Comparative Biochemistry and Physiology Part B125(2000)255–264
Identi?cation of a myo?bril-bound serine proteinase(MBSP) in the skeletal muscle of lizard?sh Saurida wanieso which
speci?cally cleaves the arginine site
Min-Jie Cao a,Kiyoshi Osatomi a,Kenji Hara b,*,Tadashi Ishihara b
a Graduate School of Marine Science and Engineering,Nagasaki Uni6ersity,Bunkyo,Nagasaki852-8521,Japan
b Department of Marine Biochemistry,Faculty of Fisheries,Nagasaki Uni6ersity,Bunkyo,Nagasaki852-8521,Japan
Received18August1999;received in revised form22October1999;accepted4November1999
Abstract
A myo?bril-bound serine proteinase(MBSP)from the skeletal muscle of lizard?sh(Saurida wanieso)was puri?ed to homogeneity by a heating treatment followed by a series of column chromatographies on DEAE-Sephacel,Sephacryl S-200,Q-Sepharose,Hydroxyapatite and Benzamidine-Sepharose6B,and characterized enzymatically.On SDS-poly-acrylamide gel electrophoresis(SDS-PAGE),the puri?ed enzyme showed a band with molecular mass of:29kDa under reducing conditions,while60kDa under non-reducing conditions.The optimum temperature of the enzyme was 50°C using t-butyloxycarbonyl-Phe-Ser-Arg-4-methylcoumaryl-7-amide(Boc-Phe-Ser-Arg-MCA)as a substrate.Sub-strate speci?city analysis both using MCA-substrates and peptides showed that MBSP speci?cally cleaved at the carboxyl side of the arginine residue.Inhibitor susceptibility analysis revealed that MBSP was inhibited effectively by Pefabloc SC, soybean trypsin inhibitor(STI)and aprotinin,indicating the characteristic of a serine proteinase.When myo?bril was incubated with the enzyme,it optically degraded myosin heavy chain at55–60°C,while a-actinin and actin were not at all hydrolyzed as detected by immunoblotting.The N-terminal amino acid sequence of MBSP was partially determined as IVGGAEXVPY-and was very homologous to other serine proteases.?2000Elsevier Science Inc.All rights reserved. Keywords:Amino acid sequence;Cleavage;Immunoblotting;Lizard?sh;MCA-substrate;Myo?bril-bound;Peptide;Puri?cation;Serine proteinase
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1.Introduction
Research with the purpose of isolating the en-zymes responsible for the so-called modori phe-nomenon(thermal gel degradation of?sh jelly products)led to the conclusion that such enzymes are serine proteinases for their myosin heavy chain degradation action was greatly suppressed by soybean trypsin inhibitor(STI)and diiso-propyl?uorophosphate(DFP),and they showed trypsin-like substrate speci?cities(Toyohara and Shimizu,1988;Toyohara et al.,1991;Osatomi et al.,1997).In many species of?sh,such modori-in-ducing proteinases are different from those de-tected in the sarcoplasmic fractions,since they could not be extracted from myo?bril even in the presence of detergents(Toyohara et al.,1991).It
Abbre6iations:Boc-,t-butyloxycarbonyl-;Suc-,succinyl;Z-,
carbobenzoxy;MCA,4-methylcoumaryl-7-amide;OBzl-,ben-
zyl-;Pyr-,pyroglutamyl-;HPLC,high performance liquid chromatography.
*Corresponding author.Tel.:+81-95-847-1111;fax:+81-
95-844-3516.
E-mail address:hara@net.nagasaki-u.ac.jp(K.Hara)
0305-0491/00/$-see front matter?2000Elsevier Science Inc.All rights reserved. PII:S0305-0491(99)00176-5
M.-J.Cao et al./Comparati6e Biochemistry and Physiology,Part B125(2000)255–264 256
was also presumed that such proteinases are rarely distributed in the sarcoplasmic fraction, but unevenly in the myo?brillar,especially myosin fraction(Shimizu et al.,1986).We desig-nated these proteinases as myo?bril-bound serine proteinases(MBSPs)and such a serine proteinase was puri?ed to homogeneity from the skeletal muscle of fresh water?sh carp(Cyprinus carpio) (Osatomi et al.,1997).Its N-terminal amino acid sequence was partially determined and revealed high identity with other serine proteinases,its cleavage speci?cities both toward MCA-sub-strates and peptides were investigated(Cao et al., 1999b).The carp MBSP(MBP)exhibited a hy-drolysis preference for myosin heavy chain when it was incubated with myo?brillar proteins, though a-actinin,actin,tropomyosin were also degraded to some extent(Cao et al.,1999a).Re-cently,a neutral serine protease that speci?cally cleaved myosin light chain2was puri?ed from the skeletal muscle of a myopathic hamster and its entire primary structure was determined by cDNA cloning and its tissue location was iden-ti?ed within sarcomeres(Holt et al.,1998;Levine et al.,1999).These results indicated the possibility that MBSPs ubiquitously exist in muscle cells. Taking into account such modori-relative serine proteinases in?sh,practically,it is very important to investigate these enzymes in marine?sh for?sh jelly products are mainly processed from marine ?sh.For a long period of time,MBSPs responsi-ble for the modori phenomenon have been de-tected in few marine?shes(Shimizu et al.,1986; Toyohara and Shimizu,1988;Toyohara et al., 1990,1991),however,their puri?cation have never been successfully carried out.The dif?culty in separating such proteinases from myo?brillar proteins and/or their low concentrations may be the main factors that hamper their puri?cation.In most cases,even though such enzymes can be initially separate,they could associate with my-o?brillar proteins again in the next step.
In this study,we describe the puri?cation of a MBSP from the muscle of lizard?sh and partially characterize it.The cleavage speci?cities of the enzyme to MCA-substrates and peptides were in-vestigated,and its N-terminal amino acid se-quence was analyzed.These data indicated that the present enzyme was a novel trypsin-type ser-ine proteinase.2.Materials and methods
2.1.Materials
Fresh lizard?sh,Saurida wanieso(body weight :1kg)was purchased in early October from a local commercial supply in Nagasaki,Japan.The ?sh was decapitated,muscle be collected and im-mediately stored at?35°C until use.
DEAE-Sephacel,Sephacryl S-200,Q-Sepharose and Benzamidine-Sepharose6B were purchased from Pharmacia(Uppsala,Sweden).t-Butyloxy-carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide (Boc-Phe-Ser-Arg-MCA),antipain,leupeptin, neurotensin,bovine adrenal medulla dodecapep-tide(BAM-12P),dynorphin A1–13,vasoactive intestinal peptide(VIP),a-neoendorphin,lysyl-bradykinin,methionyl-lysyl-bradykinin,and other synthetic?uorogenic peptide substrates(MCA-substrates)were obtained from Peptide Institute, (Osaka,Japan).p-Chloromercuribenzoic acid (pCMB)and benzamidine were products of Sigma(St Louis,MO,USA).4-(2-aminoethyl)-benzenesulfonyl?uoride hydrochloride(Pefabloc SC)was from Merck(Darmstadt,Germany).Hy-droxyapatite,antipain,aprotinin,5,5%-dithiobis (2-nitrobenzoic acid)(DTNB),EDTA,STI and bovine serum albumin were purchased from Wako Pure Chemicals(Osaka,Japan).p-Tosyl-L-lysyl-chloromethylketone(TLCK),p-tosyl-L-phenylalanyl-chloromethylketone(TPCK)and acetonitrile were products of Nacalai Tesque(Ky-oto,Japan).Protein standards for SDS-polyacry-lamide gel electrophoresis(SDS-PAGE)and immunoblotting were from Bio-Rad(Richmond, CA,USA).Polyclonal antibodies against red sea bream(Pagrus major)actin and a-actinin were raised in rats.All other chemicals used were of reagent grade.
