异均有统计学意义（P〈0.01,P〈0.05）。结论肉桂醛能上调宫颈癌HeLa细胞P21蛋白表达和下调CDK4蛋白表达,可能是促进HeLa细胞凋亡的机制之一。     

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

Summary. The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48h and was approximately two times higher after 72h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H₂O₂ production was observed both 48 and 72h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H₂O₂ production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H₂O₂ was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H₂O₂, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia.     

Antiaflatoxigenic activity of eugenol is due to inhibition of lipid peroxidation

Eugenol inhibited aflatoxin production by Aspergillus parasiticus NRRL 2999 in a dose-dependent manner up to a concentration of 0.75 mmol l-1 without inhibiting growth. When the mould was grown for 3 d in the presence of 0.45 mmol l-1 eugenol (concentration inhibiting aflatoxin production by 50%), in vivo activities of components of polysubstrate monooxygenase were decreased at idiophase, concomitant with decreased activities of enzymes involved in free radical scavenging, lipid peroxidation and maintenance of redox potential. These results indicate that antiaflatoxigenic actions of eugenol may be related to inhibition of the ternary steps of aflatoxin biosynthesis involving lipid peroxidation and oxygenation.     

The inhibition of capacitative calcium entry due to ATP depletion but not due to glucosamine is reversed by staurosporine.

The capacitative Ca2+ entry pathway in J774 macrophages is rapidly inhibited by the amino sugar glucosamine. This pathway is also inhibited by treatments such as 2-deoxy-D-glucose (2dGlc) or glucose deprivation that inhibit glycolysis and lead to significant decreases in cellular ATP and other trinucleotides. We sought to determine whether glucosamine's effect on capacitative Ca2+ entry was also due to ATP depletion, as has been suggested recently for its link to insulin resistance. In contrast to brief treatments with 2dGlc, there was no significant decrease in ATP following exposure to glucosamine. In addition, the 2dGlc-mediated inhibition of capacitative Ca2+ influx was reversed by staurosporine, a microbial alkaloid that inhibits a broad range of protein kinases. Staurosporine was also able to reverse the inhibition of capacitative Ca2+ entry seen following other treatments that decreased cellular ATP levels, including cytochalasin B and iodoacetic acid. Other inhibitors of protein kinase C, including bisindolylmaleimide, K252a, H-7, and calphostin C, were unable to mimic this effect of staurosporine. However, the inhibition of capacitative Ca2+ influx in the presence of glucosamine was not reversed by staurosporine. These data indicate that the inhibitory action on capacitative Ca2+ entry of glucosamine is distinct from that caused by ATP depletion.     

Polyamine-mediated inhibition of in-vitro cell proliferation is not due to acrolein

The influence of acrolein or spermine on the viability and growth of phytohaemagglutinin-stimulated rat thymic lymphocytes in cultures supplemented with foetal calf serum have been investigated. Acrolein (greater than 20 microM) was cytotoxic; spermine had little effect on viability, but inhibited [3H]TdR incorporation at low concentrations (approximately 10 microM). Cells treated with greater than 8 microM acrolein 3 hr before stimulation exhibited irreversible inhibition of protein synthesis, whereas 50 microM spermine had no effect, even when cells were treated for 24 hr before stimulation. However, addition of 25 microM spermine after stimulation did inhibit both [3H]-uridine incorporation and protein synthesis: this was reversible if cells were freed of polyamine within 4 hr, but not if washed after 24 hr. These results show that, contrary to several previous reports, in-vitro inhibition of cell proliferation by spermine is not due to the formation and action of acrolein.     

Creatine phosphate inhibition of adenylate deaminase is mainly due to pyrophosphate.

Inhibition of rat skeletal muscle adenylate deaminase by creatine phosphate reported previously is due to inorganic pyrophosphate present as a contaminant in commercial preparations of creatine phosphate. This conclusion is based on the following evidence: a compound that inhibits adenylate deaminase can be separated from commercially prepared creatine phosphate by ion exchange chromatography; the inhibition by "creatine phosphate" and by the separated inhibitory compound is relieved by treatment with inorganic pyrophosphatase; inhibition by inorganic pyrophosphate is similar to that produced by unpurified creatine phosphate; and pyrophosphate is present in commercially available creatine phosphate in amounts sufficient to account for the inhibition. Some commercial preparations of creatine phosphate contain much less pyrophosphate than others; these preparations are only weakly inhibitory. Inorganic triphosphate is a more powerful inhibitor of the enzyme than pyrophosphate; it may also be present as a contaminant in creatine phosphate.     

Creatine phosphate inhibition of heart lactate dehydrogenase and muscle pyruvate kinase is due to a contaminant

Previously reported inhibitions of heart lactate dehydrogenase (Guppy, M., and Hochachka, P.W. (1978) J. Biol. Chem. 253, 8465-8469) and muscle pyruvate kinase (Kemp, R.G. (1973) J. Biol. Chem. 248, 3963-3967) by creatine phosphate are due to oxalate which is a contaminant found in some commercial preparations of creatine phosphate.     

Cessation of growth to prevent cell death due to inhibition of phosphatidylcholine synthesis is impaired at 37 degrees C in Saccharomyces cerevisiae

Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1 (CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitive PCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7-8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 degrees C and not 25 degrees C. The inhibition of phosphatidylcholine synthesis at 37 degrees C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect.     

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

Outgrowth and vegetative growth inhibition of Bacillus subtilis spores after transient exposure to polymyxin B.

Polymyxin B combined with the resting spores of Bacillus subtilis and inhibited outgrowth and vegetative growth after germination. The antibiotic was released from the resting spores and its inhibitory action was reversed by the addition of di- and trivalent metallic cations.     

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.