Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2

beta-Defensins are small antimicrobial peptides of the innate immune system produced in response to microbial infection of mucosal tissue and skin. We demonstrate that murine beta-defensin 2 (mDF2beta) acts directly on immature dendritic cells as an endogenous ligand for Toll-like receptor 4 (TLR-4), inducing up-regulation of costimulatory molecules and dendritic cell maturation. These events, in turn, trigger robust, type 1 polarized adaptive immune responses in vivo, suggesting that mDF2beta may play an important role in immunosurveillance against pathogens and, possibly, self antigens or tumor antigens.     

MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha

Phosphorylation of mitogen-activated protein kinases (MAPKs) on specific tyrosine and threonine sites by MAP kinase kinases (MAPKKs) is thought to be the sole activation mechanism. Here, we report an unexpected activation mechanism for p38alpha MAPK that does not involve the prototypic kinase cascade. Rather it depends on interaction of p38alpha with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38alpha. We detected formation of a TRAF6-TAB1-p38alpha complex and showed stimulus-specific TAB1-dependent and TAB1-independent p38alpha activation. These findings suggest that alternative activation pathways contribute to the biological responses of p38alpha to various stimuli.     

A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase

Arachidonate and other unsaturated long-chain fatty acids were found to activate protein kinase C from human neutrophils. Kinase activation by arachidonate required calcium and was enhanced by diolein but did not require exogenous phosphatidylserine. Submaximal levels of arachidonate also enhanced the affinity of the kinase for calcium during activation by phosphatidylserine. Thus the release of arachidonate, which is triggered in many cell types by ligand-receptor interactions, could play a second messenger role in the regulation of cellular function by activation of protein kinase C.     

Interaction of brain synaptic vesicles induced by endogenous Ca2+ -dependent phospholipase A2

Endogenous phospholipase A2 activity of brain synaptic vesicles was Ca2+ -dependent and was increased by prostaglandin F2 alpha, calmodulin, adenosine 3', 5' -monophosphate, and adenosine triphosphate, whereas the activity was inhibited by prostaglandin E2 in the absence or presence of calmodulin. Light-scattering measurements demonstrated that stimulation of the enzyme's activity correlated with the induction of vesicle-vesicle aggregation. The effects of these compounds on endogenous synaptic vesicle phospholipase A2 activity may imply a common end point of their purported neuromodulatory actions, and indicate that synaptic vesicle phospholipase A2 may play a central role in presynaptic neurotransmission.     

谷氨酰胺合成酶催化合成茶氨酸条件的优化

在不同诱导条件下,对谷氨酰胺合成酶催化合成茶氨酸体系进行优化,确定最适诱导表达条件,为微生物发酵合成茶氨酸提供技术依据.研究结果表明:基因工程菌最佳培养温度为30 ℃,最佳诱导剂异丙基硫代-β-D半乳糖苷浓度为0.1 mmol·L-1,最佳诱导温度为28 ℃.合成茶氨酸的最适pH值为9.5,适合的缓冲液为100 mmol·L-1咪唑;反应体系中咪唑缓冲液优于磷酸钾缓冲液;低浓度L-谷氨酸钠、高浓度盐酸乙胺和高浓度三磷酸腺苷对茶氨酸的合成均有一定的促进作用,可以提高茶氨酸的生成量.     

不同来源及脱乙酰度壳聚糖对铜离子吸附特性的研究

从虾、蟹壳中提取甲壳素并分别制备不同脱乙酰度壳聚糖,比较不同来源、不同脱乙酰度壳聚糖对铜离子的吸附特性。结果表明：蟹源壳聚糖对铜的吸附能力明显高于虾源壳聚糖,高脱乙酰度壳聚糖吸附铜离子的能力明显低于低脱乙酰度壳聚糖,除高脱乙酰度虾源壳聚糖外,其余3种壳聚糖等温吸附规律均符合Langmuir模型,4种壳聚糖等温吸附规律均不符合Freundlich模型。     

Restoration by calmodulin of a Ca2+-dependent K+ current missing in a mutant of Paramecium

A combination of genetics, biochemistry, and biophysics was used to show that calmodulin is involved in the regulation of an ion channel. Calmodulin restored the Ca2+-dependent K+ current in pantophobiac, a mutant in Paramecium that lacks this current. The restoration of the current occurred within 2 hours after the injection of 1 picogram of wild-type calmodulin into the mutant. The current remained for approximately 30 hours before the mutant phenotype returned. The injection of calmodulin isolated from pantophobiac had no effect. These results imply that calmodulin is required for the function or regulation of the Ca2+-dependent K+ current in Paramecium.     

Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase

A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) has been found to be required for virulence and survival within macrophages. Here, SPI2 was shown to allow Salmonella typhimurium to avoid NADPH oxidase-dependent killing by macrophages. The ability of SPI2-mutant bacteria to survive in macrophages and to cause lethal infection in mice was restored by abrogation of the NADPH oxidase-dependent respiratory burst. Ultrastructural and immunofluorescence microscopy demonstrated efficient localization of the NADPH oxidase in the proximity of vacuoles containing SPI2-mutant but not wild-type bacteria, suggesting that SPI2 interferes with trafficking of oxidase-containing vesicles to the phagosome.     