2.2.Acti6ity towards MCA substrates
Routinely,the proteolytic activity assays were performed in duplicate using Boc-Phe-Ser-Arg-MCA as a substrate.Variation between duplicate samples was always B10%.Appropriately di-luted enzyme(100m l)was added to800m l of0.1 M borate buffer(Na2B4O7-KH2PO4,pH8).The reaction was immediately initiated by the addition of100m l of50m M substrate and incubated at 50°C for10min.To stop the reaction,1.5ml of the stopping agent(methyl alcohol:n-butyl alco-
M.-J.Cao et al./Comparati6e Biochemistry and Physiology,Part B125(2000)255–264257
hol:distilled water=35:30:35,v/v)was added. The?uorescence intensity of the liberated7-amino-4-methylcoumarin was measured by a ?uorescence spectrophotometer at an excitation wavelength of380nm and an emission wave-length of450nm.One unit of enzyme activity was de?ned as the amount of the enzyme to release1 nmol of7-amino-4-methylcoumarin per min.
2.3.Puri?cation of the myo?bril-bound serine proteinase
All puri?cation steps were carried out at0–4°C.Freshly defrosted lizard?sh muscle(500g) was minced and homogenized with3-fold(w/v)20 mM borate buffer,pH7.5(buffer A).The ho-mogenate was centrifuged at8000×g for20min. The supernatant was discarded and the pellet resuspended in4-fold distilled water,homoge-nized,adjusted to pH6and centrifuged.The above washing operation was repeated three more times with the resulting pellet to elute sarcoplas-mic enzymes completely.The resulting pellet was further homogenized with4-fold distilled water and adjusted to pH6.The homogenate was con-sequently heated in boiling water while stirring until the temperature reached55°C and further incubated at the same temperature for5min then immediately cooled in ice water.After centrifuga-tion,the supernatant,which was regarded as crude MBSP,was lyophilized.The lyophilized powder from1kg of muscle was dissolved in200 ml of distilled water,homogenized and adjusted to pH 4.The suspension was centrifuged at 10000×g for20min and the supernatant titrated immediately to pH6,and then dialyzed against buffer A before it was applied to a DEAE-Sep-hacel column,which was previously equilibrated with the dialysis buffer.A linear gradient of NaCl elution from0to0.5M was performed and the active fractions in the?rst peak were pooled, concentrated by ultra?ltration to20ml on an Amicon membrane YM-10and loaded on a Sep-hacryl S-200column equilibrated with buffer A containing0.2M NaCl.Active fractions were pooled,dialyzed against buffer A and submitted to a column of Q-Sepharose equilibrated with the same buffer.The column was eluted with buffer A and then with a linear gradient of NaCl from0to 0.5M.Pooled active fractions were dialyzed against1mM borate buffer(pH7.5)and loaded on a hydroxyapatite column previously equili-brated with the same buffer.Elution was carried out with a gradient elution of the same buffer from1mM to0.1M.Active fractions were consequently applied to a Benzamidine-Sepharose 6B column equilibrated with10mM borate buffer,pH8.MBSP was eluted with10mM borate buffer(pH8)containing0.5M NaCl. Active fractions from the?nal stage were used for enzymatic property analyses.
2.4.Assay of protein concentration and preparation of Pefabloc SC-treated myo?bril Protein concentration was determined by mea-suring the absorbance at280nm of the sample solution or with the methods of Lowry et al. (1951)and Smith et al.(1985)with bovine serum albumin as a standard.Pefabloc SC-treated my-o?bril was prepared from lizard?sh muscle ac-cording to Yanagihara et al.(1991)with minor modi?cations.
2.5.SDS-polyacrylamide gel electrophoresis and immunoblotting
SDS-PAGE was performed in10–20%gradient polyacrylamide gel according to the method of Laemmli(1970)and stained with silver staining or Coomassie Brilliant Blue R as indicated.Im-munoblotting detection of the degradation of a-actinin and actin by MBSP was carried out with the method described by Towbin et al.(1979)with
a slight modi?cation.
2.6.Effects of pH and temperature
The effect of pH on the activity of puri?ed MBSP was determined at50°C using Boc-Phe-Ser-Arg-MCA as a substrate and0.1M of the following buffers:sodium acetate buffer(pH4–6),sodium phosphate buffer(pH6–8),Tris–HCl buffer(pH8–9)and Na2CO3–borate buffer(pH 9–11).The effect of temperature on the enzyme activity was measured by using0.1M borate buffer(pH8)at different temperatures(30–65°C).
2.7.Degradation of peptides and analysis of the resulting peptide fragments
A50m l aliquot of puri?ed enzyme(0.25unit) was incubated with each peptide(10nmol)at
M.-J.Cao et al./Comparati6e Biochemistry and Physiology,Part B125(2000)255–264 258
37°C for2h in0.1ml of0.1M Tris–HCl buffer (pH8).The reaction mixture was then immedi-ately subjected to reverse phase HPLC on a m bondapak C18column(3.9×300mm,Waters). Elution was carried out with a linear gradient of acetonitrile(0–70%)containing0.1%tri?-uoroacetic acid for40min at a?ow rate of1.0 ml/min by monitoring the absorbance at215nm. The respective hydrolyzed peptide products from reverse phase HPLC were identi?ed by collecting corresponding fractions,and their sequences were analyzed using a Protein Sequencer(Applied Biosystems,model492)with chemicals and pro-gram supplied by the manufacturer.
2.8.Hydrolysis of myo?brillar proteins by MBSP at6arious temperatures
Myo?bril solution was prepared by dissolving Pefabloc SC treated myo?bril in0.1M phosphate buffer(pH7)containing0.5M NaCl.Reaction mixtures consisting of150m l myo?bril solution(2 mg protein)and50m l of puri?ed MBSP(0.25 unit)were incubated at the following tempera-tures for1h:40,50,55,60and70°C.A control test was performed by incubating the myo?bril solution at55°C for1h using water in place of MBSP.Reactions were terminated by adding1ml of20mM Tris–HCl buffer(pH8)containing5% SDS and8M urea.The mixtures were then boiled for5min in the presence of5%2-mercaptoethanol.
2.9.Determination of the N-terminal amino acid sequence of the enzyme
To determine the N-terminal amino acid se-quence,the puri?ed enzyme was applied to SDS-PAGE under reducing conditions,and the sample was successively electrophoretically transferred to a polyvinylidenedi?uoride(PVDF)membrane (Millipore,USA)and stained with Coomassie Brilliant Blue R.The protein band with a molecu-lar mass of29kDa was cut out and submitted to amino acid sequencing using a Protein Sequencer.
3.Results
To obtain the pure MBSP without contaminat-ing of sarcoplasmic serine proteinase,lizard?sh skeletal muscle was initially extensively washed until the serine proteinase activity in the soluble fraction could not be detected.Myo?bril-bound serine proteinase was then separated from my-o?brillar proteins with a heating treatment at 55°C,pH6for5min accompanied by the obvious denaturation of some myo?brillar proteins. MBSP was?nally puri?ed to homogeneity by a series of column chromatographies on DEAE-Sephacel,Sephacryl S-200,Q-Sepharose,Hydrox-yapatite and Benzamidine-Sepharose6B(Fig.1). The puri?cation results of the enzyme are sum-marized in Table 1.The speci?c activity was increased:180-fold with a1%recovery from the crude MBSP.On SDS-PAGE,the enzyme gave a single band corresponding to29kDa under reduc-ing conditions and a band of:60kDa under non-reducing conditions,suggesting that MBSP exists as a homodimer connected by disul?de-bond(s)(Fig.2).
The optimum pH and temperature of the en-zyme toward Boc-Phe-Ser-Arg-MCA were deter-mined(Fig.3).The enzyme showed optimum pH from7to8.Optimum temperature was50°C, which explained the latent ability of MBSP to cause the modori phenomenon.
Table1
Puri?cation result of MBSP from the skeletal muscle of lizard?sh
Puri?cation
Total protein Yield
Speci?c activity
Step Total activity
(fold)(%)
(units)
(mg)(units/mg)
1027365
Crude enzyme0.41100 DEAE-Sephacel3640.1 1.1 2.811
5.8
14.2 3.8
Sephacryl S-200 1.5
21.3
2.3
17.8
7.1
Q-Sepharose8.5
1.2
0.1 4.545113 1.2 Hydroxyapatite
B0.05 3.6721801 Benzamidine Sepharose
M.-J.Cao et al./Comparati6e Biochemistry and Physiology,Part B125(2000)255–264259
Fig.1.Chromatographic puri?cation of MBSP.(A)DEAE-Sephacel chromatography.The dialyzed crude MBSP was loaded on a DEAE-Sephacel column(2.6×45cm)equilibrated with20mM borate buffer,pH7.5.After washing to the absorbance at280nm reached the baseline,the column was eluted with a linear gradient of NaCl(0.05–0.5M)in the same buffer with a total volume of600 ml.(B)Q-Sepharose chromatography.Active fractions from Sephacryl S-200were dialysed against20mM borate buffer,pH7.5then applied to Q-Sepharose column(1×12cm).A linear gradient elution of NaCl(0–0.5M)in the same buffer was performed.(C) Benzamidine-Sepharose6B chromatography.The sample from Hydroxyapatite chromatography was applied to Benzamidine-Sepharose 6B column,active fractions were eluted with10mM borate buffer,pH8containing0.5M NaCl.
