A new cyclopeptide with antifungal
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A new cyclopeptide with antifungal activity from the co-culture broth of two marine mangrove fungi
Chunyuan Li a
, Jinhua Wang a
, Cuiping Luo a
, Weijia Ding a
& Daniel G. Cox
b
a
College of Science, Institute of Biomaterial, South China
Agricultural University , Guangzhou, 510642 Guangdong, P .R. China
b
Department of Pharmacognosy and Research Institute of
Pharmaceutical Sciences, School of Pharmacy , The University of Mississippi, 38677 MS, USA
Published online: 26 Feb 2014.
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D o w n l o a d e d b y [W u h a n U n i v e r s i t y ] a t 17:27 16 D e c e m b e r 2014
A new cyclopeptide with antifungal activity from the co-culture broth of two marine mangrove fungi
Chunyuan Li a *,Jinhua Wang a ,Cuiping Luo a ,Weijia Ding a and Daniel G.Cox b
a
College of Science,Institute of Biomaterial,South China Agricultural University,Guangzhou,510642Guangdong,P.R.China;b Department of Pharmacognosy and Research Institute of Pharmaceutical Sciences,School of Pharmacy,The University of Mississippi,38677MS,USA
(Received 26December 2013;?nal version received 21January 2014)
A new cyclic tetrapeptide,cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(1),was isolated from the co-culture broth of two mangrove fungi Phomopsis sp.K38and Alternaria sp.E33.The structure of 1was determined by analysis of spectroscopic data and Marfey’s analytic method.Primary bioassay demonstrated that compound 1exhibited moderate to high inhibitory activity against four crop-threatening fungi including Gaeumannomyces graminis ,Rhizoctonia cerealis ,Helminthosporium sativum and Fusarium graminearum as compared with triadimefon.
Keywords:mangrove fungi;cyclopeptide;antifungal activity;co-culture
1.Introduction
Natural products (NPs)obtained from micro-organisms are historical source of valuable medicinal lead compounds (Newman &Cragg 2012),but the dif?culties involved in working with complex mixtures and the rediscovery of the same bioactive compounds diminish the attractiveness of such NPs (Wolfender et al.2010).This suggests that it is necessary to develop more ef?cient approaches to activate the cryptic biosynthetic pathways of micro-organisms.According to the hypothesis that some special microbial secondary metabolites may only be produced due to the activation of complex regulatory mechanisms,the method of co-cultivating two micro-organisms in a single con?ned environment has been developed to generate novel NPs in recent years (Bertrand et al.2013).Moreover,these stress-induced molecules exhibited speci?c antimicrobial (Cueto et al.2001;Oh et al.2007;Li et al.2011;Zhu et al.2011;Wang et al.2013)and anticancer activities (Oh et al.2005;Li et al.2010;Zuck et al.2011).
During our continuous search for structurally new bioactive compounds from mangrove fungi,we investigated the metabolites of Phomopsis sp.K38and Alternaria sp.E33,which were both isolated from the coast of South China Sea (Li et al.2006,2007,2008).Then,we investigated the secondary metabolites produced by their co-cultivation,which yielded three new bioactive molecules (Li et al.2010,2011;Wang et al.2013).In this study,we describe the isolation,structural elucidation and antifungal activity of another new cyclopeptide,cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(1),from the co-culture broth of the same fungi.
q 2014Taylor &
Francis
*Corresponding author.Email:chunyuanli@https://www.wendangku.net/doc/ca15224272.html,
Natural Product Research ,2014
Vol.28,No.9,616–621,https://www.wendangku.net/doc/ca15224272.html,/10.1080/14786419.2014.887074
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2.Results and discussion
Cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(1)had [a ]D 240.08(c 0.01,MeOH)and revealed a pseudomolecular ion [M tH]tat m /z 453.2706corresponding to a molecular formula of C 22H 36N 4O 6(calcd for C 22H 36N 4O 6453.2704),indicating seven degrees of unsaturation.Detailed analyses of the 1H and 13C NMR spectral data for 1,with the aid of COSY,HMQC and HMBC spectra (Table S1),established the presence of two 4-OH-proline (4-OH-Pro)and two leucine (Leu)residues.The amino acid composition accounted for 6out of the 7degrees of unsaturation,indicating that 1is a cyclic tetrapeptide.The signals at lowest ?eld (d 170.7and 168.5)in the 13C NMR spectrum were assigned to the carbonyls of the 4-OH-Pro units,while the remaining signals (d 166.5and 166.3)to the two Leu units.The HMBC correlations (Table S1)from the H-2(d 4.40)of L -4-OH-Pro1to the carbonyl at d 170.7(4-OH-L -Pro1C-1),the Z NH group (d 7.99)and H-2(d 4.05)of Leu1to the carbonyls at d 166.3(Leu1C-1)and d 170.7(4-OH-L -Pro1C-1),the H-5(d 3.25)of L -4-OH-Pro1to the carbonyl at d 166.5(Leu2C-1),the H-2(d 4.23)of L -4-OH-Pro2to the carbonyl at d 168.5(4-OH-L -Pro2C-1),the H-5(d 3.48)of L -4-OH-Pro2to the carbonyl at d 166.3(Leu1C-1),the H-2(d 3.62)of Leu2to the carbonyls at d 166.5(Leu2C-1)and d 168.5(4-OH-L -Pro2C-1)de?ned the amino acid sequence.The planar structure of 1was the same as the known cyclo-(L -leucyl-cis -4-hydroxy-D -prolyl-L -leucyl-trans -4-hydroxy-L -proline)reported in the previous literature (Mitova et al.2003),but their speci?c rotations were quite different.The relative con?guration of 4-OH-L -Pro1was determined to be the trans -con?guration of H-4and H-2by NOE correlation between the 4-OH at d 5.10and H-2at d 4.40.Similarly,NOE correlation also con?rmed the trans -con?guration of H-4and H-2in 4-OH-L -Pro2.Thus,the stereochemistry of compound 1was different from the known cyclo-(L -leucyl-cis -4-hydroxy-D -prolyl-L -leucyl-trans -4-hydroxy-L -proline)which contained both trans -4-hydroxy-L -proline and cis -4-hydroxy-L -proline.
