The International Journal of Biochemistry &Cell Biology 33(2001)865–876
Ligand speci?city and conformational stability of human
fatty acid-binding proteins
Aukje W.Zimmerman *,Herman T.B.van Moerkerk,Jacques H.Veerkamp
Department of Biochemistry ,Uni 6ersity Medical Center Nijmegen ,P .O .Box 9101,6500HB Nijmegen ,The Netherlands
Received 2March 2001;accepted 4May 2001
Abstract
Fatty acid binding proteins (FABPs)are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids.At least eight different types of FABP occur,each with a speci?c tissue distribution and possibly with a distinct function.To de?ne the functional characteristics of all eight human FABPs,viz.heart (H),brain (B),myelin (M),adipocyte (A),epidermal (E),intestinal (I),liver (L)and ileal lipid-binding protein (I-LBP),we studied their ligand speci?city,their conformational stability and their immunological crossreactivity.Additionally,binding of bile acids to I-LBP was studied.The FABP types showed differences in fatty acid binding af?nity.Generally,the af?nity for palmitic acid was lower than for oleic and arachidonic acid.All FABP types,except E-FABP,I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sul-phonic acid (ANS).Only L-FABP,I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sul-fonyl)amino)undecanoic acid (DAUDA).I-LBP showed increasing binding of bile acids in the order taurine-conjugated \glycine-conjugated \unconjugated bile acids.A hydroxylgroup of bile acids at position 7decreased and at position 12increased the binding af?nity to I-LBP.The fatty acid-binding af?nity and the conformation of FABP types were differentially affected in the presence of urea.Our results demonstrate signi?cant differences in ligand binding,conformational stability and surface properties between different FABP types which may point to a speci?c function in certain cells and tissues.The preference of I-LBP (but not L-FABP)for conjugated bile acids is in accordance with a speci?c role in bile acid reabsorption in the ileum.?2001Elsevier Science Ltd.All rights reserved.
Keywords :Brain FABP;Epidermal FABP;Fluorescence;Immunological cross-reactivity;Ileal lipid-binding protein;Bile acid
https://www.doczj.com/doc/5b12177236.html, /locate /ijbcb
Abbre 6iations :ANS,1-anilinonaphtalene-8-sulfonic acid;DAUDA,(11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)unde-canoic acid;FABP,fatty acid-binding protein;A-FABP,adipocyte FABP;B-FABP,brain FABP;E-FABP,epidermal FABP;H-FABP,heart FABP;I-FABP,intestinal FABP;I-LBP,ileal lipid-binding protein;L-FABP,liver FABP;M-FABP,myelin FABP.*Corresponding author.Tel.:+31-24-361-4303;fax:+31-24-354-0339.E -mail address :a.zimmerman@bioch.kun.nl (A.W.Zimmerman).1357-2725/01/$-see front matter ?2001Elsevier Science Ltd.All rights reserved.PII:S 1357-2725(01)00070-X
A.W.Zimmerman et al./The International Journal of Biochemistry&Cell Biology33(2001)865–876 866
1.Introduction
Fatty acid-binding proteins(FABPs)are mem-bers of a family of14–15kDa cytosolic proteins. Nine different FABP types,named after the?rst tissue of isolation,have been identi?ed up to now [reviewed in[1,2]].Some types(L-FABP,H-FABP)occur in more than one tissue whereas others(I-FABP,A-FABP,M-FABP,B-FABP) are limited to only one tissue.Some organs con-tain more FABP types,located either in different cell types(brain,kidney,ovary,stomach),or in the same cell(enterocyte).FABPs show22–73% similarity of amino acid sequence and have a similar clam shell-like structure,comprising of ten antiparallel b-strands and two nearly parallel a-helices.The conformation of the bound fatty acid differs among the different FABP types[3,4].
All members of the FABP family,except ileal lipid-binding protein(I-LBP),have a high af?nity for fatty acids.However,differences in ligand speci?city have been observed.I-FABP and H-FABP are speci?c binders of fatty acids,whereas L-FABP and I-LBP bind more bulky,hydropho-bic ligands as lysophospholipids,bile acids,ei-cosanoids,and some drugs[2,5–7].L-FABP has a unique stoichiometry since it can bind two fatty acids simultaneously[4].
Although the FABP structure has been studied extensively,the speci?c physiological functions of FABPs are still unknown.A role in fatty acid uptake and transfer has been proposed,but also modulation of fatty acid concentration and regu-lation of various cellular systems and processes (signal transduction,gene transcription)have been indicated[1,2].Fatty acid binding character-istics and protein stability are important parame-ters in this respect.Several studies have been performed on fatty acid binding to different FABP types[reviewed in[1,2]].However,direct comparison of binding parameters is dif?cult due to different techniques used.Simultaneous studies on all FABP types using the same procedure are scarce.Only Richieri et al.[8]studied the fatty acid binding characteristics of four FABP types with the ADIFAB procedure,and we reported some preliminary data for?ve FABP types[9].