M .-J .Cao et al ./Comparati 6e Biochemistry and Physiology ,Part B 125(2000)255–264
260Fig.2.SDS-PAGE of puri?ed MBSP.The enzyme was elec-trophoresed on a 10–20%gradient gel under reducing (lane 2)and non-reducing conditions (lane 3)followed by silver stain-ing.The protein applied in lane 3was about one tenth of that in lane 2.Positions of molecular mass standards are shown in kDa;the arrow head indicates the protein band under non-re-ducing conditions.
lysine residue,revealing good agreement with the results of the hydrolysis of MCA-substrates.
Since the present enzyme is a MBSP,its degra-dation action on myo?brillar proteins was of much interest to us.As shown in Fig.4,it opti-cally hydrolyzed myosin heavy chain at 55–60°C,a temperature higher than that toward MCA-sub-strates (50°C).This may be due to the difference of substrates that affected the optimum tempera-ture of the enzyme.From the result of im-munoblotting,a -actinin and actin were not at all hydrolyzed by MBSP under the same reaction conditions (Fig.5).
Fig.3.Effects of (A)pH and (B)temperature on the activity of puri?ed MBSP.
Inhibition susceptibility studies revealed that the enzyme activity was strongly inhibited by serine proteinase inhibitors,such as Pefabloc SC,STI,aprotinin,benzamidine,leupeptin and an-tipain (Table 2).Though cysteine proteinase in-hibitors such as DTNB and pCMB also suppressed the activity to great degrees,it was not affected by E-64,a typical cysteine protease in-hibitor.Neither did pepstatin A and EDTA,clas-sical inhibitors of aspartic proteinase and metalloproteinase show obvious inhibitory effect on the enzyme.Calcium slightly activated the enzyme activity.
Substrate speci?city of the enzyme toward vari-ous MCA-substrates is shown in Table 3.MBSP speci?cally cleaved substrates with arginine residue at the P 1position,while those with lysine at the same position (Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA)were merely affected.Substrates for chymotrypsin-type serine proteinase (Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA)and aminopeptidase (Arg-MCA)were not at all hydrolyzed.These results indicate that the present enzyme is a trypsin-type serine proteinase but strictly arginine-speci?c.The action of MBSP toward several pep-tides containing monobasic and dibasic residues was also examined and the cleavage results are summarized in Table 4.Except a minor cleavage at the carboxyl site of the Arg-Arg pair in dynor-phin A 1–13,all cleavages speci?cally occurred after arginine residue of the bonds between two consecutive residues (Arg-Arg and Arg-Lys).No cleavage was identi?ed after the lysine residue,whether in the Lys-Lys,Lys-Arg pairs or a single
M .-J .Cao et al ./Comparati 6e Biochemistry and Physiology ,Part B 125(2000)255–264
261
Table 2
Effect of chemicals on the enzyme activity a Final concentration Chemicals
Relative activity (mM)
(%)None
1001
Pefabloc SC 50.1mg /ml STI
010m M Aprotinin 01Benzamidine 6TLCK 1251TPCK 33Leupeptin 0.10Antipain 0.101DTNB 130.1
PCMB 370.1mg /ml E-64
100Pepstatin A 0.1mg /ml 841EDTA 110CaCl 2
1
132
a
Puri?ed MBSP was preincubated with each reagent in 50mM borate buffer (pH 8.0)at room temperature for 20min.Remaining activity was determined by the addition of Boc-Phe-Ser-Arg-MCA to a ?nal concentration of 5m M and reacted at 50°C for 10min.Control tests were performed under identical conditions in the absence of chemicals.Table 4
Summary of cleavage sites of peptides by puri?ed MBSP a
a
The sites and extents of cleavage by puri?ed MBSP in each peptide are shown.A larger arrowhead indicates a major cleavage site,and a small arrowhead,a minor cleavage site.
Table 3
Substrate speci?city of puri?ed MBSP toward MCA-substrates Relative activity (%)Substrate (5m M)
100Boc-Phe-Ser-Arg-MCA Boc-Val-Pro-Arg-MCA 111106Boc-Gln-Gly-Arg-MCA Boc-Gln-Arg-Arg-MCA
10188Boc-Asp(OBzl)-Pro-Arg-MCA Boc-Leu-Lys-Arg-MCA 64Boc-Leu-Arg-Arg-MCA 49Boc-Gly-Lys-Arg-MCA 4947Boc-Leu-Ser-Thr-Arg-MCA 34Boc-Gly-Arg-Arg-MCA 32Boc-Ile-Glu-Gly-Arg-MCA Boc-Leu-Gly-Arg-MCA 2522Boc-Ala-Gly-Pro-Arg-MCA 19Boc-Arg-Val-Arg-Arg-MCA Boc-Glu-Lys-Lys-MCA 3Boc-Val-Leu-Lys-MCA 17Z-Phe-Arg-MCA Z-Arg-Arg-MCA 60Arg-MCA
Suc-Leu-Leu-Val-Tyr-MCA 00
Suc-Ala-Ala-Pro-Phe-MCA
Fig.4.Degradation of myo?brillar proteins by puri?ed MBSP at different temperatures.Pefablos SC-treated myo?bril,dis-solved in 0.1M phosphate buffer (pH 7)containing 0.5M NaCl was incubated with puri?ed MBSP at different tempera-tures for 1h.After incubation,20mM Tris–HCl buffer (pH 8)containing 5%SDS and 8M urea was added into the reaction mixtures and boiled for 5min.Samples (15m g proteins /lane)were applied to a 10–20%gradient gel followed by staining with Coomassie Brilliant Blue https://www.doczj.com/doc/8a16152716.html,ne 1,0h;2,40°C;3,50°C;4,55°C;5,60°C;6,70°C;7,55°C control (without MBSP).MHC,myosin heavy chain.
In order to know the structural information of MBSP,its primary structure was analyzed by sequencing with automated Edman degradation.Because merely a little amount of puri?ed enzyme
M .-J .Cao et al ./Comparati 6e Biochemistry and Physiology ,Part B 125(2000)255–264
262Fig.5.Immunoblotting of the degradation of (A)a -actinin and (B)actin.Myo?bril was ?rst incubated with puri?ed MBSP for 1h as described in Fig.4,and then applied to two 10–20%gradient gels.After electrophoresis,the gels were transferred to clear blot membrane-p and immunoblotted with anti-red sea bream a -actinin and anti-red sea bream actin antibodies,https://www.doczj.com/doc/8a16152716.html,ne 1,prestained marker;2,0h;3,40°C;4,50°C;5,55°C;6,60°C;7,70°C;8,55°C control (without MBSP).
search in the SwissProt and GenBank database also shows that MBSP is different from any known proteins or gene product sequences,suggesting MBSP is a novel serine proteinase.4.Discussion
In this study,a unique MBSP was dissociated from myo?bril by the heating treatment and was ?nally puri?ed to homogeneity.A heating treat-ment at 55°C for a short time accompanied by the denaturation of some myo?brillar proteins effec-tively solubilized the enzyme.To determine whether a similar enzyme also exists in the sar-coplasmic fraction,the supernatant of the muscle homogenate from the ?rst centrifugation step was treated with ammonium sulfate fractionation and applied to a DEAE-Sephacel column after dialysis against 20mM borate buffer (pH 7.5).The par-tially puri?ed sarcoplasmic enzyme (with much lower activity toward Boc-Phe-Ser-Arg-MCA)was compared with MBSP in both substrate speci?c-ities and inhibitor susceptibilities.The results,however,showed that they were different (data not shown).Any enzymatic activity similar to MBSP could not be detected by same heating treatment of the sarcoplasmic fraction.Therefore,it is obvious that a similar enzyme does not exist in the sar-coplasmic fraction.