The absolute con?gurations of the amino acid residues were established by acid hydrolysis of the peptide,derivation with 1-?uoro-2,4-dinitrophenyl-5-L -alaninamide (FDAA,Marfey’s reagent)and HPLC analysis of the derivatives (Marfey 1984;Fujii et al.1998).The retention times of FDAA derivatives of the amino acid residues of compound 1were observed as 40.08,66.54and https://www.wendangku.net/doc/ca15224272.html,pared with the retention times of FDAA derivatives of standard 4-OH-D -proline,4-OH-L -proline,D -leucine and L -leucine which were observed as 44.21,40.08,66.55,and 71.63,respectively,showed the two 4-OH-proline residues of compound 1both have the L -con?guration,while the two leucine residues of compound 1were L -con?gured and D -con?gured,respectively.Hence,the structure of compound 1was established as cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(Figure 1).
Compound 1was evaluated for in vitro antifungal activity against four crop-threatening fungi (Gaeumannomyces graminis ,Rhizoctonia cerealis ,Helminthosporium sativum and Fusarium graminearum ).The results are summarised in Table 1.It indicated moderate to high antifungal activities as compared with the positive control.Especially,the fungus H.sativum was more sensitive to compound 1than the other tested micro-organisms.The minimum inhibitory concentration (MIC)values of compound 1against H.sativum were close to that of the positive control,triadimefon.Thus,the result suggests that compound 1is worthy of consideration for the research and development as an antifungal agent used in agriculture.3.Experimental
3.1.General experimental procedures
Optical rotation was measured on a PL341spectropolarimeter (PerkinElmer Inc.,Waltham,MA,USA).IR spectra were measured using a FT-IR NICOLET spectrophotometer (Thermo Electron Corporation,Madison,WI,USA).Nuclear magnetic resonance (NMR)data were recorded on a
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Bruker AVANCE 400spectrometer (Bruker BioSpin GmbH Company,Rheinstetten,Germany),using deuterated dimethyl sulfoxide (DMSO-d 6)as solvent and residual DMSO-d 6resonance as internal standard,and coupling constants (J )are in Hz.Electrospray ionisation mass (ESI-MS)and high-resolution electrospray ionisation mass (HR-ESI-MS)were operated on LCQ-DECA-XP (Thermo,Madison,WI,USA)and LCMS-IT-TOF (Shimadzu,Kyoto,Japan)mass spectrometers,respectively.The compounds were puri?ed on an Agilent 1100HPLC system equipped with a quaternary pump,a diode array detector and a reverse-phase silica gel column (Phenomenex Synergi Hydro-RP,250mm ￡100mm,5m m)at a ?ow rate of 1.0mL/min.Pre-fractionation of the extract was carried out using silica gel column (200–300mesh;Qingdao Haiyang Chemicals Co.,Ltd,Qingdao,China)and Supelclean TM C-18solid-phase extraction (SPE)tubes.TLC was performed on pre-coated silica gel 60F254plates (Merck).Microplate reader was from TECAN Inc.Ninety-six well ?at-bottom microtitre plates were from Falcon (NJ,USA).All the solvents were of HPLC grade and used without further puri?cation.3.2.Fungal strains and fermentation
The Phomopsis sp.K38and Alternaria sp.E33strains were isolated from the mangrove in Leizhou Peninsula,Guangdong Province,China.Stock cultures were maintained in slants of solid cornmeal seawater agar.The small agar slices bearing mycelia were cut and transferred aseptically to a 250mL Erlenmeyer ?ask containing 100mL of liquid medium (glucose 10g/L,peptone 2g/L,yeast extract 1g/L,NaCl 30g/L).The ?ask was incubated at 308C on a rotary
Table 1.Antifungal activity of compound 1(MIC,m g/mL).
Samples
Fungi
Compound 1
Positive control a
G.graminis 220150R.cerealis 160100H.sativum
130120F.graminearum
250
150
a
Triadimefon is used as positive control.
5
L-Leu1
Figure 1.The structure of compounds 1.
618 C.Li et al.
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shaker for 7days.The mycelium of each fungus was transferred to 500mL Erlenmeyer ?asks containing culture liquid (200mL),at the same time.The ?asks were then incubated at 308C for 25days.Four crop-threatening fungi (G.graminis ,R.cerealis ,H.sativum and F.graminearum )for bioassay were obtained from the College of Science,South China Agricultural University.3.3.Extraction and isolation
The cultures (60L)were ?ltered through cheesecloth.The ?ltrate was concentrated to 3L in vacuo below 508C and extracted ?ve times using an equal volume of ethyl acetate and n -butyl alcohol in sequence,respectively.The combined n -butyl alcohol extract (11.8g)was subjected to silica gel column chromatography and eluted with petroleum ether–ethyl acetate–methanol (70:30:0,50:50:0,0:100:0,0:90:10,0:70:30,0:50:50,0:0:100)to afford fractions 1–7(0.011,0.021,0.032,0.35,0.67,2.8,5.5g,respectively).Fraction 5(0.67g)was fractionated using C-18SPE cartridges eluted with a stepwise gradient of 20%,40%,50%,60%,70%,80%,90%and 100%of methanol in water to yield eight fractions (SubF1–SubF8).Fraction SubF1(0.019g)was puri?ed by reverse-phase HPLC (Synergi Hydro 250￡10mm column,isocratic elution of 25%methanol–75%distilled water,UV detector at 254nm,?ow of 1.0mL/min)to afford 3.2mg of cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(1).
Cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)(1):colour-less needles;?a 20D 240.0(c 0.01,MeOH);IR (neat):3437,3260,2957,2872,1663,1437,1302,1051,1026cm 21;1
H NMR (400MHz,DMSO-d 6)d H :trans -4-OH-L -Pro1:4.40(1H,dd,6.8,10.4Hz,H-2),2.05(1H,m,H-3a),1.96(1H,m,H-3b),4.28(1H,m,H-4),3.25(2H,dd,4.2,11.6Hz,H-5),5.10(1H,d,2.4Hz,Z OH),L -Leu1:7.99(1H,br s,Z NH),4.05(1H,t,6.0Hz,H-2),1.37(1H,m,H-3a),1.79(1H,m,H-3b),1.88(1H,m,H-4),0.86(3H,d,4.2Hz,3H),0.87(3H,d,4.2Hz,3H),trans -4-OH-L -Pro2:4.23(1H,dd,4.2,6.8Hz,H-2),2.06(1H,m,H-3a),2.26(1H,m,H-3b),4.25(1H,m,H-4),3.48(2H,m,H-5),5.00(1H,d,4.0Hz,Z OH),D -Leu2:8.31(1H,br s,Z NH),3.62(1H,m,4.8,9.6Hz,H-2),1.45(1H,m,H-3a),1.56(1H,m,H-3b),1.68(1H,m,H-4),0.88(3H,d,4.2Hz,H-5),0.91(3H,d,4.2Hz,H-6);13C NMR (100MHz,DMSO-d 6)d C :trans -4-OH-L -Pro1:170.7(s,C-1),57.0(d,C-2),36.6(t,C-3),67.0(d,C-4),53.7(t,C-5),L -Leu1:166.3(s,C-1),52.5(d,C-2),37.7(t,C-3),24.1(d,C-4),22.73(q,C-5),22.75(q,C-6),trans -4-OH-L -Pro2:168.5(s,C-1),55.7(d,C-2),36.4(t,C-3),66.7(d,C-4),52.8(t,C-5),L -Leu2:166.5(s,C-1),55.0(d,C-2),41.9(t,C-3),23.9(d,C-4),21.5(q,C-5),21.8(q,C-6).HMBC and 1H–1H COSY data are summarised in Table S1.HR-ESI-MS m /z 453.2706[M tH]t(calcd for 453.2704).3.4.Determination of absolute con?guration
The absolute con?guration was determined by Marfey’s analytic method (Marfey 1984;Fujii et al.1998).LC–MS analysis of FDAA-derivatised hydrolysates and standard FDAA-derivatised amino acids was carried out using C8(4.6￡150mm column)with a linear gradient of H 2O (0.05%HCOOH)–MeOH (0.05%HCOOH)60:40to 10:90in 75min at 0.6mL/min.3.5.Antifungal activity assays
Dilution method (Yang et al.2006)was used to screen antifungal activity in vitro ,against G.graminis ,R.cerealis ,H.sativum and F.graminearum .The test sample was dissolved in DMSO and diluted using 20%DMSO in sterile water,and then transferred in duplicate to 96-well ?at-bottom microplates.One to three small colonies of the test plant pathogens were picked from agar plates and suspended in 5mL sterile water,and ?ltered through Miracloth.The optical density at 630nm of the suspensions was compared with the 0.5McFarland standard.The suspensions were diluted in Sabouraud dextrose culture broth to afford a ?nal concentration of 104CFU/mL.
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The fungal inocula were added to the solutions in the microplate to achieve a ?nal volume of 200m L and ?nal sample concentrations from 500to 20m g/mL.Positive (triadimefon)and blank (the ?nal solution without test sample)controls were included on each test plate.All results were observed at 630nm using the EL-340Biokinetics reader before and after incubation (288C for 72h).The MIC value was the lowest test concentration that allowed no detectable growth.4.Conclusion
One new cyclic tetrapeptide,Cyclo-(L -leucyl-trans -4-hydroxy-L -prolyl-D -leucyl-trans -4-hydroxy-L -proline)was isolated from the co-culture broth of two mangrove fungi Phomopsis sp.K38and Alternaria sp.E33.
Supplementary material
Supplementary material relating to this article is available online,alongside Table S1and Figures S1–S8.Acknowledgements
This work was supported by the National Natural Science Foundation of China (21102049),the Natural Science Foundation of Guangdong Province (9451064201003751),the Science and Technology Project of Guangzhou City (11C12100771),the Key Academic Program of the 3rd Phase ‘211Project’of South China Agricultural University (2009B010100001)and the national scholarship fund of China for studying abroad (for Dr Chunyuan Li,No.201208440268).
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数据结构第三章栈和队列3习题
第三章栈和队列试题 一、单项选择题 1.栈的插入和删除操作在（）进行。 A. 栈顶 B. 栈底 C. 任意位置 D. 指定位置 2.当利用大小为n的数组顺序存储一个栈时，假定用top==n表示栈空，则向这个栈插入一个元素时， 首先应执行（）语句修改top指针。 A. top++; B. top--; C. top = 0; D. top; 3.若让元素1,2,3依次进栈，则出栈次序不可能出现（）种情况。 A. 3, 2, 1 B. 2, 1, 3 C. 3, 1, 2 D. 1, 3, 2 4.在一个顺序存储的循环队列中，队头指针指向队头元素的（）位置。 A. 前一个 B. 后一个 C. 当前 D. 后面 5.当利用大小为n的数组顺序存储一个队列时，该队列的最大长度为（）。 A. n-2 B. n-1 C. n D. n+1 6.从一个顺序存储的循环队列中删除一个元素时，需要（）。 A. 队头指针加一 B. 队头指针减一 C. 取出队头指针所指的元素 D. 取出队尾指针所指的元素 7.假定一个顺序存储的循环队列的队头和队尾指针分别为front和rear，则判断队空的条件为（）。 A. front+1 == rear B. rear+1 == front C. front == 0 D. front == rear 8.假定一个链式队列的队头和队尾指针分别为front和rear，则判断队空的条件为（）。 A. front == rear B. front != NULL C. rear != NULL D. front == NULL 9.设链式栈中结点的结构为（data, link），且top是指向栈顶的指针。若想在链式栈的栈顶插入一 个由指针s所指的结点，则应执行操作（）。 A. top->link = s; B.s->link = top->link; top->link = s; C. s->link = top; top = s; D. s->link = top; top = top->link; 10.设链式栈中结点的结构为（data, link），且top是指向栈顶的指针。若想摘除链式栈的栈顶结点， 并将被摘除结点的值保存到x中，则应执行操作（）。 A. x = top->data; top = top->link; B. top = top->link; x = top->data; C. x = top; top = top->link; D. x = top->data; 11.设循环队列的结构是 #define MaxSize 100 typedef int ElemType;
