Induction of cell arrest in G2: structural specificity of trigonelline

Synthetic analogues of N-methyl nicotinic acid, trigonelline, were prepared to test the structural features necessary for the induction of cellular arrest in G2 in Pisum sativum. Analogues that (1) were regioisomers of trigonelline, (2) possessed different 1,3-substituents, and (3) contained additional substituents on the pyridine ring were tested for their ability to induce cell arrest in G2 and to antagonize trigonelline induced arrest in G2. Only N-methyi-3-quinoline-carboxylic acid and 1-methyl nicotinamide induced cell arrest in G2, and 1-methyl-4-pyridine carboxylic acid and 1-methyl-2-pyridine carboxylic acid were effective trigonelline antagonists. These data further support a specific role for trigonelline in the induction of cell arrest in G2.     

Nicotinic acid and nicotinamide metabolism and promotion of cell arrest in G2 in Pisum sativum

Nicotinic acid and nicotinamide are immediate precursors of trigonelline, a hormone present in cotyledons of Pisum sativum L. which promotes cell arrest in G2 during cell maturation in roots and shoots. All three compounds are members of the pyridine nucleotide pathway for the synthesis of NAD and NADP. Concentrations of nicotinic acid and nicotinamide in excised roots grown for 3 days in White's medium with sucrose were determined by HPLC. Results suggest that nicotinamide is rapidly converted first to nicotinic acid and then trigonelline. High nicotinic acid concentrations may occur in excised roots. Conversion of trigonelline to nicotinic acid in excised roots did not occur in these experiments. The concentrations of either nicotinamide or nicotinic acid in roots are not related to the proportions of cells arrested in G2. Trigonelline promotes cell arrest in G2, and nicotinic acid and nicotinamide are active only because they are converted to trigonelline.     

Relationship between trigonelline concentration and promotion of cell arrest in G2 in cultured roots of Pisum sativum

Trigonelline, G2 Factor, present in cotyledons of Pisum sativum is transported to roots and shoots after germination. This hormone promotes prefere     

Comparison of Salt-Induced Osmotic Adjustment and Trigonelline Accumulation in Two Soybean Cultivars

21-d-old seedlings of the soybean (Glycine max) cvs. Essex and Forrest were treated with NaCl in a step-wise manner over 9 d (3 d 30 mM, 3 d 70 mM, and 3 d 100 mM) and maintained under 100 mM NaCl for an additional 14 d. During salt treatment, osmotic potential decreased more in cv. Forrest relative to cv. Essex. In non-stressed leaf tissue, cv. Forrest contained more trigonelline (TRG) relative to cv. Essex. During salt treatment, TRG amounts increased in cv. Forrest but were unchanged in cv. Essex. Both cvs. osmotically adjusted in response to salt stress; the maximal osmotic adjustment was 0.80 and 0.18 MPa in cv. Forrest and cv. Essex, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Degradation of nicotinamide adenine dinucleotide in cell suspension cultures

Metabolites of [carbonyl-¹⁴C]-NAD in cell suspension cultures of mung bean, soybean and garbanzo bean are trigonelline and compounds of the pyridine nucleotide cycle. Degradation of nicotinate does not occur. In parsley cell cultures nicotinate degradation and formation of nicotinic acid N-α-l-arabinoside were observed. These conjugates are alternative reservoir forms of nicotinic acid. The adenine moiety of NAD is degraded in cell cultures via hypoxanthine-xanthine-allantoin-allantoic acid, with accumulation of the latter two compounds.     

Leghaemoglobin biosynthesis in a new single cell system from soybean root nodules

Single cells were effectively released from 35–45-day-old soybean ( Glycine max L. cv. Yaefusanari) nodules by treatment with an enzymic solution containing 1 mg/ml maceration enzyme (Pectolyase Y-23), 0.5 M mannitol, 2% (w/v) sucrose and 0.5% (w/v) potassium dextran sulfate. Bacteroid-containing cells were purified by Percoll density gradient centrifugation. Electron microscopic observation showed that these cells were protoplasts enclosed by a thin wall and with well preserved internal structures including bacteroids. The single cells obtained were stable against centrifugation and vigorous pipetting. The cells retained the ability to synthesize proteins including leghaemoglobin. The ratio of leghaemoglobin components synthesized in the single cells was similar to that of components synthesized in the nodules. The bacteroidal cell fraction was further separated into three fractions by a Percoll density gradient centrifugation. Comparison of the absolute and relative leghaemoglobin content, the activity of glutamine synthetase in the cytoplasm and the activity of 3-hydroxybutyrate dehydrogenase in the bacteroid suggests that these fractions contained cells in different stages of symbiosis. This new single cell system should provide a useful experimental system for analyzing events in the root nodule.     