The cleavage speci?city of the enzyme toward MCA-substrates revealed that only those with arginine residue at P 1were hydrolyzed,while sub-strates with lysine residue at the same site (Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA)were rarely cleaved.Among MCA-substrates tested,the enzyme revealed relatively high activity toward Boc-Val-Pro-Arg-MCA,Boc-Gln-Gly-Arg-MCA,Boc-Gln-Arg-Arg-MCA and Boc-Phe-Ser-Arg-MCA.When substrates with the amino acid residue changed at P 2and P 3were compared,the P 3residue was much important in determining the enzymatic cleavage ef?ciency (Table 3).The hydrolysis of peptides showed that the enzyme selectively cleaved at the arginine residue of the bonds between two consecutive residues (Arg-Ar-gand Arg-Lys)whereas Lys-Arg pair in lysyl-bradykinin,methionyl-lysyl-bradykinin and Lys-Lys pair in VIP were not at all affected which further exhibited its arginine-speci?c cleavage characteristic.This result corresponded well with those membrane-associated processing serine proteases puri?ed from yeast,rat liver and
was obtained in the puri?cation and the protein loss in the transfer from polyacrylamide gel to the PVDF membrane,only a short sequence of the ?rst 10residues (Ile-Val-Gly-Gly-Ala-Glu-Xaa-Val-Pro-Tyr-)was determined.Though the sequence was short,as it was in the conserved region-1of serine proteases,and showed high homology,it was not identical with those of other serine proteases (Mikes et al.,1966;Vu et al.,1997;Holt et al.,1998;Cao et al.,1999b)(Fig.6).A homology
https://www.doczj.com/doc/8a16152716.html,parison of the N -terminal amino acid sequence of MBSP with other serine proteases.Residues identical to MBSP are boxed.
M.-J.Cao et al./Comparati6e Biochemistry and Physiology,Part B125(2000)255–264263
porcine intestinal mucosa(Mizuno and Matsuo, 1984;Takahashi et al.,1991;Tamanoue et al.,1993; Tsuchiya et al.,1994).These proteases,however, were assumed to be involved in the limited endopro-teolysis of inactive precursor proteins at dibasic or multiple basic amino acids to produce biologically active peptides and proteins.On the other hand, advances in molecular biology have revealed that in organisms from yeast to mammals,a broad spectrum of biologically active peptides and proteins are produced by cleavage of inactive precursors,mainly at dibasic pairs by Kex2family serine proteases,including Furin,PC2,PC1/PC3, PC4,PACE4,PC5/PC6,LPC/PC7/PC8/SPC7. Nakayama(1997)and Steiner et al.(1992)have reviewed such enzymes in detail.MBSP shows similar cleavage selectivity to those membrane-asso-ciated serine proteases,therefore it is reasonable to presume that it physiologically acts as a processing enzyme.
The degradation of myo?brillar proteins by MBSP showed that myosin heavy chain was opti-cally hydrolyzed at around55–60°C.Its original band was obviously degraded in1h incubation, while other myo?brillar proteins such as a-actinin and actin were not affected as detected by im-munoblotting.Recently,a neutral serine protease (mekratin)was puri?ed from the skeletal muscle of myopathic hamster.Mekratin speci?cally cleaves myosin light chain2and causing idiopathic dilated cardiomyopathy(Holt et al.,1998).We have also reported the degradation action of a myo?bril-bound serine proteinase from carp muscle on myo?brillar proteins,which showed optical hydrol-ysis to myosin heavy chain at around55°C.a-Ac-tinin,actin,tropomyosin were also cleaved by the carp enzyme to some extent at the same temperature range(Cao et al.,1999a).The present enzyme, however,showed much more preference to myosin heavy chain than carp MBP.Different from mekratin,which did not show degradation action on myosin heavy chain as crude enzyme was tested, MBSPs from carp and lizard?sh optically cleaved it.Previous researches have indicated that the modori phenomenon is due to the breakdown of myosin heavy chain(the major constitute in my-o?bril and provides gel forming ability in the preparation of?sh jelly products)at:60°C(Toy-ohara et al.,1991;Osatomi et al.,1997;Cao et al., 1999a).The degradation action of MBSP on myosin heavy chain revealed its similarity to carp MBP and is most probably responsible for the modori effect.
It has been proposed by Kay et al.(1982a,b)that the initial stages of the degradation of myosin and other myo?brillar proteins take place outside the lysosomes in rat muscle by a trypsin-like serine proteinase.However,because of the low concentra-tion of such trypsin-like enzyme in rat muscle,its puri?cation was not successfully carried out. Though the tissue distributions of MBSP and carp MBP have not been investigated,a similar enzyme was not detected in the sarcoplasmic fraction which excluded the possible existence of such serine proteinases in lysosomes.Recent results have shown that the tissue location of a myo?bril-associated serine proteinase(mekratin)was within sarcomeres, where it could readily cleave the contractile proteins such as troponin T,troponin I,myosin light chain 2and titin(Levine et al.,1999).The carp MBP cleaved myosin heavy chain and other myo?brillar proteins such as a-actinin,actin and tropomyosin optically at neutral pH(Cao et al.,1999a)while MBSP showed more speci?city toward myosin heavy chain.These facts indicate the possibility that myo?bril-associated serine proteinases act as non-lysosomal enzymes and may involve in the initial stages of the degradation of myosin and other myo?brillar proteins.
Serine proteases hold relatively high sequence homology not only around the active site region but also in other conserved regions.Because of the low yield in the puri?cation process and the protein loss in the electrophoretical transfer operation,the N-terminal amino acid sequence of MBSP was only determined to the tenth amino acid residue.How-ever,it showed high homology but not identical with other serine proteases in the same region.Since MBSP has never been reported to be puri?ed from marine?sh,and its primary sequence and strict arginine-speci?c cleavage speci?cities both toward MCA-substrates and peptides differed it from the MBP puri?ed from carp muscle(Osatomi et al., 1997;Cao et al.,1999b),we come to the conclusion that MBSP is a novel serine proteinase.The com-plete primary sequence,tissue location and physio-logical functions of MBSP are of much interest in further study.