with的复合结构和独立主格结构
1. with+宾语+形容词。比如：. The boy wore a shirt with the neck open, showing his bare chest. 那男孩儿穿着一件衬衫，颈部敞开，露出光光的胸膛。Don’t talk with your mouth full. 嘴里有食物时不要讲话。 2. with+宾语+副词。比如：She followed the guide with her head down. 她低着头，跟在导游之后。 What a lonely world it will be with you away. 你不在，多没劲儿呀！ 3. with+宾语+过去分词。比如：He was listening to the music with his eyes half closed. 他眼睛半闭着听音乐。She sat with her head bent. 她低着头坐着。 4. with+宾语+现在分词。比如：With winter coming, it’s time to buy warm clothes. 冬天到了，该买些保暖的衣服了。 He soon fell asleep with the light still burning. 他很快就睡着了，（可）灯还亮着。 5. with+宾语+介词短语。比如：He was asleep with his head on his arms. 他的头枕在臂膀上睡着了。 The young lady came in, with her two- year-old son in her arms. 那位年轻的女士进来了，怀里抱着两岁的孩子。 6. with+宾语+动词不定式。比如： With nothing to do in the afternoon, I went to see a film. 下午无事可做，我就去看了场电影。Sorry, I can’t go out with all these dishes to wash. 很抱歉，有这么多盘子要洗，我不能出去。 7. with+宾语+名词。比如： He died with his daughter yet a school-girl.他去逝时，女儿还是个小学生。 He lived a luxurious life, with his old father a beggar . 他过着奢侈的生活，而他的老父亲却沿街乞讨。(8)With so much work to do ,I can't go swimming with you. (9)She stood at the door,with her back towards us. (10)He entered the room,with his nose red with cold. with复合结构与分词做状语有啥区别 [ 标签：with, 复合结构, 分词状语] Ciro Ferrara 2009-10-18 16:17 主要是分词形式与主语的关系 满意答案好评率：100%
第三章栈和队列习题_数据结构电子教案
习题三栈和队列 一单项选择题 1. 在作进栈运算时,应先判别栈是否(① ),在作退栈运算时应先判别栈是否(② )。当栈中元素为n个,作进栈运算时发生上溢,则说明该栈的最大容量为(③ )。 ①, ②: A. 空 B. 满 C. 上溢 D. 下溢 ③: A. n-1 B. n C. n+1 D. n/2 2．若已知一个栈的进栈序列是1，2，3，…，n，其输出序列为p1，p2，p3，...，pn，若p1＝3，则p2为( )。 A 可能是2 B 一定是2 C 可能是1 D 一定是1 3. 有六个元素6，5，4，3，2，1 的顺序进栈，问下列哪一个不是合法的出栈序列？（） A. 5 4 3 6 1 2 B. 4 5 3 1 2 6 C. 3 4 6 5 2 1 D. 2 3 4 1 5 6 4.设有一顺序栈S，元素s1,s2,s3,s4,s5,s6依次进栈，如果6个元素出栈的顺序是s2,s3,s4, s6, s5,s1,则栈的容量至少应该是（） A.2 B. 3 C. 5 D.6 5. 若栈采用顺序存储方式存储，现两栈共享空间V[1..m]，top[i]代表第i个栈( i =1,2)栈顶，栈1的底在v[1]，栈2的底在V[m]，则栈满的条件是（）。 A. |top[2]-top[1]|=0 B. top[1]+1=top[2] C. top[1]+top[2]=m D. top[1]=top[2] 6. 执行完下列语句段后，i值为：（） int f(int x) { return ((x>0) ? x* f(x-1):2);} int i ; i =f(f(1)); A．2 B. 4 C. 8 D. 无限递归 7. 表达式3* 2^(4+2*2-6*3)-5求值过程中当扫描到6时，对象栈和算符栈为（），其中^为乘幂。 A. 3,2,4,1,1；(*^(+*- B. 3,2,8；(*^- C. 3,2,4,2,2；(*^(- D. 3,2,8；(*^(- 8. 用链接方式存储的队列，在进行删除运算时（）。 A. 仅修改头指针 B. 仅修改尾指针 C. 头、尾指针都要修改 D. 头、尾指针可能都要修改 9. 递归过程或函数调用时，处理参数及返回地址，要用一种称为（）的数据结构。 A．队列 B．多维数组 C．栈 D. 线性表 10．设C语言数组Data[m+1]作为循环队列SQ的存储空间， front为队头指针，rear为队尾指针，则执行出队操作的语句为（） A.front=front+1 B. front=（front+1）% m C.rear=(rear+1)%(m+1) D. front=(front+1)%(m+1) 11.循环队列的队满条件为 ( ) A. (sq.rear+1) % maxsize ==(sq.front+1) % maxsize; B. (sq.front+1) % maxsize ==sq.rear C. (sq.rear+1) % maxsize ==sq.front D.sq.rear ==sq.front
高中英语独立主格结构、with的复合结构专项练习测试40题(有答案)
一、选择题 1、With time ____ by , they got to know each other better. A. passes B. passing C. passed D. to be passed 2、 the economic crisis getting more and more serious, the government is searching for ways to improve people’s life. A. As B. With C. When D. If 3John received an invitation to dinner, and with his work ____, he gladly accepted it. A. finished B. finishing C. having finished D. was finished 4、With all flights___, they had to come by bus. A. had canceled B.canceled C.have been canceled D. having canceled 5、With a large number of people _______ camping, it has now become one of the most popular activities in the UK. A. take part in B. took part in C. taking part in D. to be taking part in 6、None of us had expected that the middle﹣aged engineer died with his design _________（） A．.to uncomplete B．.uncompleted C．uncompleting．D．.uncomplete 7、______，we managed to get out of the forest．（） A．The guide led the way B．The guide leading the way C．With the guide to lead the way D．Having led the way 8、Will all his work ,he could have a good rest. A.to do B.doing C.did D.done 9、 ______, her suggestion is of greater value than yours. A. All things considering B. All things considered C. All things were considered D. With all things were considered 10、With the kind boy ________ the way, we found the park soon. A. leads B. to lead C. led D. leading 11、 She stood there, ______ from her cheeks. A. tears' rolling down B. tears rolled down C. with tears rolled down D. tears rolling down 12、 While watching television, __________. A. the doorbell rang B. the doorbell rings C. we heard the doorbell ring D. we heard the doorbell rings 13、The murderer was brought in, with his hands______ behind his back. A. be tied B. having tied C. to be tied D. tied 14、 With a lot of difficult problem _____, the newly-elected president is having a hard time.
