球孢白僵菌高渗适应性相关基因Bbmpd的克隆与表达分析

【目的】克隆与球孢白僵菌(Beauveria bassiana)的高渗适应性相关基因,并对其功能进行分析,以揭示球孢白僵菌对高渗等逆境适应的分子机理。【方法】利用YADE法克隆T-DNA的侧翼序列并进行基因组步行,获得突变基因的全长及上游序列;利用RT-PCR技术分析突变基因的表达特性以及与Bbhog1的关系;采用同源重组技术敲除Bbmpd基因。【结果】克隆得到插入突变基因及其上、下游序列全长3037bp。该基因与编码球孢白僵菌的1-磷酸甘露醇脱氢酶基因相似性为98%。Bbmpd的表达受高渗环境(0.8mol/L NaCl)的诱导,受Bbhog1信号途径的激活调节,Bbhog1缺失导致Bbmpd表达下调。Bbmpd缺失突变体在高渗胁迫下的生长受到明显抑制。Bbmpd缺失不影响球孢白僵菌在查氏培养基上的生长和产孢。【结论】由T-DNA突变体克隆了编码球孢白僵菌1-磷酸甘露醇脱氢酶基因Bbmpd,该基因的表达受高渗环境的诱导和Bbhog1的调控,与球孢白僵菌高渗适应性相关。     

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G₁ phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB.     

Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr(31), Thr(51), Thr(129) and Thr(143). Substitution of Thr(31) with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr(51), Thr(129) or Thr(143) with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr(31) is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr(31). Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.     

Arginine bi-directional translocation and breakdown into ornithine along the arbuscular mycorrhizal mycelium

Bi-directional translocation and degradation of Arginine (Arg) along the arbuscular mycorrhizal (AM) fungal mycelium were testified through ¹⁵N and/or ¹³C isotopic labeling. In vitro mycorrhizas of Glomus intraradices and Ri T-DNA-transformed carrot roots were grown in dual compartment Petri dishes. [¹⁵N- and/or¹³C]Arg was supplied to either the fungal compartment or the mycorrhizal compartment or separate dishes containing the uncolonized roots. The levels and labeling of free amino acids (AAs) in the mycorrhizal roots and in the extraradical mycelia(ERM) were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The ERM of AM fungi exposed in either NH₄ ⁺ or urea as sole external nitrogen source had much higher ¹⁵N enrichment of Arg, compared with those in nitrate or exogenous Arg; however, glycerol supplied as an external carbon source to the ERM had no significant effect on the level of Arg in the ERM. Meanwhile, Arg biosynthesized in the ERM could be translocated intact to the mycorrhizal roots and thereby the level of Arg in the mycorrhizal roots increased to about 20% after culture of ERM in 4 mmol/L NH₄ ⁺ for 6 weeks. Also Arg was found to be bi-directionally transported along the AM fungal mycelium through [U-¹³C]Arg labeling either in the mycorrhizal compartment or in the fungal compartment. Once Arg was translocated to the potential N-limited sites, it would be further degraded into ornithine (Orn) and urea since either [U-¹³C] or [U-¹⁵N/U-¹³C]Orn was apparently shown up in the mycorrhizal root tissues when [U-¹³C] or [U-¹⁵N/U-¹³C]Arg was labeled in the fungal compartment, respectively. Evidently Orn formation indicated the ongoing activities of Arg translocation and degradation through the urea cycle in AM fungal mycelium. Supported by Science and Technology Department of Zhejiang Province (Grant No. 2006C22009).     

Animal movements in fire‐prone landscapes

Movement is a trait of fundamental importance in ecosystems subject to frequent disturbances, such as fire‐prone ecosystems. Despite this, the role of movement in facilitating responses to fire has received little attention. Herein, we consider how animal movement interacts with fire history to shape species distributions. We consider how fire affects movement between habitat patches of differing fire histories that occur across a range of spatial and temporal scales, from daily foraging bouts to infrequent dispersal events, and annual migrations. We review animal movements in response to the immediate and abrupt impacts of fire, and the longer‐term successional changes that fires set in train. We discuss how the novel threats of altered fire regimes, landscape fragmentation, and invasive species result in suboptimal movements that drive populations downwards. We then outline the types of data needed to study animal movements in relation to fire and novel threats, to hasten the integration of movement ecology and fire ecology. We conclude by outlining a research agenda for the integration of movement ecology and fire ecology by identifying key research questions that emerge from our synthesis of animal movements in fire‐prone ecosystems.     

A ratiometric fluorescent detection of Zn(II) in aqueous solutions using pyrene-appended histidine

A fluorescent chemosensor, Py-His, based on histidine was easily synthesized in solid phase synthesis. Py-His displayed a highly sensitive ratiometric response to Zn(II) with potent binding affinity (K_a = 1.17 × 10¹³ M^?2) in aqueous solutions. The detection limit of Py-His for Zn(II) was calculated as 80.8 nM. Moreover, Py-His distinguished Zn(II) and Hg(II) by different ratiometric response type; the chemosensor showed a more enhanced increase of excimer emission intensity to Zn(II) than Hg(II). Upon addition of Ag(I) and Cu(II), Py-His showed a turn-off response mainly due to the quenching effect of these metal ions. The binding stoichiometry (2:1 or 1:1) of Py-His to target metal ions played a critical role in the fluorescent response type (ratiometric and turn off response) to target metal ions. The role of imidazole group of Py-His for ratiometric detection of Zn(II) was proposed by pH titration experiments.