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从实践的角度探讨在日语教学中多媒体课件的应用 在今天中国的许多大学，为适应现代化，信息化的要求，建立了设备完善的适应多媒体教学的教室。许多学科的研究者及现场教员也积极致力于多媒体软件的开发和利用。在大学日语专业的教学工作中，教科书、磁带、粉笔为主流的传统教学方式差不多悄然向先进的教学手段而进展。 一、多媒体课件和精品课程的进展现状 然而，目前在专业日语教学中能够利用的教学软件并不多见。比如在中国大学日语的专业、第二外語用教科书常见的有《新编日语》（上海外语教育出版社）、《中日交流标准日本語》（初级、中级）（人民教育出版社）、《新编基础日语（初級、高級）》（上海译文出版社）、《大学日本语》（四川大学出版社）、《初级日语》《中级日语》（北京大学出版社）、《新世纪大学日语》（外语教学与研究出版社）、《综合日语》（北京大学出版社）、《新编日语教程》（华东理工大学出版社）《新编初级（中级）日本语》（吉林教育出版社）、《新大学日本语》（大连理工大学出版社）、《新大学日语》（高等教育出版社）、《现代日本语》（上海外语教育出版社）、《基础日语》（复旦大学出版社）等等。配套教材以录音磁带、教学参考、习题集为主。只有《中日交流標準日本語（初級上）》、《初級日语》、《新编日语教程》等少数教科书配备了多媒体DVD视听教材。 然而这些试听教材，有的内容为日语普及读物，并不适合专业外语课堂教学。比如《新版中日交流标准日本语（初级上）》，有的尽管DVD视听教材中有丰富的动画画面和语音练习。然而，课堂操作则花费时刻长，不利于教师重点指导，更加适合学生的课余练习。比如北京大学的《初级日语》等。在这种情形下，许多大学的日语专业致力于教材的自主开发。 其中，有些大学的还推出精品课程，取得了专门大成绩。比如天津外国语学院的《新编日语》多媒体精品课程为2007年被评为“国家级精品课”。目前已被南开大学外国语学院、成都理工大学日语系等全国40余所大学推广使用。
Unit1 Americans believe no one stands still. If you are not moving ahead, you are falling behind. This attitude results in a nation of people committed to researching, experimenting and exploring. Time is one of the two elements that Americans save carefully, the other being labor. "We are slaves to nothing but the clock,” it has been said. Time is treated as if it were something almost real. We budget it, save it, waste it, steal it, kill it, cut it, account for it; we also charge for it. It is a precious resource. Many people have a rather acute sense of the shortness of each lifetime. Once the sands have run out of a person’s hourglass, they cannot be replaced. We want every minute to count. A foreigner’s first impression of the U.S. is li kely to be that everyone is in a rush -- often under pressure. City people always appear to be hurrying to get where they are going, restlessly seeking attention in a store, or elbowing others as they try to complete their shopping. Racing through daytime meals is part of the pace
新视野三版读写B2U4Text A College sweethearts 1I smile at my two lovely daughters and they seem so much more mature than we,their parents,when we were college sweethearts.Linda,who's21,had a boyfriend in her freshman year she thought she would marry,but they're not together anymore.Melissa,who's19,hasn't had a steady boyfriend yet.My daughters wonder when they will meet"The One",their great love.They think their father and I had a classic fairy-tale romance heading for marriage from the outset.Perhaps,they're right but it didn't seem so at the time.In a way, love just happens when you least expect it.Who would have thought that Butch and I would end up getting married to each other?He became my boyfriend because of my shallow agenda:I wanted a cute boyfriend! 2We met through my college roommate at the university cafeteria.That fateful night,I was merely curious,but for him I think it was love at first sight."You have beautiful eyes",he said as he gazed at my face.He kept staring at me all night long.I really wasn't that interested for two reasons.First,he looked like he was a really wild boy,maybe even dangerous.Second,although he was very cute,he seemed a little weird. 3Riding on his bicycle,he'd ride past my dorm as if"by accident"and pretend to be surprised to see me.I liked the attention but was cautious about his wild,dynamic personality.He had a charming way with words which would charm any girl.Fear came over me when I started to fall in love.His exciting"bad boy image"was just too tempting to resist.What was it that attracted me?I always had an excellent reputation.My concentration was solely on my studies to get superior grades.But for what?College is supposed to be a time of great learning and also some fun.I had nearly achieved a great education,and graduation was just one semester away.But I hadn't had any fun;my life was stale with no component of fun!I needed a boyfriend.Not just any boyfriend.He had to be cute.My goal that semester became: Be ambitious and grab the cutest boyfriend I can find. 4I worried what he'd think of me.True,we lived in a time when a dramatic shift in sexual attitudes was taking place,but I was a traditional girl who wasn't ready for the new ways that seemed common on campus.Butch looked superb!I was not immune to his personality,but I was scared.The night when he announced to the world that I was his girlfriend,I went along
Unit 1 1学习外语是我一生中最艰苦也是最有意义的经历之一。虽然时常遭遇挫折，但却非常有价值。 2我学外语的经历始于初中的第一堂英语课。老师很慈祥耐心，时常表扬学生。由于这种积极的教学方法，我踊跃回答各种问题，从不怕答错。两年中，我的成绩一直名列前茅。 3到了高中后，我渴望继续学习英语。然而，高中时的经历与以前大不相同。以前，老师对所有的学生都很耐心，而新老师则总是惩罚答错的学生。每当有谁回答错了，她就会用长教鞭指着我们，上下挥舞大喊：“错！错！错！”没有多久，我便不再渴望回答问题了。我不仅失去了回答问题的乐趣，而且根本就不想再用英语说半个字。 4好在这种情况没持续多久。到了大学，我了解到所有学生必须上英语课。与高中老师不。大学英语老师非常耐心和蔼，而且从来不带教鞭！不过情况却远不尽如人意。由于班大，每堂课能轮到我回答的问题寥寥无几。上了几周课后，我还发现许多同学的英语说得比我要好得多。我开始产生一种畏惧感。虽然原因与高中时不同，但我却又一次不敢开口了。看来我的英语水平要永远停步不前了。 5直到几年后我有机会参加远程英语课程，情况才有所改善。这种课程的媒介是一台电脑、一条电话线和一个调制解调器。我很快配齐了必要的设备并跟一个朋友学会了电脑操作技术，于是我每周用5到7天在网上的虚拟课堂里学习英语。 6网上学习并不比普通的课堂学习容易。它需要花许多的时间，需要学习者专心自律，以跟上课程进度。我尽力达到课程的最低要求，并按时完成作业。 7我随时随地都在学习。不管去哪里，我都随身携带一本袖珍字典和笔记本，笔记本上记着我遇到的生词。我学习中出过许多错，有时是令人尴尬的错误。有时我会因挫折而哭泣，有时甚至想放弃。但我从未因别的同学英语说得比我快而感到畏惧，因为在电脑屏幕上作出回答之前，我可以根据自己的需要花时间去琢磨自己的想法。突然有一天我发现自己什么都懂了，更重要的是，我说起英语来灵活自如。尽管我还是常常出错，还有很多东西要学，但我已尝到了刻苦学习的甜头。 8学习外语对我来说是非常艰辛的经历，但它又无比珍贵。它不仅使我懂得了艰苦努力的意义，而且让我了解了不同的文化，让我以一种全新的思维去看待事物。学习一门外语最令人兴奋的收获是我能与更多的人交流。与人交谈是我最喜欢的一项活动，新的语言使我能与陌生人交往，参与他们的谈话，并建立新的难以忘怀的友谊。由于我已能说英语，别人讲英语时我不再茫然不解了。我能够参与其中，并结交朋友。我能与人交流，并能够弥合我所说的语言和所处的文化与他们的语言和文化之间的鸿沟。 III. 1. rewarding 2. communicate 3. access 4. embarrassing 5. positive 6. commitment 7. virtual 8. benefits 9. minimum 10. opportunities IV. 1. up 2. into 3. from 4. with 5. to 6. up 7. of 8. in 9. for 10.with V. 1.G 2.B 3.E 4.I 5.H 6.K 7.M 8.O 9.F 10.C Sentence Structure VI. 1. Universities in the east are better equipped, while those in the west are relatively poor. 2. Allan Clark kept talking the price up, while Wilkinson kept knocking it down. 3. The husband spent all his money drinking, while his wife saved all hers for the family. 4. Some guests spoke pleasantly and behaved politely, while others wee insulting and impolite. 5. Outwardly Sara was friendly towards all those concerned, while inwardly she was angry. VII. 1. Not only did Mr. Smith learn the Chinese language, but he also bridged the gap between his culture and ours. 2. Not only did we learn the technology through the online course, but we also learned to communicate with friends in English. 3. Not only did we lose all our money, but we also came close to losing our lives.