Cholic acid and chenodeoxycholic acid are secreted by the liver into the bile as N-acyl conju-gates of glycine or taurine and mainly reabsorbed in the terminal ileum.Bile acids bind to I-LBP and L-FABP[10,11].In L-FABP and I-LBP,the four residue gap in the region between b-strands G and H may result in a wider opening which may enable the binding of bile acids[12,13].The greater?exibility of the backbone may allow a bile acid to enter the I-LBP binding cavity[13]. The binding of some bile acids to I-LBP was investigated by NMR[10],titration calorimetry [14]and gel?ltration[15],but quantitative and comparative data are lacking.
We performed comparative studies on ligand binding,conformational characteristics and sur-face properties of eight human FABP types,to provide more insight in their speci?c localization and possible functions.We describe the fatty acid-binding characteristics of H-FABP,L-FABP,I-FABP,A-FABP,M-FABP,E-FABP,B-FABP and I-LBP.Additionally,interactions with the ?uorescent probes(11-((5-dimethylaminonaphtal-ene-1-sulfonyl)amino)undecanoic acid(DAUDA) and1-anilinonaphtalene-8-sulfonic acid(ANS) were studied.The bile acid binding to I-LBP was investigated for unconjugated and glyco-and tau-roconjugated deoxycholic,chenodeoxycholic and cholic acid by?uorescence.We studied the stabil-ity of the binding centre of each FABP type on basis of its fatty acid binding activity in the presence of denaturant.Their conformational sta-bility was determined on basis of tryptophan ?uorescence.Finally,surface properties of the FABP types were examined by measuring their reactivity with antibodies raised against the differ-ent types in rabbit.
2.Materials and methods
2.1.Materials
All human FABP preparations were obtained by recombinant cDNA technology as described [9,16,17].Human brain(B-)FABP cDNA in pBluescript was a kind gift of Dr F.Shimidzu (Otsuka GEN Research Institute,Tokushima,
A.W.Zimmerman et al./The International Journal of Biochemistry&Cell Biology33(2001)865–876867
Japan).B-FABP cDNA was cloned into the pET3d expression vector via Bam HI and Nco I restriction sites.Extraction from inclusion bodies and puri?cation were performed as described for H-FABP mutants[18],except that the anion ex-change step was omitted.Human I-LBP cDNA in the pET3d vector was kindly provided by Pro-fessor J.C.Sacchettini(Texas A&M,University College Station,TX).Expression and puri?cation of human I-LBP was performed as described for H-FABP[17].Human epidermal(E-)FABP preparations were a kind gift from Professor F. Spener(University of Mu¨nster,Germany).
1-14C-labeled preparations of palmitic acid, oleic acid,arachidonic acid,and[1-14C]glyco-cholic acid and[carboxyl-14C]chenodeoxycholic acid were obtained from Amersham Pharmacia Biotech;unlabeled bile acids from Sigma(St. Louis,MO);1,8-ANS and DAUDA from Molec-ular Probes,Leiden,The Netherlands.
All protein concentrations are given in m g/ml, based on the Lowry procedure with bovine serum albumin as a standard.Standardization to molar concentrations was not applied,since except for H-FABP and L-FABP correction factors are not known.
2.2.Binding assays
Prior to binding assays,FABP preparations were delipidated using the Lipidex procedure[19]. Fatty acid-binding assays were performed at 37°C in0.5ml10mM Tris–HCl(pH8.0),using 10m g FABP and0-3m M1-14C-labeled oleic acid, palmitic acid or arachidonic acid,respectively.K d and B max values were determined by Scatchard analysis.At least three independent assays were performed in triplicate.
Emission spectra and?uorescence enhancement of1m M DAUDA or ANS were measured in1 ml10mM Tris–HCl(pH8.0)/1%ethanol after addition of90m g FABP.Excitation wavelengths used were335and369nm for DAUDA and ANS,respectively.Assays were carried out at 25°C(3.0nm slits)with a Shimadzu RF-5301 PC spectro?uorophotometer.2.3.Bile acid binding assay
Binding of nine different bile acids and bile acid conjugates,viz.(tauro-or glyco-)cholic acid, (tauro-or glyco-)deoxycholic acid,and(tauro-or glyco-)chenodeoxycholic acid to I-LBP was de-termined by?uorescence spectroscopy.Increasing concentrations(0–8mM)of the different bile acids were added stepwise to15m g I-LBP in20 mM K2HPO4/0.05%NaN3,pH8.0.Emission spectra were recorded from310to360nm,at an excitation wavelength of280nm,with 3.0nm slits.Fluorescence intensity at maximal wave-length was plotted against the bile acid concen-tration to obtain binding curves.Curve?tting was used to determine the bile acid concentration giving half-maximal?uorescence.
