COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.     

凤尾蕨内生真菌的研究Ⅱ——1株产芦丁内生真菌初步研究

报道了从凤尾蕨属井栏边草(Pteris multifida)根状茎中分离出一内生真菌——菌株JJF006,对其形态特征进行了初步研究,并用TLC对该菌株培养物进行了分析。初步结果表明:该真菌液体培养基中含有芦丁成分。     

柴达木沙漠链霉菌S10T阿糖腺苷的大孔树脂分离工艺优化与结构鉴定

为了获得具有药用价值的活性天然产物,采用4种大孔吸附树脂对柴达木沙漠链霉菌(Streptomyces qaidemensis)S10^T发酵液进行静态吸附和解吸实验,优化分离工艺。结果显示,AB-8型树脂具有良好的吸附和解吸性能,该树脂对柴达木沙漠链霉菌S10^T发酵液中的活性天然产物吸附工艺为发酵液pH值9,吸附时间4 h,洗脱液70%甲醇溶液。经正向硅胶、反相硅胶和葡聚糖凝胶Sephadex LH-20分离得到了一个化合物,¹H-NMR和¹³C-NMR结合高分辨质谱(LC-HR-MS)鉴定该化合物为阿糖腺苷(vidarabine),是一种具有抗病毒活性的核苷类抗生素,并简单探究了其在柴达木沙漠链霉菌中的生物合成过程。     

Eosinophil peroxidase catalyzes bromination of free nucleosides and double-stranded DNA

Chronic parasitic infections are a major risk factor for cancer development in many underdeveloped countries. Oxidative damage of DNA may provide a mechanism linking these processes. Eosinophil recruitment is a hallmark of parasitic infections and many forms of cancer, and eosinophil peroxidase (EPO), a secreted hemoprotein, plays a central role in oxidant production by these cells. However, mechanisms through which EPO may facilitate DNA oxidation have not been fully characterized. Here, we show that EPO effectively uses plasma levels of bromide as a cosubstrate to brominate bases in nucleotides and double-stranded DNA, forming several stable novel brominated adducts. Products were characterized by HPLC with on-line UV spectroscopy and electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). Ring assignments for brominated purine bases as their 8-bromo adducts were identified by NMR spectroscopy. Using stable isotope dilution LC/ESI/MS/MS, we show that while guanine is the preferred purine targeted for bromination as a free nucleobase, 8-bromoadenine is the major purine oxidation product generated following exposure of double-stranded DNA to either HOBr or the EPO/H(2)O(2)/Br(-) system. Bromination of nucleobases was inhibited by scavengers of hypohalous acids such as the thioether methionine, but not by a large molar excess of primary amines. Subsequently, N-monobromoamines were demonstrated to be effective brominating agents for both free nucleobases and adenine within intact DNA. A rationale for selective modification of adenine, but not guanine, in double-stranded DNA based upon stereochemical criteria is presented. Collectively, these results suggest that specific brominated DNA bases may serve as novel markers for monitoring oxidative damage of DNA and the nucleotide pool by brominating oxidants.     

Calcium-dependent stabilization of the central sequence between Met(76) and Ser(81) in vertebrate calmodulin

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met(76) and Ser(81) in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met(76) and Ser(81) in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met(76) and Asp(80) in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768-776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met(76) and Ser(81) and modulates the structural coupling between the opposing domains of CaM.     

Rejection of S-heteroallelic pollen by a dual-specific s-RNase in Solanum chacoense predicts a multimeric SI pollen component

S-heteroallelic pollen (HAP) grains are usually diploid and contain two different S-alleles. Curiously, HAP produced by tetraploids derived from self-incompatible diploids are typically self-compatible. The two different hypotheses previously advanced to explain the compatibility of HAP are the lack of pollen-S expression and the "competition effect" between two pollen-S gene products expressed in a single pollen grain. To distinguish between these two possibilities, we used a previously described dual-specific S(11/13)-RNase, termed HVapb-RNase, which can reject two phenotypically distinct pollen (P(11) and P(13)). Since the HVapb-RNase does not distinguish between the two pollen types (it recognizes both), P(11)P(13) HAP should be incompatible with the HVapb-RNase in spite of the competition effect. We show here that P(11)P(13) HAP is accepted by S(11)S(13) styles, but is rejected by the S(11/13)-RNase, which demonstrates that the pollen-S genes must be expressed in HAP. A model involving tetrameric pollen-S is proposed to explain both the compatibility of P(11)P(13) HAP on S(11)S(13)-containing styles and the incompatibility of P(11)P(13) HAP on styles containing the HVapb-RNase.     

Conformational adjustments of SNase R and its N-terminal fragments probed by monoclonal antibodies

Two monoclonal antibodies specific for staphylococcal nuclease R (SNase R) (McAb2C9 and McAb1B8) were prepared and used to probe protein folding during peptide elongation, by measuring antibody binding to seven N-terminal fragments (SNR141, SNR135, SNR121, SNR110, SNR102, SNR79 and SNR52) of SNase R. Comparative studies of the conformations of the N-terminal fragments have shown that all seven fragments of SNase R have a certain amount of residual structure, indicating that folding may occur during elongation of the nascent peptide chain. We show that the binding abilities of the intact enzyme and its seven fragments to the monoclonal antibodies are not simply proportional to the length of the peptide chain, suggesting that there may be continuous conformational adjustment in the nascent peptide chain as new C-terminal amino acids are added. A folding intermediate close in structure to the native state but with structural features in common with SNR121 is highly populated in 0.6 M GuHCl, and is also formed transiently during folding.     

Downregulation of K(+) channel genes expression in type I diabetic cardiomyopathy

Type I diabetic cardiomyopathy has consistently been shown to be associated with decrease of repolarising K(+) currents, but the mechanisms responsible for the decrease are not well defined. We investigated the streptozotocin (STZ) rat model of type I diabetes. We utilized RNase protection assay and Western blot analysis to investigate the message expression and protein density of key cardiac K(+) channel genes in the diabetic rat left ventricular (LV) myocytes. Our results show that message and protein density of Kv2.1, Kv4.2, and Kv4.3 are significantly decreased as early as 14 days following induction of type I diabetes in the rat. The results demonstrate, for the first time, that insulin-deficient type I diabetes is associated with early downregulation of the expression of key cardiac K(+) channel genes that could account for the depression of cardiac K(+) currents, I(to-f) and I(to-s). These represent the main electrophysiological abnormality in diabetic cardiomyopathy and is known to enhance the arrhythmogenecity of the diabetic heart. The findings also extend the extensive list of gene expression regulation by insulin.