Geranium thunbergii extract-induced G1 phase cell cycle arrest and apoptosis in gastric cancer cells

ABSTRACT

Geranium thunbergii is a traditional East Asian medicine for stomach diseases including dysentery and stomach ulcers in East Asia and has been reported to possess biological activity. The benefits of G. thunbergii in gastric cancer are unknown. In this study, we demonstrate that G. thunbergii extract suppresses proliferation and induces death and G1/S cell cycle arrest of gastric cancer cells. Proliferation was significantly inhibited in a time- and dose-dependent manner. Cell cycle arrest was associated with significant decreases in CDK4/cyclinD1 complex and CDK2/cyclinE complex genes expression. In addition, the protein expression of caspase-3 was decreased and that of activated poly (ADP-ribose) polymerase (PARP) was increased, which indicated apoptosis. The expressions of the Bax and Bcl-2, which are apoptosis related proteins, were upregulated and down-regulated, respectively. The results indicate that G. thunbergii extract can inhibit proliferation and induce both G/S cell cycle arrest and apoptosis of gastric cancer cells. Also, the induction of apoptosis involved the intrinsic pathways of the cells. Take the results, we suggest that G. thunbergii extract has anti-gastric cancer activity and may be a potential therapeutic candidate for gastric cancer.     

Bisphenol A induces apoptosis and G2-to-M arrest of ovarian granulosa cells

We investigated the impact of bisphenol A (BPA) on murine ovarian granulosa cells. Ovarian granulosa cells were cultured with 100 fM to 100 microM BPA for 24 h to 72 h. BPA decreased granulosa cell viability in a dose- and time-dependent manner. The lowest concentration that induced a significant decrease was 100 pM (89.2 +/- 4.0% of the control). TUNEL analysis demonstrated that treatment with BPA increased apoptosis of granulosa cells in a dose- and time-dependent manner. In addition, flow cytometry analyses revealed that treatment with BPA resulted in G2-to-M arrest, which was most prominent at 48 h. BPA increased the expression of Bax and concomitantly decreased the expression of Bcl2 at both protein and mRNA levels of granulosa cells. These findings suggest that low, presumably environmentally relevant doses of BPA, decrease the viability of granulosa cells by inducing apoptosis and G2-to-M arrest. Up-regulation of Bax and down-regulation of Bcl2 were suggested to be involved in this apoptotic effect.     

Cantharidin decreased viable cell number in human osteosarcoma U-2 OS cells through G2/M phase arrest and induction of cell apoptosis

ABSTRACT

Cantharidin (CTD), a sesquiterpenoid bioactive substance, has been reported to exhibit anticancer activity against various types of cancer cells. The aim of the present study was to investigate the apoptosis effects and the underlying mechanisms of CTD on osteosarcoma U-2 OS cells. Results showed that CTD induced cell morphologic changes, reduced total viable cells, induced DNA damage, and G₂/M phase arrest. CTD increased the production of reactive oxygen species and Ca²⁺, and elevated the activities of caspase-3 and ?9, but decreased the level of mitochondrial membrane potential. Furthermore, CTD increased the ROS- and ER stress-associated protein expressions and increased the levels of pro-apoptosis-associated proteins, but decreased that of anti-apoptosis-associated proteins. Based on these observations, we suggested that CTD decreased cell number through G₂/M phase arrest and the induction of cell apoptosis in U-2 OS cells and CTD could be a potential candidate for osteosarcoma treatments.     

Biochemical and morphological identification of ceramide-induced cell cycle arrest and apoptosis in cultured granulosa cells

We have investigated the effects of ceramide on the progression of cell cycle and on apoptotic cell death in ovarian cultured granulosa cells. Rates of cellular proliferation were measured by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) and flow cytometric cell cycle analysis. We also examined for morphological and biochemical signs of apoptosis. The PCNA expression was downregulated in a dose-dependent manner after treatment with C6-ceramide. Flow cytometric analysis demonstrated that the exposure of granulosa cells to C6-ceramide markedly decreased the population associated with G0/G1 DNA content and the reduction of cell numbers in G0/G1 phase was accompanied by the elevation of the A0 phase. The exposure of granulosa cells to exogenous C6-ceramide induced drastic morphological changes including cytoplasmic- or nuclear condensation and typical apoptotic DNA degradation. We also observed that phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, significantly inhibited the ceramide-induced apoptosis. These results suggested that ceramide might block the progression of cell cycle at G0/G1 phase and as a consequence, granulosa cells would be committed to apoptosis. Our findings also indicated that down-regulation of the PKC activity might be involved in the ceramide-induced apoptosis in cultured granulosa cells.     