2.4.Protein denaturation studies
In order to determine the stability of the fatty acid-binding center,fatty acid binding was mea-sured in the presence of various urea concentra-tions at0.8nmol[1-14C]oleic acid and10m g protein in400m l10mM Tris/HCl,pH8.0using the Lipidex procedure.We used urea stock solu-tions of9.6and11.2M(pre-warmed at37°C)to obtain urea concentrations of0–7M.The mix-ture was prepared and incubated as described previously[20].Curve?tting was used to deter-mine the concentration of urea giving half the initial binding activity(midpoint).
Equilibrium unfolding as a function of urea concentration was monitored by?uorescence spectroscopy.The tryptophan?uorescence was measured by the shift of the maximum emission wavelength at an excitation wavelength of283 nm(1.5nm slits).Emission spectra were recorded from300to400nm.For L-FABP,tyrosine ?uorescence was measured at an excitation wave-length of278nm(3.0nm slits),and emission spectra were recorded from290to315nm.Mix-tures of different urea concentrations,10mM Tris/HCl,pH8.0and15m g/ml protein were incubated for15min at37°C prior to?uores-cence measurements.All values were corrected for the?uorescence of the same mixture of urea/ Tris–HCl(pH8.0)without protein.The emission
A.W.Zimmerman et al./The International Journal of Biochemistry&Cell Biology33(2001)865–876 868
wavelength yielding maximum?uorescence was plotted against the urea concentration for each preparation to visualize the unfolding pro?le. Curve?tting was used to determine[D]50,the concentration of denaturant at the midpoint of the transition.
2.5.Cross-reacti6ity of anti-FABP antisera Antisera against all FABP types were raised in rabbits as described previously[21].Cross-reactiv-ities of FABP types with the antisera were deter-mined in ELISA with2ng protein coated per well (6wells for each FABP type).Monoclonal perox-idase-conjugated goat-anti-rabbit IgG(g-chain speci?c)was used as a secondary antibody,and the reaction was visualized using ortho-phenylene diamine.Antisera were applied at a concentration giving an extinction value of1.0–1.2at492nm with its own FABP type.Cross-reactivity was de?ned as the reciprocal of the serum dilution required to half maximal absorbance and ex-pressed as percentage of the value obtained with the speci?c antigen-antiserum combination[21].
3.Results and discussion
3.1.Binding assays
First we studied the binding for various fatty acids with the Lipidex assay.We observed differ-ences in fatty acid binding af?nities between the various types of FABP.Generally,the af?nity for palmitic acid was lower than for oleic and arachi-donic acid(Table1).B-FABP shows,like H-FABP,M-FABP and I-FABP,a higher af?nity for both oleic acid and palmitic acid than L-FABP and A-FABP.Arachidonic acid is bound with high af?nity by B-FABP,comparable to H-FABP,M-FABP and L-FABP,whereas I-FABP,A-FABP and E-FABP show higher K d values.Maximal binding(B max)values were gener-ally20-30nmol/mg protein except for A-FABP (10nmol/mg).Binding data on some FABP types agree with previous results on human and rat FABPs[9,16,21,22].In a competition assay with ANS,Simpson et al.[23],found2–3-fold lower af?nity of oleic and palmitic acid for murine E-FABP than for A-FABP.In contrast,we found that human E-FABP binds these fatty acids and arachidonic acid with higher af?nity than A-FABP(Table1).With the Lipidex procedure, human E-FABP was shown to bind stearic acid with high af?nity,but oleic acid and arachidonic acid with lower af?nity[24].Bovine E-FABP showed high af?nity for palmitic acid[25].Our data are generally in agreement with these?nd-ings,although our K d value of E-FABP for oleic acid is about2-fold lower.Murine B-FABP showed very high af?nity for docosahexaenoic acid but no af?nity for palmitic acid[26].In contrast,human B-FABP shows high af?nity for palmitic acid as well as for oleic and arachidonic acid(Table1).These data does not support the idea that speci?c binding properties of B-FABP are necessary for the realization of the characteris-tic fatty acid composition of brain tissue. Although the Lipidex assay gives rise to higher dissociation constants than the acrylodated intes-tinal fatty acid binding protein(ADIFAB)proce-dure[8],due to possible disturbance of the equilibrium after Lipidex addition,it is still a useful comparative method.Recently,Richieri et
Table1
K d values of human FABP types
Arachidonic FABP type Oleic acid
Palmitic acid
acid
Heart0.9690.050.3790.02
0.4490.05
0.3890.020.1790.06
0.3790.05
Brain
Myelin0.3790.02
0.3190.03
0.6290.16
2.8690.59
Adipocyte 1.5690.25 2.4990.22 Epidermal 1.0490.160.8290.02 1.1790.07 Intestinal0.6090.08 1.4990.16
0.5790.04
Liver0.8990.030.4490.04
4.0290.11
K d values(in m M)were determined from Scatchard plots with 0–3m M[1-14C]fatty acid and10m g protein by the Lipidex procedure.Values are means9S.D.of at least three indepen-dent experiments.I-LBP did not show signi?cant binding to any fatty acid used.Values for H-FABP,M-FABP,A-FABP, I-FABP and L-FABP are derived from[9],and given for comparison.