Rad6-dependent ubiquitination of histone H2B in yeast

Although ubiquitinated histones are present in substantial levels in vertebrate cells, the roles they play in specific biological processes and the cellular factors that regulate this modification are not well characterized. Ubiquitinated H2B (uH2B) has been identified in the yeast Saccharomyces cerevisiae, and mutation of the conserved ubiquitination site is shown to confer defects in mitotic cell growth and meiosis. uH2B was not detected in rad6 mutants, which are defective for the ubiquitin-conjugating enzyme Ubc2, thus identifying Rad6 as the major cellular activity that ubiquitinates H2B in yeast.     

Akt-mediated phosphorylation of EZH2 suppresses methylation of lysine 27 in histone H3

Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase that plays an important role in many biological processes through its ability to trimethylate lysine 27 in histone H3. Here, we show that Akt phosphorylates EZH2 at serine 21 and suppresses its methyltransferase activity by impeding EZH2 binding to histone H3, which results in a decrease of lysine 27 trimethylation and derepression of silenced genes. Our results imply that Akt regulates the methylation activity, through phosphorylation of EZH2, which may contribute to oncogenesis.     

Structural basis of Smad2 recognition by the Smad anchor for receptor activation

The Smad proteins mediate transforming growth factor-beta (TGFbeta) signaling from the transmembrane serine-threonine receptor kinases to the nucleus. The Smad anchor for receptor activation (SARA) recruits Smad2 to the TGFbeta receptors for phosphorylation. The crystal structure of a Smad2 MH2 domain in complex with the Smad-binding domain (SBD) of SARA has been determined at 2.2 angstrom resolution. SARA SBD, in an extended conformation comprising a rigid coil, an alpha helix, and a beta strand, interacts with the beta sheet and the three-helix bundle of Smad2. Recognition between the SARA rigid coil and the Smad2 beta sheet is essential for specificity, whereas interactions between the SARA beta strand and the Smad2 three-helix bundle contribute significantly to binding affinity. Comparison of the structures between Smad2 and a comediator Smad suggests a model for how receptor-regulated Smads are recognized by the type I receptors.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation

Suspensions of thymocytes from young rats were incubated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which resulted in a sustained increase in cytosolic free Ca2+ concentration followed by DNA fragmentation and loss of cell viability. Both the Ca2+ increase and DNA fragmentation were prevented in cells treated with the inhibitor of protein synthesis, cycloheximide, and DNA fragmentation and cell killing were not detected when cells were incubated in a "Ca2+-free" medium or pretreated with high concentrations of the calcium probe, quin-2 tetraacetoxymethyl ester. These results indicate that TCDD can kill immature thymocytes by initiating a suicide process similar to that previously described for glucocorticoid hormones.     

瘤胃微生物体外利用赖氨酸对有关酶和尿素氮的影响

为了测定体外培养条件下瘤胃微生物的赖氨酸消化率及赖氨酸降解过程中谷氨酸脱氢酶(GDH)、γ-谷氨酰转肽酶(γ-GT)、谷草转氨酶(GOT)、谷丙转氨酶(GPT)和尿素氮(UN)的变化及其相关关系,经瘤胃瘘管取成年山羊瘤胃液混匀后分装至12个血清瓶中,每瓶40 mL,同时每瓶加入淀粉20 mg;血清瓶随机均分为2组,其中一组每瓶再注入8 mL0.25 mmol/L的L-赖氨酸作为赖氨酸组,另一组每瓶再注入等体积的去离子水作为对照,一并放入39℃培养箱培养16 h,并于培养的0,8和16 h取培养液测定GDH、γ-GT、GOT、GPT、UN和游离氨基酸。结果表明,底物中添加赖氨酸时,培养液中UN浓度可保持稳定,否则培养16 h后的UN浓度极显著升高;GDH活性在赖氨酸的降解代谢过程中随培养时间的延长而增加;培养时间的长短显著影响GDH、γ-GT活性及UN的含量(P≤0.05)。在不添加赖氨酸的条件下,培养16 h的γ-GT与16 h的GPT和UN均呈极显著正相关(R=0.95;R=0.92)。当底物中添加赖氨酸时,培养0 h的GDH与培养8 h的γ-GT显著相关(R=0.88);而培养8 h的γ-GT又与8 h的UN显著相关(R=0.86);培养0,8和16 h的赖氨酸浓度与培养0 h的GDH呈负相关,与培养8 h的GDH呈极显著负相关(R=-0.94)。对照组培养8和16 h的赖氨酸消化率分别为31.64%和63.59%,赖氨酸组培养8和16h的赖氨酸消化率则分别为49.24%和74.55%,均极显著高于对照组培养8 h的消化率。提示在氮源缺乏的条件下,瘤胃微生物可能通过γ-GT、GPT和GOT的共同作用增加尿素氮的积累以维持生长,瘤胃微生物的赖氨酸降解本质上属于酶解。     

Prolonged Ca2+-dependent afterhyperpolarizations in hippocampal neurons of aged rats

The possibility that calcium is elevated in brain neurons during aging was examined by quantifying afterhyperpolarizations induced by spike bursts in CAl neurons of hippocampal slices from young and aged rats. The afterhyperpolarizations result from Ca2+-dependent K+ conductance increases and are blocked in medium low in Ca2+ and prolonged in medium high in Ca2+. The afterhyperpolarization and associated conductance increases were considerably prolonged in cells from aged rats, although inhibitory postsynaptic potentials did not differ with age. Since elevated intracellular Ca2+ can exert deleterious effects on neurons, the data suggest that altered Ca2+ homeostasis may play a significant role in normal brain aging.