新大学日语简明教程课文翻译 第21课 一、我的留学生活 我从去年12月开始学习日语。已经3个月了。每天大约学30个新单词。每天学15个左右的新汉字，但总记不住。假名已经基本记住了。 简单的会话还可以，但较难的还说不了。还不能用日语发表自己的意见。既不能很好地回答老师的提问，也看不懂日语的文章。短小、简单的信写得了，但长的信写不了。 来日本不久就迎来了新年。新年时，日本的少女们穿着美丽的和服，看上去就像新娘。非常冷的时候，还是有女孩子穿着裙子和袜子走在大街上。 我在日本的第一个新年过得很愉快，因此很开心。 现在学习忙，没什么时间玩，但周末常常运动，或骑车去公园玩。有时也邀朋友一起去。虽然我有国际驾照，但没钱，买不起车。没办法，需要的时候就向朋友借车。有几个朋友愿意借车给我。 二、一个房间变成三个 从前一直认为睡在褥子上的是日本人，美国人都睡床铺，可是听说近来纽约等大都市的年轻人不睡床铺，而是睡在褥子上，是不是突然讨厌起床铺了？ 日本人自古以来就睡在褥子上，那自有它的原因。人们都说日本人的房子小，从前，很少有人在自己的房间，一家人住在一个小房间里是常有的是，今天仍然有人过着这样的生活。 在仅有的一个房间哩，如果要摆下全家人的床铺，就不能在那里吃饭了。这一点，褥子很方便。早晨，不需要褥子的时候，可以收起来。在没有了褥子的房间放上桌子，当作饭厅吃早饭。来客人的话，就在那里喝茶；孩子放学回到家里，那房间就成了书房。而后，傍晚又成为饭厅。然后收起桌子，铺上褥子，又成为了全家人睡觉的地方。 如果是床铺的话，除了睡觉的房间，还需要吃饭的房间和书房等，但如果使用褥子，一个房间就可以有各种用途。 据说从前，在纽约等大都市的大学学习的学生也租得起很大的房间。但现在房租太贵，租不起了。只能住更便宜、更小的房间。因此，似乎开始使用睡觉时作床，白天折小能成为椅子的、方便的褥子。
新视野大学英语第一册Unit 1课文翻译 学习外语是我一生中最艰苦也是最有意义的经历之一。 虽然时常遭遇挫折，但却非常有价值。 我学外语的经历始于初中的第一堂英语课。 老师很慈祥耐心，时常表扬学生。 由于这种积极的教学方法，我踊跃回答各种问题，从不怕答错。 两年中，我的成绩一直名列前茅。 到了高中后，我渴望继续学习英语。然而，高中时的经历与以前大不相同。 以前，老师对所有的学生都很耐心，而新老师则总是惩罚答错的学生。 每当有谁回答错了，她就会用长教鞭指着我们，上下挥舞大喊：“错！错！错！” 没有多久，我便不再渴望回答问题了。 我不仅失去了回答问题的乐趣，而且根本就不想再用英语说半个字。 好在这种情况没持续多久。 到了大学，我了解到所有学生必须上英语课。 与高中老师不同，大学英语老师非常耐心和蔼，而且从来不带教鞭！ 不过情况却远不尽如人意。 由于班大，每堂课能轮到我回答的问题寥寥无几。 上了几周课后，我还发现许多同学的英语说得比我要好得多。 我开始产生一种畏惧感。 虽然原因与高中时不同，但我却又一次不敢开口了。 看来我的英语水平要永远停步不前了。 直到几年后我有机会参加远程英语课程，情况才有所改善。 这种课程的媒介是一台电脑、一条电话线和一个调制解调器。 我很快配齐了必要的设备并跟一个朋友学会了电脑操作技术，于是我每周用5到7天在网上的虚拟课堂里学习英语。 网上学习并不比普通的课堂学习容易。 它需要花许多的时间，需要学习者专心自律，以跟上课程进度。 我尽力达到课程的最低要求，并按时完成作业。 我随时随地都在学习。 不管去哪里，我都随身携带一本袖珍字典和笔记本，笔记本上记着我遇到的生词。 我学习中出过许多错，有时是令人尴尬的错误。 有时我会因挫折而哭泣，有时甚至想放弃。 但我从未因别的同学英语说得比我快而感到畏惧，因为在电脑屏幕上作出回答之前，我可以根据自己的需要花时间去琢磨自己的想法。 突然有一天我发现自己什么都懂了，更重要的是，我说起英语来灵活自如。 尽管我还是常常出错，还有很多东西要学，但我已尝到了刻苦学习的甜头。 学习外语对我来说是非常艰辛的经历，但它又无比珍贵。 它不仅使我懂得了艰苦努力的意义，而且让我了解了不同的文化，让我以一种全新的思维去看待事物。 学习一门外语最令人兴奋的收获是我能与更多的人交流。 与人交谈是我最喜欢的一项活动，新的语言使我能与陌生人交往，参与他们的谈话，并建立新的难以忘怀的友谊。 由于我已能说英语，别人讲英语时我不再茫然不解了。 我能够参与其中，并结交朋友。
习惯与礼仪 我是个漫画家，对旁人细微的动作、不起眼的举止等抱有好奇。所以，我在国外只要做错一点什么，立刻会比旁人更为敏锐地感觉到那个国家的人们对此作出的反应。 譬如我多次看到过，欧美人和中国人见到我们日本人吸溜吸溜地出声喝汤而面露厌恶之色。过去，日本人坐在塌塌米上，在一张低矮的食案上用餐，餐具离嘴较远。所以，养成了把碗端至嘴边吸食的习惯。喝羹匙里的东西也象吸似的，声声作响。这并非哪一方文化高或低，只是各国的习惯、礼仪不同而已。 日本人坐在椅子上围桌用餐是1960年之后的事情。当时，还没有礼仪规矩，甚至有人盘着腿吃饭。外国人看见此景大概会一脸厌恶吧。 韩国女性就座时，单腿翘起。我认为这种姿势很美，但习惯于双膝跪坐的日本女性大概不以为然，而韩国女性恐怕也不认为跪坐为好。 日本等多数亚洲国家，常有人习惯在路上蹲着。欧美人会联想起狗排便的姿势而一脸厌恶。 日本人常常把手放在小孩的头上说“好可爱啊！”，而大部分外国人会不愿意。 如果向回教国家的人们劝食猪肉和酒，或用左手握手、递东西，会不受欢迎的。当然，饭菜也用右手抓着吃。只有从公用大盘往自己的小盘里分食用的公勺是用左手拿。一旦搞错，用黏糊糊的右手去拿，
会遭人厌恶。 在欧美，对不受欢迎的客人不说“请脱下外套”，所以电视剧中的侦探哥隆波总是穿着外套。访问日本家庭时，要在门厅外脱掉外套后进屋。穿到屋里会不受欢迎的。 这些习惯只要了解就不会出问题，如果因为不知道而遭厌恶、憎恨，实在心里难受。 过去，我曾用色彩图画和简短的文字画了一本《关键时刻的礼仪》（新潮文库）。如今越发希望用各国语言翻译这本书。以便能对在日本的外国人有所帮助。同时希望有朝一日以漫画的形式画一本“世界各国的习惯与礼仪”。 练习答案 ５、 （１）止める並んでいる見ているなる着色した （２）拾った入っていた行ったしまった始まっていた
新视野大学英语第三版第二册读写课文翻译 Unit 1 Text A 一堂难忘的英语课 1 如果我是唯一一个还在纠正小孩英语的家长，那么我儿子也许是对的。对他而言，我是一个乏味的怪物：一个他不得不听其教诲的父亲，一个还沉湎于语法规则的人，对此我儿子似乎颇为反感。 2 我觉得我是在最近偶遇我以前的一位学生时，才开始对这个问题认真起来的。这个学生刚从欧洲旅游回来。我满怀着诚挚期待问她：“欧洲之行如何？” 3 她点了三四下头，绞尽脑汁，苦苦寻找恰当的词语，然后惊呼：“真是，哇！” 4 没了。所有希腊文明和罗马建筑的辉煌居然囊括于一个浓缩的、不完整的语句之中！我的学生以“哇！”来表示她的惊叹，我只能以摇头表达比之更强烈的忧虑。 5 关于正确使用英语能力下降的问题，有许多不同的故事。学生的确本应该能够区分诸如their/there/they're之间的不同，或区别complimentary 跟complementary之间显而易见的差异。由于这些知识缺陷，他们承受着大部分不该承受的批评和指责，因为舆论认为他们应该学得更好。 6 学生并不笨，他们只是被周围所看到和听到的语言误导了。举例来说，杂货店的指示牌会把他们引向stationary（静止处），虽然便笺本、相册、和笔记本等真正的stationery（文具用品）并没有被钉在那儿。朋友和亲人常宣称They've just ate。实际上，他们应该说They've just eaten。因此，批评学生不合乎情理。 7 对这种缺乏语言功底而引起的负面指责应归咎于我们的学校。学校应对英语熟练程度制定出更高的标准。可相反，学校只教零星的语法，高级词汇更是少之又少。还有就是，学校的年轻教师显然缺乏这些重要的语言结构方面的知识，因为他们过去也没接触过。学校有责任教会年轻人进行有效的语言沟通，可他们并没把语言的基本框架——准确的语法和恰当的词汇——充分地传授给学生。
新视野大学英语1课文翻译 1下午好！作为校长，我非常自豪地欢迎你们来到这所大学。你们所取得的成就是你们自己多年努力的结果，也是你们的父母和老师们多年努力的结果。在这所大学里，我们承诺将使你们学有所成。 2在欢迎你们到来的这一刻，我想起自己高中毕业时的情景，还有妈妈为我和爸爸拍的合影。妈妈吩咐我们：“姿势自然点。”“等一等,”爸爸说，“把我递给他闹钟的情景拍下来。”在大学期间，那个闹钟每天早晨叫醒我。至今它还放在我办公室的桌子上。 3让我来告诉你们一些你们未必预料得到的事情。你们将会怀念以前的生活习惯，怀念父母曾经提醒你们要刻苦学习、取得佳绩。你们可能因为高中生活终于结束而喜极而泣，你们的父母也可能因为终于不用再给你们洗衣服而喜极而泣！但是要记住：未来是建立在过去扎实的基础上的。 4对你们而言，接下来的四年将会是无与伦比的一段时光。在这里，你们拥有丰富的资源：有来自全国各地的有趣的学生，有学识渊博又充满爱心的老师，有综合性图书馆，有完备的运动设施，还有针对不同兴趣的学生社团——从文科社团到理科社团、到社区服务等等。你们将自由地探索、学习新科目。你们要学着习惯点灯熬油，学着结交充满魅力的人，学着去追求新的爱好。我想鼓励你们充分利用这一特殊的经历，并用你们的干劲和热情去收获这一机会所带来的丰硕成果。 5有这么多课程可供选择，你可能会不知所措。你不可能选修所有的课程，但是要尽可能体验更多的课程！大学里有很多事情可做可学，每件事情都会为你提供不同视角来审视世界。如果我只能给你们一条选课建议的话，那就是：挑战自己！不要认为你早就了解自己对什么样的领域最感兴趣。选择一些你从未接触过的领域的课程。这样，你不仅会变得更加博学，而且更有可能发现一个你未曾想到的、能成就你未来的爱好。一个绝佳的例子就是时装设计师王薇薇，她最初学的是艺术史。随着时间的推移，王薇薇把艺术史研究和对时装的热爱结合起来，并将其转化为对设计的热情，从而使她成为全球闻名的设计师。 6在大学里，一下子拥有这么多新鲜体验可能不会总是令人愉快的。在你的宿舍楼里，住在你隔壁寝室的同学可能会反复播放同一首歌，令你头痛欲裂！你可能喜欢早起，而你的室友却是个夜猫子！尽管如此，你和你的室友仍然可能成