A .W .Zimmerman et al ./The International Journal of Biochemistry &Cell Biology 33(2001)865–876
869
Table 2
ANS and DAUDA binding to different FABP types ANS
FABP type
DAUDA
Fluorescence Maximal emission wavelength Maximal emission wavelength Fluorescence (arbitrary units)(nm)
(nm)(arbitrary units)461Heart 36.296.6536 1.190.8Brain 47141.695.6539 1.290.625.694.5517472 6.693.3Myelin 467Adipocyte 44.797.3531 1.890.51.690.4530Epidermal 3.090.2466 5.691.049847431.899.0Intestinal 471Liver 30.891.850536.497.7Ileal
–
536
0.490.6
10m g of FABP was incubated with 1m M ligand in 1ml 10mM Tris /HCl,pH 8.0/0.1%ethanol.Fluorescence is directly related to ligand binding and is linear up to 30m g protein [9].Fluorescence values (in arbitrary units)at maximal emission wavelength were corrected for the values with only ligand at the corresponding wavelength,and are shown as means 9S.D.for three independent experiments.
al.[27]showed with the ADIFAB procedure that no FABP type revealed a high degree of selectivity for a particular fatty acid.Af ?nities of all FABPs decrease with decreasing chain length and increas-ing double bond number.Binding af ?nities varied with FABP type according to brain $myelin $heart \liver \intestine \adipocyte [27].Our present data are generally in agreement with these ?ndings.Both the fatty acid-protein interaction and the aqueous solubility of fatty acids appear to play a marked role in determining the fatty acid binding af ?nity [27].In a recent study [22]we showed that all FABP types except I-LBP in-creased FA transfer from immobilized liposomes to mitochondria to a similar extent.
We were not able to detect any fatty acid binding to I-LBP using the Lipidex procedure.In earlier studies,binding of oleate and palmitate to I-LBP was found by 13C NMR spectroscopy [10]and titration calorimetry [14].I-LBP was shown to bind oleate and palmitate in a 1:1molar ratio [10],but the af ?nities for these fatty acids were much lower with rat I-LBP than with rat L-FABP [15].Titration calorimetry revealed a K d value of 36m M for oleate [14].
DAUDA was used as a ligand to study L-FABP [16,28].We previously showed that I-FABP also binds this ?uorescent ligand well,whereas H-FABP,M-FABP and A-FABP do not [9].We now report that neither B-FABP,E-FABP nor I-LBP have af ?nity for DAUDA (Table 2).Binding of DAUDA to L-FABP and I-FABP caused a marked shift in the maximum ?uores-cence emission wavelength from 533to 505and to 498nm,respectively,and an increase in ?uores-cence intensity.A shift and ?uorescence enhance-ment were not or hardly observed for the other FABP types.Remarkably,I-LBP did not bind DAUDA,although it closely resembles L-FABP on basis of its structure and its binding of bile acids.
ANS is a commonly used probe employed to observe the character of different regions of glob-ular and membrane proteins.It has been shown to be a competitive inhibitor for binding of fatty acids [23,29].Like fatty acids,it binds in the internal cavity although in a different orientation as shown for A-FABP [30].Previously we ob-served ANS binding to human H-FABP,M-FABP,A-FABP and L-FABP,but marginally to I-FABP [9].B-FABP also binds ANS,whereas E-FABP and I-LBP do not (Table 2).Maximal ?uorescence with this probe was reached at wave-lengths of 461to 474nm.ANS showed a K d value of 16m M to rat I-FABP and of 0.41and 0.53m M to murine A-FABP and E-FABP,respectively [29,31,32].In contrast to murine E-FABP [29],
A.W.Zimmerman et al./The International Journal of Biochemistry&Cell Biology33(2001)865–876 870
two human E-FABP preparations we used did not bind ANS.We found however very low B max values of these E-FABP preparations with fatty acids(about5nmol/mg protein in contrast to20 nmol/mg for the E-FABP preparation used in the Lipidex assay).
