Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

多型炭角菌的培养及多糖提取

多型炭角菌在4%葡萄糖,1%蛋白胨,250mL三角瓶装培养液80mL,pH6.5,25℃的培养条件下生长最好,发酵终点为168小时。正交试验得到K+, Mg2+,Ca2+, Zn2+, Fe2+, Mn2+和Cu2+等金属离子的最适浓度分别为0.15%, 0.03%, 0.05%, 0.0015%, 0.001%, 0.00005%和 0.0002%。对培养得到的菌丝体进行多糖提取,得到2种粗多糖:X-1(水溶性多糖)和X-2(碱溶性多糖);从发酵液提取的粗多糖为X-3。经Sephadex G-100和 Sephadex G-150凝胶柱层析,收集、合并的单一峰位部分经聚丙烯酰胺凝胶电泳及纸层析均呈现为单一斑点。多糖酸水解后均能与茚三酮显色,蛋白质组成分析表明其分子中富含16种氨基酸。     

Suppression of androgen receptor-mediated transactivation and cell growth by the glycogen synthase kinase 3 beta in prostate cells

Androgens play important roles in the growth of normal prostate and prostate cancer via binding to the androgen receptor (AR). In addition to androgens, AR activity can also be modulated by selective growth factors and/or kinases. Here we report a new kinase signaling pathway by showing that AR transactivation was repressed by wild type glycogen synthase kinase 3beta (GSK3 beta) or constitutively active S9A-GSK3 beta in a dose-dependent manner. In contrast, the catalytically inactive kinase mutant GSK3 beta showed little effect on the AR transactivation. The suppression of AR transactivation by GSK3 beta was abolished by the GSK3 beta inhibitor lithium chloride. The in vitro kinase assay showed that GSK3 beta prefers to phosphorylate the amino terminus of AR that may lead to the suppression of activation function 1 activity located in the NH(2)-terminal region of AR. GSK3 beta interrupted the interaction between the NH(2) and COOH termini of AR, and overexpression of the constitutively active form of GSK3 beta, S9A-GSK3 beta, reduced the androgen-induced prostate cancer cell growth in stably transfected CWR22R cells. Together, our data demonstrated that GSK3 beta may function as a repressor to suppress AR-mediated transactivation and cell growth, which may provide a new strategy to modulate the AR-mediated prostate cancer growth.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Plasma Adiponectin Levels and Metabolic Factors in Nondiabetic Adolescents

Objectives: The relationship of plasma adiponectin levels with various anthropometric and metabolic factors has been surveyed extensively in adults. However, how plasma adiponectin levels are related to various anthropometric indices and cardiovascular risk factors in adolescents is not as vigorously studied. In this study, we investigated this among healthy nondiabetic adolescents. Research Methods and Procedures: Two hundred thirty nondiabetic subjects (125 boys and 105 girls, ～10 to 19 years old) were included. The plasma adiponectin, fasting plasma glucose, insulin, lipids and anthropometric indices including body height, weight, waist circumference, and hip circumference were examined. Body fat mass (FM) and percentage were obtained from DXA scan. The homeostasis model assessment was applied to estimate the degree of insulin resistance. Results: The plasma adiponectin levels were significantly higher in girls (30.79 ± 14.48 μg/mL) than boys (22.87 ± 11.41 μg/mL). The plasma adiponectin levels were negatively related to BMI, FM, FM percentage, waist circumference, waist‐to‐hip ratio, insulin resistance, plasma insulin, triglycerides, and uric acid levels, but positively with high‐density lipoprotein cholesterol (HDL‐C) with the adjustment for age and gender. Using different multivariate linear regression models, only age and HDL‐C were consistently related to the plasma adiponectin levels after adjustment for the other variables. Discussion: The relationship between plasma adiponectin and various anthropometric indices and metabolic factors, especially HDL‐C, previously reported in adults was present in the healthy nondiabetic adolescents. Whether variation of plasma adiponectin levels in healthy nondiabetic adolescents may influence their future coronary artery disease risk warrants further investigation.     

Effect of exogenous spermidine on polyamine metabolism in water hyacinth leaves under mercury stress

The influence of exogenous spermidine (Spd) on arginine decarboxylase (ADC), ornithine decarboxylase (ODC), polyamine oxidase (PAO) activities and polyamines (PAs), proline contents in water hyacinth leaves under Mercury (Hg) stress was investigated after 6 days treatment. The results showed that free putrescine (Put) content increased, the contents of free spermidine (Spd) and spermine (Spm) and the (Spd + Spm)/Put ratio in water hyacinth leaves decreased significantly with the increase of the Hg concentrations. Hg stress also disturbed the activities of ADC, ODC and PAO and caused changes on proline content. Compared to the Hg-treatment only, exogenous Spd (0.1 mM) significantly reduced the accumulation of free Put, increased the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in water hyacinth leaves. Furthermore, exogenous Spd enhanced the activities of ADC, ODC and PAO and significantly increased proline content. The PS-conjugated PAs and PIS-bound PAs changed in the same trend as free PAs. These results suggest that exogenous Spd can alleviate the metabolic disturbance of polyamines caused by Hg in water hyacinth leaves.     