5种基本句型和独立主格结构讲解
英语中的五种基本句型结构 一、句型1：Subject (主语) ＋Verb (谓语) 这种句型中的动词大多是不及物动词，所谓不及物动词，就是这种动词后不可以直接接宾语。常见的动词如：work, sing, swim, fish, jump, arrive, come, die, disappear, cry, happen等。如： 1) Li Ming works very hard.李明学习很努力。 2) The accident happened yesterday afternoon.事故是昨天下午发生的。 3）Spring is coming. 4) We have lived in the city for ten years. 二、句型2：Subject (主语) ＋Link. V(系动词) ＋Predicate(表语) 这种句型主要用来表示主语的特点、身份等。其系动词一般可分为下列两类： (1)表示状态。这样的词有：be, look, seem, smell, taste, sound, keep等。如： 1) This kind of food tastes delicious.这种食物吃起来很可口。 2) He looked worried just now.刚才他看上去有些焦急。 (2)表示变化。这类系动词有：become, turn, get, grow, go等。如： 1) Spring comes. It is getting warmer and warmer.春天到了，天气变得越来越暖和。 2) The tree has grown much taller than before.这棵树比以前长得高多了。 三、句型3：Subject(主语) ＋V erb (谓语) ＋Object (宾语) 这种句型中的动词一般为及物动词, 所谓及物动词，就是这种动词后可以直接接宾语，其宾语通常由名词、代词、动词不定式、动名词或从句等来充当。例： 1) He took his bag and left.（名词）他拿着书包离开了。 2) Li Lei always helps me when I have difficulties. (代词)当我遇到困难时，李雷总能给我帮助。 3) She plans to travel in the coming May Day.（不定式）她打算在即将到来的“五一”外出旅游。 4) I don’t know what I should do next. (从句)我不知道下一步该干什么。 注意：英语中的许多动词既是及物动词，又是不及物动词。 四、句型4：Subject(主语)＋Verb(谓语)＋Indirect object(间接宾语)＋Direct object (直接宾语) 这种句型中，直接宾语为主要宾语，表示动作是对谁做的或为谁做的，在句中不可或缺，常常由表示“物”的名词来充当；间接宾语也被称之为第二宾语，去掉之后，对整个句子的影响不大，多由指“人”的名词或代词承担。引导这类双宾语的常见动词有：buy, pass, lend, give, tell, teach, show, bring, send等。如： 1) Her father bought her a dictionary as a birthday present.她爸爸给她买了一本词典作为生日礼物。 2）The old man always tells the children stories about the heroes in the Long March. 老人经常给孩子们讲述长征途中那些英雄的故事。上述句子还可以表达为： 1）Her father bought a dictionary for her as a birthday present. 2）The old man always tells stories about the heroes to the children in the Long March. 五、句型5：Subject(主语)＋Verb (动词)＋Object (宾语)＋Complement(补语) 这种句型中的“宾语＋补语”统称为“复合宾语”。宾语补足语的主要作用或者是补充、说明宾语的特点、身份等；或者表示让宾语去完成的动作等。担任补语的常常是名词、形容词、副词、介词短语、分词、动词不定式等。如： 1）You should keep the room clean and tidy. 你应该让屋子保持干净整洁。（形容词） 2) We made him our monitor.(名词)我们选他当班长。 3) His father told him not to play in the street.(不定式)他父亲告诉他不要在街上玩。
第三章栈和队列练习题
第三章栈和队列练习题 一、单项选择题 1．一个顺序栈一旦被声明，其占用空间的大小（）。 A．已固定B．可以改变C．不能固定D．动态变化 2．链栈和顺序栈相比，有一个比较明显的缺点，即（）。 A．插入操作更加方便B．通常不会出现栈满的情况 C．不会出现栈空的情况D．删除操作更加方便 3．用单链表表示的链式队列的队头在链表的（）位置。 A．链头B．链尾C．链中D．任意位置 4．在解决计算机主机与打印机之间速度不匹配问题时通常设置一个打印数据缓冲区，主机将要输出的数据依次写入缓冲区中，而打印机则从缓冲区中取出数据打印，该缓冲区应该是一个（）结构。 A．堆栈B．队列C．数组D．先性表 5．若已知一个栈的入栈序列是1，2，3，…，30，其输出序列是p1,p2,p3,…p n，若p1=30，则p10为（）。 A．11 B．20 C．19 D．21 6．循环队列A[m] 存放其元素，用front和rear分别表示队头及队尾，则循环队列满的条件是（）。 A．（rear+1)%m=front B．rear =front+1 C．rear=front D．（rear+1)%m-1=front 7．在一个栈顶指针为top的链栈中，将一个p指针所指的结点入栈，应执行（）。 A．top->next=p; B．p->next=top->next; top->next=p; C．p->next=top; top=p; D．p->next=top->next; top=top->next; 8．在一个栈顶指针为top的链栈中删除一个结点时，用x保存被删结点的值，则执行（）。 A．x=top;top=top->next; B．x=top->data;
with独立主格结构
with独立主格结构(即with复合结构) with独立主格结构是英语中一种重要的句法现象，在句子结构方面具有相对独立的特点。多年来也一直是命题的热点、重点，因此应该引起我们的高度重视。众所周知，with引导的独立主格结构非常活跃，虽然它在句子中只作状语，但是可以表示伴随、方式、原因、结果等各种复杂的情况。 现将with引导的独立主格结构总结如下。 一、句法结构 【结构一】 with ＋名词（代词）＋介词短语 例１ He sat there thinking, with his chin on his hand． 他手托下巴，坐在那儿沉思。 【结构二】 with ＋名词（代词）＋形容词 例２ He stared at his friend with his mouth wide open． 他张大嘴巴凝视着他的朋友。 【结构三】with ＋名词（代词）＋副词 例３ With production up by 60％, the company has had another excellent year． 产量上升了60％, 公司又是一个好年景。 【结构四】 with ＋名词（代词）＋名词 例４ She used to sit reading in the evening with her pet dog her only companion． 她从前总爱在晚上坐着看书，她的宠物狗便是她唯一的伙伴。 【结构五】with ＋名词（代词）＋现在分词 例５ She stood there chatting with her friend, with her child playing beside her． 她站在那儿跟朋友闲聊，孩子在旁边玩。 【结构六】with ＋名词（代词）＋过去分词