Taken together,these data and previously re-ported results[27]indicate that the different bind-ing af?nities of FABP types may re?ect tissue-speci?c differences in fatty acid metabolism and function.The highly conserved af?nities dis-played by FABPs of the same type but different species support such a relationship[27].Differ-ences in fatty acid af?nity could in?uence the intracellular free fatty acid levels.For example, the occurrence of FABP types with lower af?nity such as A-FABP and E-FABP could cause higher intracellular fatty acid levels resulting in fatty acid export.In contrast,FABP types with high af?nity like H-FABP and B-FABP might be able to con-centrate fatty acids from the serum into the cell. Interactions with(signalling)molecules as perox-isomal proliferator-activated receptors may be de-pendent on FABP type and independent of fatty acid binding[33].
3.2.Bile acid binding
We compared the binding of nine different conjugated and non-conjugated bile acids to hu-man I-LBP,since their binding to L-FABP previ-ously showed marked differences[7,11].Other FABP types were suggested not to bind bile acids. Previously,chenodeoxycholic acid binding to porcine I-LBP was shown using13C NMR spec-troscopy[10,13].Binding of glycocholate and gly-cochenodeoxycholate to rat I-LBP was determined by titration calorimetry[14].Since ANS and DAUDA did not bind to I-LBP,dis-placement studies with bile acids as for L-FABP [7,11]were impossible.We did not observe bind-ing of chenodeoxycholic acid or glycocholic acid to I-LBP at gel?ltration with14C-labeled bile acids.Therefore,we studied the effect of bile acids on tryptophan?uorescence of I-LBP as a measure for binding.A comparable procedure was used to establish binding af?nities of retinol and related compounds to retinol-binding protein[34].H-FABP is known not to bind bile acids.Consis-tently,this FABP type did not show a change in its intrinsic?uorescence intensity after addition of various concentrations of different bile acids(re-sults not shown).Fig.1shows the binding of the main bile acids to I-LBP.Conjugation appears to markedly increase the binding af?nity which is somewhat higher for taurine-conjugated than for glycine-conjugated bile acids.Consistent with our results,af?nity of human I-LBP with tauro-cholate,but not with chenodeoxycholate,was found by gel?ltration[15].Azotaurocholate label-ing of rat I-LBP was inhibited by bile acids in the order taurocholate\taurochenodeoxycholate\ chenodeoxycholate\cholate,whereas oleic acid had no effect[35].Preferential binding of conju-gated bile acids to I-LBP seems to be important for reabsorption,since in the bowel mainly conju-gated bile acids are present,either or not modi?ed by bacterial enzymes[36].
The af?nity for I-LBP is increasing in the order deoxycholic acid\cholic acid\chenodeoxy-cholic acid(Table3).If a hydroxyl group at the 12position is absent,as is the case with chenodeoxycholic acid and its derivatives,binding to I-LBP decreases markedly as is indicated by the relatively low?uorescence intensity and the high concentration of chenodeoxycholic acid necessary for half-maximal?uorescence intensity(Table3). Titration calorimetry experiments showed also better binding of glycocholic acid than of gly-cochenodeoxycholic acid[14].Absence of the hy-droxyl group at the7position(in deoxycholic acids and its conjugates)generally resulted in a lower concentration necessary for half-maximal ?uorescence,indicating stronger binding(Table 3).Data suggests an important role for the7a-and12a-hydroxyl groups of bile acids in binding to I-LBP.The7-a hydroxyl group decreases af?nity,and the12-hydroxyl group apparently promotes binding,possibly by its proposed inter-action with Tyr-97[37].
Two-dimensional NMR spectroscopy indicated a different orientation of chenodeoxycholate com-pared to fatty acid in the binding cavity of porcine I-LBP[13].The carboxyl group is located near the proposed entry portal while the steroid ring moiety is penetrating deeper inside.A recent
A .W .Zimmerman et al ./The International Journal of Biochemistry &Cell Biology 33(2001)865–876871
study of the solution structure of I-LBP in com-plex with glycocholate shows mainly hydrophobi-cally driven interactions between the protein and the ligand [37].
Since L-FABP contains no tryptophan residues,it was not included in our ?uorescence study on bile acid binding.Cholate,chenodeoxycholate and deoxycholate bound with low af ?nity to rat L-FABP according to a DAUDA displacement assay [7].Lithocholate and taurolithocholate showed the highest binding af ?nity [7].Displace-ment of ANS from L-FABP showed that both rat and human L-FABP bind monohydroxy bile acids with higher af ?nity than trihydroxy bile acids [11].Both assays showed a lower af ?nity of bile acids to L-FABP after conjugation [7,11]in contrast to I-LBP (Table 3).Many similarities and a high ?exibility exist in the structure of L-FABP and I-LBP,but several important differ-ences may explain the distinctions in their interac-tion with the ligands [36].Differential binding of DAUDA (previous paragraph),conjugated and unconjugated bile acids and fatty acids to L-FABP and I-LBP implies distinct roles for both proteins,which is supported by their different distribution in the intestine [10].The signi ?cance of these proteins in the intracellular transport of bile acids remains to be investigated [38].
