Aureonitols A and B,Two New C13‐Polyketides from Chaetomium globosum,an Endophytic Fungus in Salvia miltiorrhiza

Two new C₁₃‐polyketides, aureonitols A and B ( 1 and 2 ), along with five known compounds ( 3 – 7 ), were isolated from the solid fermentation culture of the plant endophytic fungus Chaetomium globosum from the aerial parts of Salvia miltiorrhiza. The structures and absolute configurations of 1 and 2 were determined by comprehensive spectroscopic data analysis and computed methods. Compound 5 was found to display the remarkable antimicrobial activities against four multidrug‐resistant bacteria (Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Staphylococcus epidermidis) with MIC values of 3.13–6.25 μg/mL (ciprofloxacin: 0.78–1.56 μg/mL), and also against all tested fungal strains with MIC values of 3.13–25 μg/mL (ketoconazole: 0.78–12.50 μg/mL).     

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.     

Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Phylogeography and population structure of the Yunnan snub-nosed monkey (Rhinopithecus bieti) inferred from mitochondrial control region DNA sequence analysis

Rhinopithecus bieti, the Yunnan snub-nosed monkey, is the nonhuman primate with the highest altitudinal distribution and is also one of the 25 most globally endangered primate species. Currently, R. bieti is found in forests between 3000 and 4500 m above sea level, within a narrow area on the Tibetan Plateau between the Yangtze and Mekong rivers, where it is suffering from loss of habitat and shrinking population size (approximately 1500). To assess the genetic diversity within this species, its population structure and to infer its evolutionary history, we sequenced 401 bp of the hypervariable I (HVI) segment from the mitochondrial DNA control region (CR) for 157 individuals from 11 remnant patches throughout the fragmented distribution area. Fifty-two variable sites were observed and 30 haplotypes were defined. Compared with other primate species, R. bieti cannot be regarded as a taxon with low genetic diversity. Phylogenetic analysis partitioned haplotypes into two divergent haplogroups (A and B). Haplotypes from the two mitochondrial clades were found to be mixed in some patches although the distribution of haplotypes displayed local homogeneity, implying a strong population structure within R. bieti. Analysis of molecular variance detected significant differences among the different geographical regions, suggesting that R. bieti should be separated into three management units (MUs) for conservation. Based on our results, it can be hypothesized that the genetic history of R. bieti includes an initial, presumably allopatric divergence between clades A and B 1.0-0.7 million years ago (Ma), which might have been caused by the Late Cenozoic uplift of the Tibetan Plateau, secondary contact after this divergence as a result of a population expansion 0.16-0.05 Ma, and population reduction and habitat fragmentation in the very recent past.     

Changes in phytochelatins and their biosynthetic intermediates in red spruce (<Emphasis Type="Italic">Picea rubens </Emphasis>Sarg.) cell suspension cultures under cadmium and zinc stress

Cell suspension cultures of red spruce (Picea rubens Sarg.) were selected to study the effects of cadmium (Cd) and zinc (Zn) on phytochelatins (PCs) and related metabolites after 24 h exposure. The PC₂ and its precursor, γ-glutamylcysteine (γ-EC) increased two to fourfold with Cd concentrations ranging from 12.5 to 200 μM as compared to the control. However, Zn-treated cells showed a less than twofold increase in γ-EC and PC₂ levels as compared to the control even at the highest concentration of 800 μM. In addition, unidentified higher chain PCs were also found in both the Cd and Zn treated cells and they increased significantly with increasing concentrations of Cd and Zn. The cellular ratio of PC₂ : Cd or Zn content clearly indicated that Cd (with ratios ranging from 0.131 to 0.546) is a more effective inducer of PC₂ synthesis/accumulation than Zn (with ratios ranging from 0.032 to 0.102) in red spruce cells. A marginal decrease in glutathione (GSH) was observed in both Cd and Zn treated cells. However, the GSH precursor, cysteine, declined twofold with all Cd concentrations while the decrease with Zn was 1.5–2-fold only at the higher treatment concentrations of Zn as compared to control. In addition, changes in other free amino acids, polyamines, and inorganic ions were also studied. These results suggest that PCs and their biosynthetic intermediates play a significant role in red spruce cells protecting against Cd and Zn toxicity.     