独立主格结构练习题及解析
独立主格结构练习题及解析 1. I have a lot of books, half of ___ novels. A. which B. that C. whom D. them 2. __ more and more forests destroyed, many animals are facing thedanger of dying out. A. because B. as C. With D. Since 3. The bus was crowded with passengers going home from market, most of __ carrying heavy bags and baskets full of fruit and vegetables they hadbought there. A. them B. who C. whom D. which 4. The largest collection ever found in England was one of about 200,000 silverpennies, all of ___ over 600 years old. A. which B. that C. them
D. it 5. The cave __ very dark, he lit some candles ___ light. A. was; given B. was; to give C. being; given D. being; to give 6. The soldier rushed into the cave, his right hand __ a gun and his face ____ with sweat.A held; covered B. holding; covering C. holding; covered D. held; covering 7. The girl in the snapshot was smiling sweetly, her long hair ___ . A. flowed in the breeze B. was flowing in the breeze C. were flowing in the breeze D. flowing in the breeze 8. The children went home from the grammar school, their lessons ____ for the day. A. finishing B. finished C. had finished D. were finished 9. On Sundays there were a lot of children playing in the park, ___ parents seated together joking.
独立主格with用法小全
独立主格篇 独立主格，首先它是一个“格”，而不是一个“句子”。在英语中任何一个句子都要有主谓结构，而在这个结构中，没有真正的主语和谓语动词，但又在逻辑上构成主谓或主表关系。独立主格结构主要用于描绘性文字中，其作用相当于一个状语从句，常用来表示时间、原因、条件、行为方式或伴随情况等。除名词/代词+名词、形容词、副词、非谓语动词及介词短语外，另有with或without短语可做独立主格，其中with可省略而without不可以。*注：独立主格结构一般放在句首，表示原因时还可放在句末；表伴随状况或补充说明时，相当于一个并列句，通常放于句末。 一、独立主格结构： 1. 名词/代词+形容词 He sat in the front row, his mouth half open. Close to the bank I saw deep pools, the water blue like the sky. 靠近岸时，我看见几汪深池塘，池水碧似蓝天。 2. 名词/代词+现在分词 Winter coming, it gets colder and colder. The rain having stopped, he went out for a walk.
The question having been settled, we wound up the meeting. 也可以The question settled, we wound up the meeting. 但含义稍有差异。前者强调了动作的先后。 We redoubled our efforts, each man working like two. 我们加倍努力，一个人干两个人的活。 3. 名词/代词+过去分词 The job finished, we went home. More time given, we should have done the job much better. *当表人体部位的词做逻辑主语时，不及物动词用现在分词，及物动词用过去分词。 He lay there, his teeth set, his hands clenched, his eyes looking straight up. 他躺在那儿，牙关紧闭，双拳紧握，两眼直视上方。 4. 名词/代词+不定式 We shall assemble at ten forty-five, the procession to start moving at precisely eleven. We divided the work, he to clean the windows and I to sweep the floor.
独立主格结构图表解析
独立主格结构 一、概念 “独立主格结构”就是由一个相当于主语的名词或代词加上非谓语动词、形容词（副）词或介词短语构成的一种独立成分。该结构不是句子，也不是从句，所以它内部的动词不能考虑其时态、人称和数的变化，它与主句之间不能通过并列连词连接，也不能由从句阴道词引导，通常用逗号与主句隔开。独立主格结构在很多情况下可以转化为相应的状语从句或者其他状语形式，但很多时候不能转化为分词形式，因为它内部动词的逻辑主语与主句主语不一致。 二、独立主格的特点
1.当独立主格结构中的being done表示“正在被做时”，being不可以被省略。 2.当独立主格结构的逻辑主语是it, there时，being不可以省略。 三、独立主格结构的用法。 一般放在句首，表示原因时还可放在句末；表伴随状况或补充说明时，相当于一个并列句，通常放于句末。