Fig.1.Binding of bile acids to ileal lipid-binding protein.Bile acid binding was studied with 0–1mM bile acid and 15m g I-LBP in 1ml 20mM phosphate buffer,pH 8.0.Nine bile acids were used:cholic acids (A),deoxycholic acids (B)and chenodeoxycholic acids (C),either unconjugated ( )or conjugated with taurine ( )or glycine ( ).Fluorescence emission spectra were recorded from 310to 360nm,at 280nm excitation wavelength.Titration curves represent maximal ?uorescence intensities at each bile acid concentration and were determined by a curve ?tting program.Typical curves out of 2–3experiments are shown.
A .W .Zimmerman et al ./The International Journal of Biochemistry &Cell Biology 33(2001)865–876
872Table 3
Binding of bile acids to ileal lipid-binding protein Concentration at Bile acid type
Maximal half-maximal
?uorescence intensity
?uorescence (m M)
(arbitrary units)Cholic acid*39.899.522096349.191.16092Taurocholic acid Glycocholic acid 1299150.290.345.494.5Deoxycholic acid 129914291Taurodeoxycholic 53.992.6acid
7791469.491.7Glycodeoxycholic acid*
402950Chenodeoxycholic 18.893.5acid*
Taurochenodeoxy-8391829.991.6cholic acid
Glycochenodeoxy-13094
28.191.1
cholic acid
Bile acid concentrations at half-maximal ?uorescence were derived from titration curves,using 0–1mM bile acid and 15m g I-LBP in 1ml 20mM phosphate buffer,pH 8.0(Fig.1).Fluorescence emission spectra were recorded from 310to 360nm,at 280nm excitation wavelength.Values are means of two (range)or three*(9S.D.)experiments.
conditions (Fig.2).Oleic acid binding to H-FABP and I-FABP is less sensitive to urea than for other FABP types.The binding centre of E-FABP shows the lowest stability,which agrees with the low B max value of the E-FABP prepara-tions.
The conformational stability of the different FABP types was evaluated by examination of the ?uorescence spectra of the proteins at different urea concentrations.Basically,the intrinsic ?uorescence of FABPs arises from two conserved Trp residues that are located outside the fatty acid-binding center.Exceptions are I-LBP which contains only one Trp residue at position 49and L-FABP which does not contain Trp residues.We found marked differences in intrinsic ?uores-cence and in conformational stability among the examined FABP types (Table 4).Treatment of H-FABP with urea resulted in a red shift of the ?uorescence emission maximum from 331to 351nm,and the unfolding transition occurs in the range 5–7M (Fig.3),which is in agreement with previous data [18,20].Data suggest a single co-operative transition between folded and unfolded forms [20],but unfolding intermediates can only be excluded by NMR.The transition pro ?les of A-FABP and I-FABP are comparable to H-FABP (Fig.3).However,the transition curves of M-FABP and E-FABP and,to less extent,B-FABP and I-LBP are shifted to the left.Their midpoints of transition are markedly lower than
3.3.Effect of urea denaturation on the fatty acid binding centre and conformational stability
The sensitivity of the fatty acid-binding centre differs among FABP types under denaturating
Fig.2.Effect of urea denaturation on the oleic acid binding activity of different FABP types.Oleic acid binding was determined with the Lipidex procedure at 0.8nmol oleic acid and 10m g protein in 0.4ml Tris /HCl,pH 8.0in the presence of 0–7M urea.Binding activity is given relative to the initial binding activity of each FABP type.Experiments were carried out in triplicate.Typical curves (out of 3–4experiments)for each FABP type are represented.
A .W .Zimmerman et al ./The International Journal of Biochemistry &Cell Biology 33(2001)865–876
873
Table 4
Stability of binding activity and conformation of different FABP types Midpoint of oleic acid binding FABP type
Intrinsic ?uorescence at 0M urea [D ]50(M urea)
(arbitrary units)(M urea)Heart 1004.3690.25 5.9590.16Brain 3.0190.631129 4.0790.2160932.7790.18 3.0090.07Myelin 2.3890.60Adipocyte 145923 5.3690.032.0890.65Epidermal 135912 2.5790.102749553.3890.16 5.2090.02Intestinal Liver n.d.
2.3790.15 1.8590.3185911
3.7890.17
n.d.