新视野大学英语2课文翻译（Unit1-Unit7） Unit 1 Section A 时间观念强的美国人 Para. 1 美国人认为没有人能停止不前。如果你不求进取，你就会落伍。这种态度造就了一个投身于研究、实验和探索的民族。时间是美国人注意节约的两个要素之一，另一个是劳力。 Para. 2 人们一直说：“只有时间才能支配我们。”人们似乎是把时间当作一个差不多是实实在在的东西来对待的。我们安排时间、节约时间、浪费时间、挤抢时间、消磨时间、缩减时间、对时间的利用作出解释；我们还要因付出时间而收取费用。时间是一种宝贵的资源，许多人都深感人生的短暂。时光一去不复返。我们应当让每一分钟都过得有意义。 Para. 3 外国人对美国的第一印象很可能是：每个人都匆匆忙忙——常常处于压力之下。城里人看上去总是在匆匆地赶往他们要去的地方，在商店里他们焦躁不安地指望店员能马上来为他们服务，或者为了赶快买完东西，用肘来推搡他人。白天吃饭时人们也都匆匆忙忙，这部分地反映出这个国家的生活节奏。工作时间被认为是宝贵的。Para. 3b 在公共用餐场所，人们都等着别人吃完后用餐，以便按时赶回去工作。你还会发现司机开车很鲁莽，人们推搡着在你身边过去。你会怀念微笑、简短的交谈以及与陌生人的随意闲聊。不要觉得这是针对你个人的，这是因为人们非常珍惜时间，而且也不喜欢他人“浪费”时间到不恰当的地步。 Para. 4 许多刚到美国的人会怀念诸如商务拜访等场合开始时的寒暄。他们也会怀念那种一边喝茶或咖啡一边进行的礼节性交流，这也许是他们自己国家的一种习俗。他们也许还会怀念在饭店或咖啡馆里谈生意时的那种轻松悠闲的交谈。一般说来，美国人是不会在如此轻松的环境里通过长时间的闲聊来评价他们的客人的，更不用说会在增进相互间信任的过程中带他们出去吃饭，或带他们去打高尔夫球。既然我们通常是通过工作而不是社交来评估和了解他人，我们就开门见山地谈正事。因此，时间老是在我们心中的耳朵里滴滴答答地响着。 Para. 5 因此，我们千方百计地节约时间。我们发明了一系列节省劳力的装置；我们通过发传真、打电话或发电子邮件与他人迅速地进行交流，而不是通过直接接触。虽然面对面接触令人愉快，但却要花更多的时间, 尤其是在马路上交通拥挤的时候。因此，我们把大多数个人拜访安排在下班以后的时间里或周末的社交聚会上。 Para. 6 就我们而言，电子交流的缺乏人情味与我们手头上事情的重要性之间很少有或完全没有关系。在有些国家, 如果没有目光接触，就做不成大生意，这需要面对面的交谈。在美国，最后协议通常也需要本人签字。然而现在人们越来越多地在电视屏幕上见面，开远程会议不仅能解决本国的问题，而且还能通过卫星解决国际问题。
新视野大学英语3第三版课文翻译 Unit 1 The Way to Success 课文A Never, ever give up! 永不言弃！ As a young boy, Britain's great Prime Minister, Sir Winston Churchill, attended a public school called Harrow. He was not a good student, and had he not been from a famous family, he probably would have been removed from the school for deviating from the rules. Thankfully, he did finish at Harrow and his errors there did not preclude him from going on to the university. He eventually had a premier army career whereby he was later elected prime minister. He achieved fame for his wit, wisdom, civic duty, and abundant courage in his refusal to surrender during the miserable dark days of World War II. His amazing determination helped motivate his entire nation and was an inspiration worldwide. Toward the end of his period as prime minister, he was invited to address the patriotic young boys at his old school, Harrow. The headmaster said, "Young gentlemen, the greatest speaker of our time, will be here in a few days to address you, and you should obey whatever sound advice he may give you." The great day arrived. Sir Winston stood up, all five feet, five inches and 107 kilos of him, and gave this short, clear-cut speech: "Young men, never give up. Never give up! Never give up! Never, never, never, never!" 英国的伟大首相温斯顿·丘吉尔爵士，小时候在哈罗公学上学。当时他可不是个好学生，要不是出身名门，他可能早就因为违反纪律被开除了。谢天谢地，他总算从哈罗毕业了，在那里犯下的错误并没影响到他上大学。后来，他凭着军旅生涯中的杰出表现当选为英国首相。他的才思、智慧、公民责任感以及在二战痛苦而黑暗的时期拒绝投降的无畏勇气，为他赢得了美名。他非凡的决心，不仅激励了整个民族，还鼓舞了全世界。 在他首相任期即将结束时，他应邀前往母校哈罗公学，为满怀报国之志的同学们作演讲。校长说：“年轻的先生们，当代最伟大的演说家过几天就会来为你们演讲，他提出的任何中肯的建议，你们都要听从。”那个激动人心的日子终于到了。温斯顿爵士站了起来——他只有5 英尺5 英寸高，体重却有107 公斤。他作了言简意赅的讲话：“年轻人，要永不放弃。永不放弃！永不放弃！永不，永不，永不，永不！” Personal history, educational opportunity, individual dilemmas - none of these can inhibit a strong spirit committed to success. No task is too hard. No amount of preparation is too long or too difficult. Take the example of two of the most scholarly scientists of our age, Albert Einstein and Thomas Edison. Both faced immense obstacles and extreme criticism. Both were called "slow to learn" and written off as idiots by their teachers. Thomas Edison ran away from school because his teacher whipped him repeatedly for asking too many questions. Einstein didn't speak fluently until he was almost nine years old and was such a poor student that some thought he was unable to learn. Yet both boys' parents believed in them. They worked intensely each day with their sons, and the boys learned to never bypass the long hours of hard work that they needed to succeed. In the end, both Einstein and Edison overcame their childhood persecution and went on to achieve magnificent discoveries that benefit the entire world today. Consider also the heroic example of Abraham Lincoln, who faced substantial hardships, failures and repeated misfortunes in his lifetime. His background was certainly not glamorous. He was raised in a very poor family with only one year of formal education. He failed in business twice, suffered a nervous breakdown when his first love died suddenly and lost eight political
