Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.     

Effect of twenty four wheat genotypes on soil biochemical and microbial properties

Plant and Soil - Understanding the effect of wheat genotype on soil properties will be crucial in breeding towards more sustainable wheat production and increased yield. We examined the...     

中华绒螯蟹蜕壳生长及其与相关基因表达的关联分析

蜕壳是甲壳动物常见的生长发育现象,但对调控蜕壳与生长的内在机制尚缺乏足够了解。本研究在室内条件下,对中华绒螯蟹(Eriocheir sinensis)一个蜕壳周期内的个体蜕壳与生长现象进行了连续观察,分析了2个蜕壳相关基因,即蜕皮激素受体基因(Ec R)和维甲类X受体基因(RXR),以及1个生长相关基因肌肉生长抑制素基因(MSTN)的表达及其与生长性状的相关性。结果发现,中华绒螯蟹在蜕壳后会出现一个跳跃式生长期,之后进入了一个缓慢持续上升过程,当营养物质积累到一定程度(肥满度达60%左右时)时启动下一次蜕壳;MSTN基因的表达与壳长(r=﹣0.450,P0.05)、壳宽(r=﹣0.410,P0.05)增长率呈显著负相关,而与肥满度呈显著正相关(r=0.450,P0.05),Ec R和RXR基因的表达与体重、壳长和壳宽的增长率均没有显著相关性;相对来说,MSTN在蜕壳后的表达量越高,则增重率越小;而Ec R和RXR在蜕壳后表达量越高,其增重率越大。本研究结果表明,中华绒螯蟹在蜕壳后其生长具有一定的规律性,肥满度可以作为衡量中华绒螯蟹体内营养积累启动蜕壳的指标,Ec R、RXR及MSTN基因表达与生长表型具有一定的相关性。     

PD-L1基因敲除小鼠构建及初步表型验证

目的 程序性死亡配体-1(PD-L1)是免疫调节途径的重要因子,是抗肿瘤免疫疗法中重要的靶标之一。利用CRISPR/Cas9技术成功构建PD-L1基因敲除小鼠模型,并初步分析其表型。方法 构建Cas9和sgRNA载体,并转录获得RNA,通过显微注射方式将RNA注射到C57BL/6小鼠受精卵中,经过鉴定获得F₀代阳性小鼠。F₀代小鼠与野生型C57BL/6小鼠交配获得F₁代杂合子小鼠,再通过F₁代小鼠自交获得F₂代纯合子小鼠品系。随后通过Real-Time PCR和流式实验分别检测PD-L1基因在mRNA和蛋白质水平上的表达情况。结果 Real-Time PCR和流式实验检测结果显示与野生型C57小鼠相比,PD-L1纯合子小鼠的PD-L1 mRNA相对表达水平和细胞上的蛋白质表达均有显著性下降,仅测定到本底的信号,证实已成功构建PD-L1基因敲除小鼠品系,为PD-L1体内基因功能研究提供了新的小鼠模型。     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Exclusion effects on vegetation characteristics and their correlation to soil factors in the semi-arid rangeland of Mu Us Sandland,China

Exclusion has been applied as a main measure for re-vegetation all over the world.This paper,by comparing the results of year-round exclusion,seasonal exclusion,and non-exclusion,quantified the vegetation variations under three different exclusion measures and their correlation to soil factors.The analysis results for community species component and plant diversity using multi-response permutation procedures (MRPPs)showed that exclusion did change the species component and increase plant diversity remarkably,while the period of exclusion had no significant influence on these two community features.The indicator species analysis and calculation of similarity indices indicated that community for year-round exclusion were becoming xerophytization and unpalatability,and showed highly spatial heterogeneity of plant species distribution,whereas community for seasonal exclusion was under stable non-equilibrium condition.Detrended correspondence analysis (DCA)and detrend canonical correspondence analysis (DCCA)results of relationship between plant species and soil variables demonstrated that soil moisture was a controlling factor for plant species component,microbiotic soil crust cover,soil organic matter,and soil bulk density had significant effects on soil moisture,among which microbiotic soil crust was a leading factor owing to its limitation to rainfall infiltration on the one hand,and its constraints to entrance of herbaceous seeds into soil or to germination of soil seeds on the other hand.As a result of long-term removal of animal grazing,crust kept intact in year-round exclusion community,which was a main reason of community xerophytization.It was also obvious from ordination results that some important environmental factors,such as tempo-spatial change of rainfall and corresponding tempo-spatial change of soil moisture,were neglected during direct gradient analysis.In addition,biodiversity was close related to soil nutrients as well as to soil moisture condition (soil water content and crust cover),and it had positive relation to available N,and negative relation to available P.Higher soil N had advantage to non-leguminous plants growth on nutrition-poor sand land definitely.The impact of P to community component was unclear and should be studied from plant physiology.Further researches on nonequilibrium theory in semi-arid rangeland will provide a scientific and flexible animal development paradigm for being implementing livestock fen-raising and grazing-forbidden policies in China.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.     

Emergence of multifunctional oxygenase activities by random priming recombination

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.     

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.