四、非谓语动词独立主格结构。 “名词或代词+非谓语动词”结构构成的独立主格结构称为非谓语动词的独立主格结构。名词或代词和非谓语动词具有逻辑上的主谓关系。 1.不定式构成的独立主格结构 不定式构成的独立主格结构往往表示还未发生的行为或状态，在句中常 作原因状语，有时做条件状语。 Lots of homework to do, I have to stay home all day. 由于很多作业要做，我只好待在家里。 So many children to look after, the mother has to quit her job. 如此多的孩子要照顾，这个妈妈不得不辞掉她的工作。 2.动词+ing形式的独立主格结构 动词-ing形式的句中作状语时，其逻辑主语必须是主句的主语，否则就 是不正确的。动词-ing形式的逻辑主语与主句的主语不一致时，就应在 动词的-ing形式前加上逻辑主语，构成动词-ing 形式的独立主格结构，逻辑主语与动词间为主谓关系，是分词的动作执行者，分词表示的动作 时逻辑主语发出的动作。 We redoubled our efforts, each man working like two. 我们加倍努力，每个人就像在干两个人的活。 The governor considering the matter, more strikers gathered across his path. 总督思考这个问题时，更多的罢工工人聚集到他要通过的路上。 The guide leading the way, we had no trouble getting out of the forest. 在向导的带领下，我们轻松地走出了森林。 3.过去分词形式的独立主格 过去分词形式的独立主格结构是由“逻辑主语+过去分词”构成。逻辑主 语与动词之间为动宾关系，它是分词的动作承受者，这一结构在句中作 时间状语，原因状语、伴随状语、条件状语等。 This done, we went home.做完这个，我们就回家了。 All our savings gone, we started looking for jobs. 积蓄用完后，我们都开始找工作。 More time and money given, we can finish the work in advance. 如果给予更多的时间和金钱，我们能提前完成这个工作。 五、其他形式的独立主格结构
第3章-栈与队列习题参考答案
习题三参考答案 备注: 红色字体标明的是与书本内容有改动的内容。 一、选择题 1.在栈中存取数据的原则是（ B ）。 A．先进先出 B. 先进后出 C. 后进后出 D. 没有限制 2．若将整数1、2、3、4依次进栈，则不可能得到的出栈序列是（ D ）。 A．1234 B. 1324 C. 4321 D. 1423 3．在链栈中，进行出栈操作时（B ）。 A．需要判断栈是否满 B. 需要判断栈是否为空 C. 需要判断栈元素的类型 D. 无需对栈作任何差别 4．在顺序栈中，若栈顶指针top指向栈顶元素的下一个存储单元，且顺序栈的最大容量是maxSize，则顺序栈的判空条件是（ A ）。 A．top==0 B.top==-1 C. top==maxSize D.top==maxSize-1 5．在顺序栈中，若栈顶指针top指向栈顶元素的下一个存储单元，且顺序栈的最大容量是maxSize。则顺序栈的判满的条件是（ C ）。 A．top==0 B.top==-1 C. top==maxSize D.top==maxSize-1 6．在队列中存取数据元素的原则是（ A ）。 A．先进先出 B. 先进后出 C. 后进后出 D. 没有限制 7．在循环顺序队列中，假设以少用一个存储单元的方法来区分队列判满和判空的条件，front和rear分别为队首和队尾指针，它们分别指向队首元素和队尾元素的下一个存储单元，队列的最大存储容量为maxSize，则队列的判空条件是（A ）。 A．front==rear B. front!=rear C. front==rear+1 D. front==(rear+1)% maxSize 8．在循环顺序队列中，假设以少用一个存储单元的方法来区分队列判满和判空的条件，front和rear分别为队首和队尾指针，它们分别指向队首元素和队尾元素的下一个存储单元，队列的最大存储容量为maxSize，则队列的判满条件是（D ）。 A．front==rear B. front!=rear C. front==rear+1 D. front==(rear+1)% maxSize 9.在循环顺序队列中，假设以少用一个存储单元的方法来区分队列判满和判空的条件，front和rear分别为队首 和队尾指针，它们分别指向队首元素和队尾元素的下一个存储单元，队列的最大存储容量为maxSize，则队列的长度是（C ）。 A．rear-front B. rear-front+1 C. (rear-front+maxSize)%maxSize D. (rear-front+1)%maxSize 10.设长度为n的链队列采用单循环链表加以表示，若只设一个头指针指向队首元素，则入队操作的时间复杂度 为（ B ）。 A．O(1) B．O(n) C．O(log2n) D．O(n2) 二、填空题 1.栈是一种操作受限的特殊线性表，其特殊性体现在其插入和删除操作都限制在表尾进行。允许插入和删除 操作的一端称为栈顶，而另一端称为栈底。栈具有后进先出的特点。 2.栈也有两种存储结构，一种是顺序存储，另一种是链式存储；以这两种存储结构存储的栈分别称为顺序 栈和链栈。 3.在顺序栈中，假设栈顶指针top是指向栈顶元素的下一个存储单元，则顺序栈判空的条件是 top==0 ; 栈顶
第三章+栈和队列(参考答案)
第三章栈和队列 一、判断题 1、链栈的初始化是指开辟足够多的结点，然后置栈顶指针为 NULL。（×） 2、递归定义的数据结构通常不需要用递归的算法来实现对它的操作。（×） 二、填空题 1、向一个链式栈插入一个新结点时，首先把栈顶指针的值赋给新结点的指针域，然后把新结点的存储位置赋给___栈顶指针_____。 2、迷宫问题是一个回溯控制的问题，最好使用____栈______的方法来解决。 3、有如下递归过程： Void Print(int w) { int i; if (w!=0) { Print(w?1); for (i=1;i<=w;i++) printf(“%3d”,w); printf(“\n”); } } 调用语句print(4)的结果是__________。 1 2 2 3 3 3 4 4 4 4 4、假设用循环单链表实现队列，若队列非空，且队尾指针为R, 则将新结点S加入队列时，需执行下面语句：_ S->next=R->next _________；___ R->next=S _______；R=S； 三、选择题 1、设有4个数据元素a1、a 2、a3和a4，对他们分别进行栈操作或队操作。在进栈或进队操作时，按a1、a2、a 3、a4次序每次进入一个元素。假设栈或队的初始状态都是空。 现要进行的栈操作是进栈两次，出栈一次，再进栈两次，出栈一次；这时，第一次出栈得到的元素是 A 2，第二次出栈得到的元素是 B 4；类似地，考虑对这四个数据元素进行的队操作是进队两次，出队一次，再进队两次，出队一次；这时，第一次出队得到的元素是 C 1，第二次出队得到的元素是 D 2。经操作后，最后在栈中或队中的元素还有 E 2个。 供选择的答案： A～D：①a1 ②a2 ③ a3 ④a4 E：①1 ②2 ③ 3 ④ 0 2、栈是一种线性表，它的特点是 A 2。设用一维数组A[1,…,n]来表示一个栈，A[n]为栈底，用整型变量T指示当前栈顶位置，A[T]为栈顶元素。往栈中推入（PUSH）一个新元素时，变量T的值 B 2；从栈中弹出（POP）一个元素时，变量T的值 C 1。设栈空时，有输入序列a，b，c，经过PUSH，POP，PUSH，PUSH，POP操作后，从栈中弹出的元素的序列是 D 6，变量T的值是 E 4。 供选择的答案： A：①先进先出②后进先出③进优于出④出优于进⑤随机进出 B，C：①加1 ②减1 ③不变④清⑤加2 ⑥减2 D：① a,b ②b,c ③c,a ④b,a ⑤ c,b ⑥a,c E：① n+1 ②n+2 ③ n ④ n-1 ⑤ n-2 3、在做进栈运算时，应先判别栈是否 A 2；在做退栈运算时，应先判别栈是否 B 1。当栈中元素为n个，做进栈运算时发生上溢，则说明该栈的最大容量为 C 2。
With 引导的独立主格结构