Text A 课文 A The humanities: Out of date? 人文学科：过时了吗？ When the going gets tough, the tough takeaccounting. When the job market worsens, manystudents calculate they can't major in English orhistory. They have to study something that booststheir prospects of landing a job. 当形势变得困难时，强者会去选学会计。当就业市场恶化时，许多学生估算着他们不能再主修英语或历史。他们得学一些能改善他们就业前景的东西。 The data show that as students have increasingly shouldered the ever-rising c ost of tuition,they have defected from the study of the humanities and toward applied science and "hard"skills that they bet will lead to employment. In oth er words, a college education is more andmore seen as a means for economic betterment rather than a means for human betterment.This is a trend that i s likely to persist and even accelerate. 数据显示，随着学生肩负的学费不断增加，他们已从学习人文学科转向他们相信有益于将来就业的应用科学和“硬”技能。换言之，大学教育越来越被看成是改善经济而不是提升人类自身的手段。这种趋势可能会持续，甚至有加快之势。 Over the next few years, as labor markets struggle, the humanities will proba bly continue theirlong slide in succession. There already has been a nearly 50 percent decline in the portion of liberal arts majors over the past generatio n, and it is logical to think that the trend is boundto continue or even accel erate. Once the dominant pillars of university life, the humanities nowplay li ttle roles when students take their college tours. These days, labs are more vi vid and compelling than libraries. 在未来几年内，由于劳动力市场的不景气，人文学科可能会继续其长期低迷的态势。在上一代大学生中，主修文科的学生数跌幅已近50%。这种趋势会持续、甚至加速的想法是合情合理的。人文学科曾是大学生活的重要支柱，而今在学生们参观校园的时候，却只是一个小点缀。现在，实验室要比图书馆更栩栩如生、受人青睐。 Here, please allow me to stand up for and promote the true value that the h umanities add topeople's lives. 在这儿，请允许我为人文学科给人们的生活所增添的真实价值进行支持和宣传。
第10课 日本的季节 日本的一年有春、夏、秋、冬四个季节。 3月、4月和5月这三个月是春季。春季是个暖和的好季节。桃花、樱花等花儿开得很美。人们在4月去赏花。 6月到8月是夏季。夏季非常闷热。人们去北海道旅游。7月和8月是暑假，年轻人去海边或山上。也有很多人去攀登富士山。富士山是日本最高的山。 9月、10月和11月这3个月是秋季。秋季很凉爽，晴朗的日子较多。苹果、桔子等许多水果在这个季节成熟。 12月到2月是冬季。日本的南部冬天不太冷。北部非常冷，下很多雪。去年冬天东京也很冷。今年大概不会那么冷吧。如果冷的话，人们就使用暖气炉。 第12课 乡下 我爷爷住哎乡下。今天，我要去爷爷家。早上天很阴，但中午天空开始变亮，天转好了。我急急忙忙吃完午饭，坐上了电车。 现在，电车正行驶在原野上。窗外，水田、旱地连成一片。汽车在公路上奔驰。 这时，电车正行驶在大桥上。下面河水在流动。河水很清澈，可以清澈地看见河底。可以看见鱼在游动。远处，一个小孩在挥手。他身旁，牛、马在吃草。 到了爷爷居住的村子。爷爷和奶奶来到门口等着我。爷爷的房子是旧房子，但是很大。登上二楼，大海就在眼前。海岸上，很多人正在全力拉缆绳。渐渐地可以看见网了。网里有很多鱼。和城市不同，乡下的大自然真是很美。 第13课 暑假 大概没有什么比暑假更令学生感到高兴的了。大学在7月初，其他学校在二十四日左右进入暑假。暑假大约1个半月。 很多人利用这个假期去海边、山上，或者去旅行。学生中，也有人去打工。学生由于路费等只要半价，所以在学期间去各地旅行。因此，临近暑假时，去北海道的列车上就挤满了这样的人。从炎热的地方逃避到凉爽的地方去，这是很自然的事。一般在1月、最迟在2月底之前就要预定旅馆。不然的话可能会没有地方住。 暑假里，山上、海边、湖里、河里会出现死人的事，这种事故都是由于不注意引起的。大概只能每个人自己多加注意了。 在东京附近，镰仓等地的海面不起浪，因此挤满了游泳的人。也有人家只在夏季把海边的房子租下来。 暑假里，学校的老师给学生布置作业，但是有的学生叫哥哥或姐姐帮忙。 第14课 各式各样的学生 我就读的大学都有各种各样的学生入学。学生有的是中国人，有的是美国人，有的是英国人。既有年轻的，也有不年轻的。有胖的学生，也有瘦的学生。学生大多边工作边学习。因此，大家看上去都很忙。经常有人边听课边打盹。 我为了学习日本先进的科学技术和日本文化来到日本。预定在这所大学学习3年。既然特意来了日本，所以每天都很努力学习。即便如此，考试之前还是很紧张。其他学生也是这
教育界的科技革命 如果让生活在年的人来到我们这个时代，他会辨认出我们当前课堂里发生的许多事情——那盛行的讲座、对操练的强调、从基础读本到每周的拼写测试在内的教学材料和教学活动。可能除了教堂以外，很少有机构像主管下一代正规教育的学校那样缺乏变化了。 让我们把上述一贯性与校园外孩子们的经历作一番比较吧。在现代社会，孩子们有机会接触广泛的媒体，而在早些年代这些媒体简直就是奇迹。来自过去的参观者一眼就能辨认出现在的课堂，但很难适应现今一个岁孩子的校外世界。 学校——如果不是一般意义上的教育界——天生是保守的机构。我会在很大程度上为这种保守的趋势辩护。但变化在我们的世界中是如此迅速而明确，学校不可能维持现状或仅仅做一些表面的改善而生存下去。的确，如果学校不迅速、彻底地变革，就有可能被其他较灵活的机构取代。 计算机的变革力 当今时代最重要的科技事件要数计算机的崛起。计算机已渗透到我们生活的诸多方面，从交通、电讯到娱乐等等。许多学校当然不能漠视这种趋势，于是也配备了计算机和网络。在某种程度上，这些科技辅助设施已被吸纳到校园生活中，尽管他们往往只是用一种更方便、更有效的模式教授旧课程。 然而，未来将以计算机为基础组织教学。计算机将在一定程度上允许针对个人的授课，这种授课形式以往只向有钱人提供。所有的学生都会得到符合自身需要的、适合自己学习方法和进度的课程设置，以及对先前所学材料、课程的成绩记录。 毫不夸张地说，计算机科技可将世界上所有的信息置于人们的指尖。这既是幸事又是灾难。我们再也无须花费很长时间查找某个出处或某个人——现在，信息的传递是瞬时的。不久，我们甚至无须键入指令，只需大声提出问题，计算机就会打印或说出答案，这样，人们就可实现即时的"文化脱盲"。 美中不足的是，因特网没有质量控制手段；"任何人都可以拨弄"。信息和虚假信息往往混杂在一起，现在还没有将网上十分普遍的被歪曲的事实和一派胡言与真实含义区分开来的可靠手段。要识别出真的、美的、好的信息，并挑出其中那些值得知晓的, 这对人们构成巨大的挑战。 对此也许有人会说，这个世界一直充斥着错误的信息。的确如此，但以前教育当局至少能选择他们中意的课本。而今天的形势则是每个人都拥有瞬时可得的数以百万计的信息源，这种情况是史无前例的。 教育的客户化 与以往的趋势不同，从授权机构获取证书可能会变得不再重要。每个人都能在模拟的环境中自学并展示个人才能。如果一个人能像早些时候那样"读法律"，然后通过计算机模拟的实践考试展现自己的全部法律技能，为什么还要花万美元去上法学院呢？用类似的方法学开飞机或学做外科手术不同样可行吗？ 在过去，大部分教育基本是职业性的：目的是确保个人在其年富力强的整个成人阶段能可靠地从事某项工作。现在，这种设想有了缺陷。很少有人会一生只从事一种职业；许多人都会频繁地从一个职位、公司或经济部门跳到另一个。 在经济中，这些新的、迅速变换的角色的激增使教育变得大为复杂。大部分老成持重的教师和家长对帮助青年一代应对这个会经常变换工作的世界缺乏经验。由于没有先例，青少年们只有自己为快速变化的"事业之路"和生活状况作准备。