Polyamine synthesis in plants. Purification and properties of amidinotransferase from soybean (Glycine max) axes

Three-day-old soybean (Glycine max) seedlings were exposed to 0.4 M sorbitol solution for 4 h to induce amidinotransferase activity, with the corresponding enzyme being purified to homogeneity by chromatographic separation on DEAE-Sephacel, Sephacryl S-300 and L-arginine Sepharose 4B. The purified enzyme used L-arginine and L-glycine as the major donor/acceptor of the amidino group, respectively, with formation of guanidinoacetic acid and ornithine products being confirmed by ESI-MS. The enzyme is a tetrameric protein having a molecular mass of 240,000 Da, whose thiol group is needed for enzymatic activity. The K(M)s for arginine and glycine were 3.8 and 0.89 mM, respectively, with optimal temperature and pH being 37 degrees C and 9.5, respectively. The soybean amidinotransferase could be indirectly involved in nitrogen metabolism, as suggested by the observation that arginine:glycine amidinotransferase in soybean axes is indirectly involved in putrescine biosynthesis and displays feedback control at high levels of an endogenous regulator, putrescine.     

Isolation and Partial Characterization of the Major Seed Protein from Nicotiana tabacum,and Accumulation during Development

During the seed development of Nicotiana tabacum, appreciable accumulation of the soluble protein fraction started to occur at around the 6th day after anthesis and finally reached 12% on the basis of dry weight when seed maturation was accomplished. In the soluble fraction of mature seeds, four protein fractions were observed on analytical ultracentrifugation, and the protein having a sedimentation coefficient of 11.7S was the major one. The 11.7S protein was isolated and SDS-polyacrylamide gel electrophoresis indicated that the protein consisted of at least five subunits with molecular weights of 49,000, 31,000, 29,000, 21,000 and 19,000. The 11.7S protein was rich in glutamic acid or glutamine and arginine, and the presence of carbohydrate was confirmed.

During development, all of the five subunits started to appear during the period between the 12th and 15th day after anthesis.     

Overexpression of a novel osteopetrosis-related gene CCDC154 suppresses cell proliferation by inducing G2/M arrest

Osteopetrosis, a disorder of skeletal bone, can cause death during childhood. We previously described a new spontaneous autosomal recessive osteopetrosis mouse mutant, “new toothless” (ntl). In this study, we reported for the first time the identification, cloning and characterization of the coiled-coil domain-containing 154 (CCDC154), a novel gene whose deletion of ~5 kb sequence including exons 1–6 was completely linked to the ntl mutant. The CCDC154 was conserved between mouse and human and is wildly expressed in mouse tissues. The cellular localization of CCDC154 was in the early endosomes. Overexpression of CCDC154 inhibited cell proliferation of HEK293 cells by inducing G₂/M arrest. CCDC154 also inhibited tumor cell growth, and the soft agar assay revealed a significant decrease of the colony size of Hela cells upon transfection of CCDC154. Our results indicate that CCDC154 is a novel osteopetrosis-related gene involved in cell cycle regulation and tumor suppression growth.     

Overexpression of a novel osteopetrosis-related gene CCDC154 suppresses cell proliferation by inducing G2/M arrest

Osteopetrosis, a disorder of skeletal bone, can cause death during childhood. We previously described a new spontaneous autosomal recessive osteopetrosis mouse mutant, “new toothless” (ntl). In this study, we reported for the first time the identification, cloning and characterization of the coiled-coil domain-containing 154 (CCDC154), a novel gene whose deletion of ~5 kb sequence including exons 1–6 was completely linked to the ntl mutant. The CCDC154 was conserved between mouse and human and is wildly expressed in mouse tissues. The cellular localization of CCDC154 was in the early endosomes. Overexpression of CCDC154 inhibited cell proliferation of HEK293 cells by inducing G₂/M arrest. CCDC154 also inhibited tumor cell growth, and the soft agar assay revealed a significant decrease of the colony size of Hela cells upon transfection of CCDC154. Our results indicate that CCDC154 is a novel osteopetrosis-related gene involved in cell cycle regulation and tumor suppression growth.     