教学参考：With引导的独立主格结构 https://www.wendangku.net/doc/ca15224272.html, 2005/03/14 09:41 英语辅导报 with独立主格结构是英语的一种重要的句法现象，在句子结构方面具有相对独立的特点。多年 来也一直是命题的热点、重点，因此应该引起我们的高度重视。众所周知，with引导的独立主格结 构非常活跃，虽然它在句子中只作状语，但是可以表示伴随、方式、原因、结果等各种复杂的情况。现将with引导的独立主格结构加以小结。 一、句法结构 【结构一】with＋名词(代词)＋介词短语 【例句】He sat there thinking, with his chin on his hand. 他手托下巴，坐在那儿沉思。 The old man stood there, with his back against the wall. 那位老人背倚着墙站在那里。 Mary was sitting near the fire, with her back towards the door. 玛丽靠近火炉坐着，背对着门。 【结构二】with＋名词(代词)＋形容词 【例句】He stared at his friend with his mouth wide open. 他张大嘴巴凝视着他的朋友。 The man raised his head with eyes full of wonder and mystery. 这人抬起头来，眼里充满了好奇。 He stood there trembling, with his face red with cold. 他站在那儿瑟瑟发抖，脸都冻红了。 【结构三】with＋名词(代词)＋副词 【例句】With production up by 60%, the company has had another excellent year. 产量上升了60%, 公司又是一个好年景。 The stupid Emperor walked in the procession with nothing on. 这位愚蠢的皇帝一丝不挂地行进在 游行队伍中。 The naughty boy stood before his teacher with his head down. 这个淘气的男孩低着头站在老师面前。 He put on his socks with the wrong side out. 他把袜子穿反了。 【结构四】with＋名词(代词)＋名词 【例句】She used to sit reading in the evening with her pet dog her only companion. 她从前总爱在 晚上坐着看书，她的宠物狗便是她惟一的伙伴。
with独立主格结构
with独立主格结构是英语的一种重要的句法现象，在句子结构方面具有相对独立的特点。多年来也一直是命题的热点、重点，因此应该引起我们的高度重视。众所周知，with 引导的独立主格结构非常活跃，虽然它在句子中只作状语，但是可以表示伴随、方式、原因、结果等各种复杂的情况。现将with引导的独立主格结构加以小结。 一、句法结构 【结构一】with ＋名词(代词)＋介词短语 【例句】He sat there thinking, with his chin on his hand. 他手托下巴，坐在那儿沉思。 The old man stood there, with his back against the wall. 那位老人背倚着墙站在那里。 Mary was sitting near the fire, with her back towards the door. 玛丽靠近火炉坐着，背对着门。 【结构二】with ＋名词(代词)＋形容词 【例句】He stared at his friend with his mouth wide open. 他张大嘴巴凝视着他的朋友。 The man raised his head with eyes full of wonder and mystery. 这人抬起头来，眼里充满了好奇。 He stood there trembling, with his face red with cold. 他站在那儿瑟瑟发抖，脸都冻红了。 【结构三】with ＋名词(代词)＋副词 【例句】With production up by 60%, the company has had another excellent year. 产量上升了60%, 公司又是一个好年景。 The stupid Emperor walked in the procession with nothing on. 这位愚蠢的皇帝一丝不挂地行进在游行队伍中。 The naughty boy stood before his teacher with his head down. 这个淘气的男孩低着头站在老师面前。 He put on his socks with the wrong side out. 他把袜子穿反了。 【结构四】with ＋名词(代词)＋名词 【例句】She used to sit reading in the evening with her pet dog her only companion. 她从前总爱在晚上坐着看书，她的宠物狗便是她惟一的伙伴。
(完整版)独立主格结构完整讲解
一、独立主格结构的概念 独立主格结构（Absolute Structure ）是由名词或代词加上分词等构成的一种独立 结构，用于修饰整个句子。独立主格结构中的名词或代词与其后的分词等构成逻辑上的主谓关系。这种结构与主句不发生句法上的联系，它的位置相当灵活，可置于主句前、主句末或主句中，常由逗号将其与主句分开。在句中作状语，相当于一个状语从句。 需特别注意的是，独 二、独立主格结构基本构成形式 名词（代词）+现在分词；过去分词；形容词；副词；不定式；名词；介词短语） 1. 名词（代词）+现在分词 例句：The storm drawing near ，the navvy decided to call it a day The storm drawi ng n ear 在句中作：原因状语 =Si nee the storm was draw ing n ear , the n avvy decided to call it a day 由于暴风雨即将来临，那个挖土小工决定收工。（call it a day 今天到此为止） 例句：Win ter comin g, it gets colder and colder. Win ter comi ng 在句中作：伴随状语 =The win ter comes, and it gets colder and colder. 冬天来了，天气越来越冷了。 造句：时间允许的话，我就和你一起走。 Time permitti ng, I will go with you. 独立主格作：条件状语 =改写：f time permits, I will go with you. ___________ 造句：那个姑娘望着他，他不知道说什么好。 The girl staring at him, he didn ' t know what to say. 独立主格作：时间状语 =改写：As the girl stared at him, he didn ' t know what to say. 2.名词（代词）+过去分词 例句：He was listening attentively in class, his eyes fixed on the blackboard. 他上课专心听讲，眼睛紧盯着黑板。 例句：The meeti ng gone over, every one tired to go home earlier. 会议结束后，每个人都想早点回家。 造句：工作完成后，他回家了。 The work done, he went back home.