Exchange of pigment-binding amino acids in light-harvesting chlorophyll a/b protein

Four amino acids in the major light-harvesting chlorophyll (Chl) a/b complex (LHCII) that are thought to coordinate Chl molecules have been exchanged with amino acids that presumably cannot bind Chl. Amino acids H68, Q131, Q197, and H212 are positioned in helixes B, C, A, and D, respectively, and, according to the LHCII crystal structure [Kühlbrandt, W., et al. (1994) Nature 367, 614-621], coordinate the Chl molecules named a(5), b(6), a(3), and b(3). Moreover, a double mutant was analyzed carrying exchanges at positions E65 and H68, presumably affecting Chls a(4) and a(5). All mutant proteins could be reconstituted in vitro with pigments, although the thermal stability of the resulting mutant versions of recombinant LHCII varied significantly. All complexes reconstituted with the mutant proteins contained fewer chlorophyll molecules per two lutein molecules than complexes reconstituted with the wild-type protein. However, the chlorophyll-binding amino acids could not be unambiguously assigned to binding either chlorophyll a or b, as in most cases more than one chlorophyll molecule was lost due to the mutation. The changes in Chl stoichiometries suggest that in LHCII some chlorophyll positions can be filled with either Chl a or b. Only some of the point mutations in LHCII affected the ability of the apoprotein to assemble into trimeric LHCII upon insertion into isolated thylakoid membranes. Among these were exchanges of H68 with either F or L, suggesting that the stability of the LHCII trimer significantly depends on this amino acid or the Chl molecule named a(5) that is attached to it and is located close to the center of the trimeric complex. The ion pair bridge between E65 and R185 in LHCII does not appear to be essential for the proper folding of the protein.     

Analysis of flanking sequences from dissociation insertion lines: a database for reverse genetics in Arabidopsis

We have generated Dissociation (Ds) element insertions throughout the Arabidopsis genome as a means of random mutagenesis. Here, we present the molecular analysis of genomic sequences that flank the Ds insertions of 931 independent transposant lines. Flanking sequences from 511 lines proved to be identical or homologous to DNA or protein sequences in public databases, and disruptions within known or putative genes were indicated for 354 lines. Because a significant portion (45%) of the insertions occurred within sequences defined by GenBank BAC and P1 clones, we were able to assess the distribution of Ds insertions throughout the genome. We discovered a significant preference for Ds transposition to the regions adjacent to nucleolus organizer regions on chromosomes 2 and 4. Otherwise, the mapped insertions appeared to be evenly dispersed throughout the genome. For any given gene, insertions preferentially occurred at the 5' end, although disruption was clearly possible at any intragenic position. The insertion sites of >500 lines that could be characterized by reference to public databases are presented in a tabular format at http://www.plantcell. org/cgi/content/full/11/12/2263/DC1. This database should be of value to researchers using reverse genetics approaches to determine gene function.     

Inhibition modifies the effects of slow calcium-activated potassium channels on epileptiform activity in a neuronal network model

Generation of epileptiform activity typically results from a change in the balance between network excitation and inhibition. Experimental evidence indicates that alterations of either synaptic activity or intrinsic membrane properties can produce increased network excitation. The slow Ca²⁺-activated K⁺ currents (sI_AHP) are important modulators of neuronal firing rate and excitability and have important established and potential roles in epileptogenesis. While the effects of changes in sI_AHP on individual neuronal excitability are readily studied and well established, the effects of such changes on network behavior are less well known. The experiments here utilize a defined small network model of multicompartment pyramidal cells and an inhibitory interneuron to study the effects of changes in sI_AHP on network behavior. The benefits of this model system include the ability to observe activity in all cells in a network and the effects of interactions of multiple simultaneous influences. In the model with no inhibitory interneuron, increasing sI_AHP results in progressively decreasing burst activity. Adding an inhibitory interneuron changes the observed effects; at modest inhibitory strengths, increasing sI_AHP in all network neurons actually results in increased network bursting (except at very high values). The duration of the burst activity is influenced by the length of delay in a feedback loop, with longer loops resulting in more prolonged bursting. These observations illustrate that the study of potential antiepileptogenic membrane effects must be extended to realistic networks. Network inhibition can dramatically alter the observations seen in pure excitatory networks.