Tamoxifen-inducible gene deletion reveals a distinct cell type associated with trabecular bone, and direct regulation of PTHrP expression and chondrocyte morphology by Ihh in growth region cartilage

Indian hedgehog (Ihh) controls multiple aspects of endochondral skeletal development by signaling to both chondrocytes and perichondrial cells. Previous efforts to delineate direct effects of Ihh on chondrocytes by Col2-Cre-mediated ablation of Smoothened (Smo, encoding a transmembrane protein indispensable for Ihh signaling) has been only partially successful, due to the inability to discriminate between chondrocytes and perichondrial cells. Here we report a transgenic line (Col2-Cre) expressing under the control of the Colalpha1(II) promoter an inert form of Cre that is activatable by exogenous tamoxifen (TM); TM administration at proper times during embryogenesis induced Cre activity in chondrocytes but not in the perichondrium. By using this mouse line, we deleted Smo within subsets of chondrocytes without affecting the perichondrium and found that Smo removal led to localized disruption of the expression of parathyroid hormone-related protein (PTHrP) and the morphology of chondrocytes. Unexpectedly, TM invariably induced Cre activity in a subset of cells associated with the trabecular bone surface of long bones. These cells, when genetically marked and cultured in vitro, were capable of producing bone nodules. Expression of the Col2-Cre transgene in these cells likely reflected the endogenous Colalpha1(II) promoter activity as similar cells were found to express the IIA isoform of Colalpha1(II) mRNA endogenously. In summary, the present study has not only provided evidence that Ihh signaling directly controls PTHrP expression and chondrocyte morphology in the growth region cartilage, but has also uncovered a distinct cell type associated with the trabecular bone that appears to possess osteogenic potential.     

IL-1beta stimulates rat cardiac fibroblast migration via MAP kinase pathways

The pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) are elevated following acute myocardial infarction (MI) and have been implicated in the pathophysiology of cardiac disease progression. The cardiac fibroblast represents an important effector cell target for cytokine actions. In particular, cytokine-directed cardiac fibroblast migration is likely to impact both myocardial repair following acute MI and pathological myocardial remodeling in the progression to heart failure. In the present study, we examined the migratory response of neonatal rat cardiac fibroblasts to pro-inflammatory cytokines using modified Boyden chamber assays. On the basis of the knowledge of migration in other cell types, we hypothesized that members of the mitogen-activated protein kinase (MAPK) family may regulate this process. This possibility was addressed with the use of immunoblot detection of active phosphorylated MAPK species and pharmacological inhibitors for individual members of the MAPK cascades. IL-1beta stimulated robust and concentration-dependent increases in migration (maximum, 20-fold over control cells). TNF-alpha had lesser effect (fourfold increase over control). IL-6 did not induce migration. Activation of all three MAPK subfamilies (extracellular signal-regulated kinases, c-Jun NH(2)-terminal kinases, and p38) was shown to occur in response to cytokine stimulation. Fibroblast migration was attenuated by pharmacological inhibition of each MAPK subfamily. Understanding the regulation of cardiac fibroblast migration may provide insights in the search for therapies aimed at enhancing the functional nature of the remodeling process.     

花粉高尔基囊泡与牛脑微管的体外结合

把从榛木（Ｃｏｒｙｌｕｓａｖｅｌｌａｎａ Ｌ．）花粉中分离得到的高尔基囊泡与经高度纯化并聚合好的牛脑微管进行体外组合,然后于１．５ ｍ ｏｌ／Ｌ的蔗糖层上进行超离心,对其沉淀物进行ＳＤＳ－聚丙烯酰胺凝胶电泳和电镜负染。结果表明,花粉高尔基囊泡可以结合到牛脑微管上,证明植物花粉的高尔基囊泡与动物细胞的某些细胞器一样,也与细胞骨架的主要组成之一——微管具有结构上的紧密联系。花粉高尔基囊泡与牛脑微管的体外结合能力,受１０ ｍ ｍ ｏｌ／ＬＡＴＰ和０．５ ｍ ｏｌ／ＬＫＣｌ的影响,但不受５ ｍ ｍ ｏｌ／Ｌ ＡＭＰ－ＰＮＰ的影响,说明两者结合可能是通过高尔基囊泡表面与ＡＴＰ有关的某种外周膜蛋白来完成的。     

脂氧合酶、茉莉酸和水杨酸对猕猴桃果实后熟软化进程中乙烯生物合成的调控

20℃下后熟果实的LOX活性增加先于自由基产生和乙烯生成;JA处理对果实后熟软化启动期(采后1d)和果实快速软化期(采后5d)果实切片中的LOX活性、自由基产生和乙烯生物合成有促进作用,至果实软化后期(采后7d)这种效应消失;亚油酸只在采后1d果实切片中促使LOX活性和乙烯生成;SA处理抑制了果实后熟进程中组织切片的LOX活性、自由基产生和乙烯的生物合成;SA和JA对LOX活性和乙烯生物合成具有明显的拮抗作用。