Ileal Oleic acid binding was studied with 0.8nmol [1-14C]oleic acid and 10m g protein in 400m l 10mM Tris –HCl (pH 8.0)at 0–7M urea.The midpoint is the concentration of urea giving 50%of the maximal binding.Equilibrium unfolding was measured by the shift of maximum emission wavelength of tryptophan ?uorescence at an excitation wavelength of 283nm (for L-FABP tyrosine ?uorescence was measured at 307nm)in 0–8M urea for 15m g protein in 1ml 10mM Tris /HCl,pH 8.0.[D ]50is the urea concentration at which 50%of the protein is unfolded.Values are means 9S.D.for at least three independent experiments.Data are derived from curves,as given in Figs.2and 3,respectively.n.d.,not determined (no fatty acid binding to I-LBP,no tryptophan ?uorescence of L-FABP).
Fig.3.Shift in the tryptophan emission maximum of different FABP types.The Trp ?uorescence of proteins was measured at 15m g in 1ml 10mM Tris /HCl,pH 8.0,and at an excitation wavelength of 283nm.Emission spectra were recorded from 300to 400nm.Results represent the means 9S.D.for at least three independent experiments.
those for H-FABP,I-FABP and A-FABP (Table 4).In spite of the presence of a disul ?de bridge,E-FABP has the lowest stability,which is in agreement with previous ?ndings [24]and our other results.The stability of L-FABP was mea-sured by monitoring the increase in tyrosine ?uorescence.Maximal ?uorescence (307nm)indi-cating complete denaturation was obtained at 4M urea.The midpoint was determined at 1.85M urea.
Comparison of the midpoints shows that the binding af ?nity is more sensitive than the confor-mation (Table 4).Only M-FABP and E-FABP show a similar sensitivity of binding and confor-
mation.H-FABP and all its mutant proteins showed also a higher sensitivity of their binding centre to urea [18,20].L-FABP shows a lower conformational stability,but this was determined by a different method.The differences in confor-mational stability may re ?ect a variable turnover in the cellular environment.
3.4.Immunological cross -reacti 6ity of anti -FABP antisera
Surface residues of several FABP types were suggested to play an important role in the transfer rate and transfer mechanisms of anthroyloxy-la-
A.W.Zimmerman et al./The International Journal of Biochemistry&Cell Biology33(2001)865–876
874
Table5
Cross-reactivity of FABP types with different anti-FABP antisera
FABP type Antiserum type
Anti-B
Anti-H Anti-M Anti-A Anti-E Anti-I Anti-L Anti-IL
8097493291
Heart591
1001911900
100190491691
Brain0
11951900
010*******
893190
Myelin1900 893
Adipocyte5891286921003921901900 993
Epidermal0391129410001910
2900190392
994100
Intestinal190190 Liver159804911903921911000
0190191190
593190
Ileal191100
Cross-reactivities were determined in ELISA with2ng protein coated per well(6wells for each FABP type).A peroxidase-conju-gated goat-anti-rabbit IgG(g-chain speci?c)was used as secondary antibody.For each FABP type,values were normalized to their own antiserum.Values(in%)are means9S.D.for at least three independent experiments.
beled fatty acids(AOFA)to membranes[39,40]. Especially lysine residues on H-FABP and A-FABP appeared to in?uence the rate and mecha-nism of AOFA transfer.Previously,the electrostatical and hydrophobic topology of the surfaces of A-FABP,M-FABP,H-FABP,I-FABP and L-FABP were studied[41]. Immunological cross-reactivity may serve as an-other indicator for surface properties of the hu-man FABP types.We now compare eight FABP types instead of?ve[9]and applied2ng FABP in each well instead of the previously used amount of400ng to increase accuracy.We used poly-clonal antibodies since they are directed against more epitopic regions than monoclonals.Remark-ably,all FABP types,except H-FABP and A-FABP showed only signi?cant reactivity with their own antiserum(Table5).There is some cross-reactivity of anti H-FABP antiserum with all the other types.H-FABP showed a marked cross-reactivity with anti B-FABP,although B-FABP did not strongly cross-react with anti H-FABP.Besides with its own antiserum,A-FABP strongly interacted with anti M-FABP and anti B-FABP.These?ndings suggest that H-FABP shares epitopes with B-FABP,and that A-FABP shares epitopes with M-FABP and B-FABP, which can be explained by their similarity on the amino acid level.LiCata and Bernlohr[41]re-ported similarity of hydrophobic residues on the surface of A-FABP,M-FABP and H-FABP,whereas I-FABP and L-FABP were unique.With respect to electrostatic topology,all these FABP types were unique[41].In agreement with these data we found cross-reactivity of A-FABP with anti M-FABP(but not vice versa).The surface properties of B-FABP have not been examined yet.Thus,immunological data suggest hetero-geneity at the outer surface of FABP types,al-though some types share epitopes.These?ndings are in agreement with the primary sequence of these parts of the proteins. Acknowledgements
The authors thank Ms S.Huang and Dr C. Lu¨cke(Institut fu¨r Biophysikalische Chemie,J.W. Goethe Universita¨t,Frankfurt a.M.,Germany) for information on the bile acid-binding assay and valuable communication.