Mutually exclusive occurrence and metabolism of trigonelline and nicotinic acid arabinoside in plant cell cultures

In 50 cell suspension cultures of wide taxonomic origin, formation of trigonelline and nicotinic acid N-α-l-arabinoside from nicotinate was strictly alternative. The arabinoside was only found in cell cultures of the subclass Asteridae and in the higher orders of the subclasses Rosidae and Dilleniidae. Degradation of nicotinic acid could only be observed in cell cultures producing the arabinoside. Nicotinic acid degradation does not involve free 6-hydroxynicotinic acid. Cross feeding experiments with both conjugates and measurements of a nicotinic acid N-arabinoside: UDP-arabinosyltransferase support the hypothesis that metabolism of these two derivatives in cell cultures may be of chemosystematic value. Finally various discrepancies between plants and cell cultures with respect to nicotinate metabolism and to the natural occurrence of the two conjugates are discussed.     

Modulation of G(2) arrest enhances cell death induced by the antitumor 1-nitroacridine derivative,Nitracrine

Nitracrine (Ledakrin) is an antitumor drug which is activated by cellular enzymes and binds covalently to DNA. Previous studies have shown that covalent binding and crosslinking of DNA is associated with the cytotoxic and antitumor activities of this compound. In this study, cell cycle perturbations, effects on DNA synthesis and the cell death process initiated by Nitracrine were studied in murine leukemia L1210 cells. We show that exposure of L1210 cells to Nitracrine at the IC₉₉ concentration delayed progression through the S phase and transiently arrested cells in G₂/M as found by flow cytometry. Higher drug concentration (2 × IC₉₉) inhibited cell cycle progression in the S phase and induced rapid cell death. Both studied concentrations of the drug produced different effects on DNA synthesis as determined by bromodeoxyuridine incorporation, with a delay in the S phase progression at EC₉₉ concentration and irreversible arrest in early S phase at the higher dose (2 × IC₉₉). At both concentrations of Nitracrine cell death occurred preferentially in the S phase as revealed by the TUNEL assay. When cells treated with the drug for 4 hours were post-incubated in the presence of 1 mM caffeine this led to rapid cell death and suppression of the G₂ arrest. This was associated with a about 10-fold increase in the cytotoxicity of Nitracrine. Similar effects were observed for another DNA crosslinking agent, cis-platinum, and to a lesser extent, for DNA topoisomerase I inhibitor, camptothecin. Together, our studies show that suppression of G₂ arrest induced by Nitracrine greatly enhances its cytotoxicity toward L1210 cells.     

Temperature and growth-induced water potential

When the steins of dark-grown soybean [Glycine max (L.) Merr.] seedlings grew rapidly at favorable temperatures in saturating humidities, a water potential of about 0·2 MPa was induced by growth ($pS_o-$pS_w, where $pS_o is the water potential of the basal nonelongating tissue and $pS_w is the water potential of the elongating tissue). If this water potential was caused by high concentrations of solute in the apoplast, as has been proposed, lowering the temperature should have little effect on the potential. On the other hand, if the water potential was caused by apoplast tensions generated by growth, then the tensions should disappear as growth is inhibited by low temperatures. We observed that the growth-induced water potential became too small to detect when growth was inhibited by temperatures as low as 13—5 °C. The disappearance was observed as a rise in apoplast water potential using a thermocouple psychrometer for intact plants, a rise in cell turgor using a miniature pressure probe and a decrease in apoplast tensions using a pressure chamber. The disappearance was not caused by a loss of solute from the apoplast because the tensions fully accounted for the growth-induced water potential at all temperatures. The results are consistent with the lack of solute measured directly in the apoplast solutions at high temperatures (Nonami & Boyer 1987). Therefore, it was concluded that little solute was present in the apoplast at any temperature, and the growth-induced water potential was associated mostly with a tension that moved water from the xylem and into the surrounding cells to meet the demand of cell enlargement.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

高效液相色谱法测定葡萄叶中甘氨酸甜菜碱、脯氨酸及葫芦巴碱

甘氨酸甜菜碱、脯氨酸及葫芦巴碱的含量与葡萄的抗逆性能密切相关,应用离子交换树脂提取葡萄叶中的这三种物质,采用Suger-pak1(waters)色谱柱,柱温:85℃;样品测定时间:70min;进样体积:100μL;流速:0.6mL/min;检测波长:195nm;流动相:50mg/LCa-EDTA的HPLC同时分析测定这三种物质,甘氨酸甜菜碱、脯氨酸及葫芦巴碱线性相关系数分别为0.9999,0.9994及0.9994;保留时间分别为20.28,25.66及30.65min;检出限分别为1.35,2.56及1.02ng/mL,回收率为93%~96%。此方法分离效果好,稳定,精确。