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如何写先进个人事迹 篇一：如何写先进事迹材料 如何写先进事迹材料 一般有两种情况：一是先进个人，如先进工作者、优秀党员、劳动模范等；一是先进集体或先进单位，如先进党支部、先进车间或科室，抗洪抢险先进集体等。无论是先进个人还是先进集体，他们的先进事迹，内容各不相同，因此要整理材料，不可能固定一个模式。一般来说，可大体从以下方面进行整理。 (1)要拟定恰当的标题。先进事迹材料的标题，有两部分内容必不可少，一是要写明先进个人姓名和先进集体的名称，使人一眼便看出是哪个人或哪个集体、哪个单位的先进事迹。二是要概括标明先进事迹的主要内容或材料的用途。例如《王鬃同志端正党风的先进事迹》、《关于评选张鬃同志为全国新长征突击手的材料》、《关于评选鬃处党支部为省直机关先进党支部的材料》等。 (2)正文。正文的开头，要写明先进个人的简要情况，包括：姓名、性别、年龄、工作单位、职务、是否党团员等。此外，还要写明有关单位准备授予他(她)什么荣誉称号，或给予哪种形式的奖励。对先进集体、先进单位，要根据其先进事迹的主要内容，寥寥数语即应写明，不须用更多的文字。 然后，要写先进人物或先进集体的主要事迹。这部分内容是全篇材料
的主体，要下功夫写好，关键是要写得既具体，又不繁琐；既概括，又不抽象；既生动形象，又很实在。总之，就是要写得很有说服力，让人一看便可得出够得上先进的结论。比如，写一位端正党风先进人物的事迹材料，就应当着重写这位同志在发扬党的优良传统和作风方面都有哪些突出的先进事迹，在同不正之风作斗争中有哪些突出的表现。又如，写一位搞改革的先进人物的事迹材料，就应当着力写这位同志是从哪些方面进行改革的，已经取得了哪些突出的成果，特别是改革前后的．经济效益或社会效益都有了哪些明显的变化。在写这些先进事迹时，无论是先进个人还是先进集体的，都应选取那些具有代表性的具体事实来说明。必要时还可运用一些数字，以增强先进事迹材料的说服力。 为了使先进事迹的内容眉目清晰、更加条理化，在文字表述上还可分成若干自然段来写，特别是对那些涉及较多方面的先进事迹材料，采取这种写法尤为必要。如果将各方面内容材料都混在一起，是不易写明的。在分段写时，最好在每段之前根据内容标出小标题，或以明确的观点加以概括，使标题或观点与内容浑然一体。 最后，是先进事迹材料的署名。一般说，整理先进个人和先进集体的材料，都是以本级组织或上级组织的名义；是代表组织意见的。因此，材料整理完后，应经有关领导同志审定，以相应一级组织正式署名上报。这类材料不宜以个人名义署名。 写作典型经验材料-般包括以下几部分： (1)标题。有多种写法，通常是把典型经验高度集中地概括出来，一
How to manage time Time treats everyone fairly that we all have 24 hours per day. Some of us are capable to make good use of time while some find it hard to do so. Knowing how to manage them is essential in our life. Take myself as an example. When I was still a senior high student, I was fully occupied with my studies. Therefore, I hardly had spare time to have fun or develop my hobbies. But things were changed after I entered university. I got more free time than ever before. But ironically, I found it difficult to adjust this kind of brand-new school life and there was no such thing called time management on my mind. It was not until the second year that I realized I had wasted my whole year doing nothing. I could have taken up a Spanish course. I could have read ten books about the stories of successful people. I could have applied for a part-time job to earn some working experiences. B ut I didn’t spend my time on any of them. I felt guilty whenever I looked back to the moments that I just sat around doing nothing. It’s said that better late than never. At least I had the consciousness that I should stop wasting my time. Making up my mind is the first step for me to learn to manage my time. Next, I wrote a timetable, setting some targets that I had to finish each day. For instance, on Monday, I must read two pieces of news and review all the lessons that I have learnt on that day. By the way, the daily plan that I made was flexible. If there’s something unexpected that I had to finish first, I would reduce the time for resting or delay my target to the next day. Also, I would try to achieve those targets ahead of time that I planed so that I could reserve some more time to relax or do something out of my plan. At the beginning, it’s kind of difficult to s tick to the plan. But as time went by, having a plan for time in advance became a part of my life. At the same time, I gradually became a well-organized person. Now I’ve grasped the time management skill and I’m able to use my time efficiently.
英语演讲稿：未来的工作 这篇《英语演讲稿范文：未来的工作》，是特地，希望对大家有所帮助！ 热门演讲推荐：竞聘演讲稿 | 国旗下演讲稿 | 英语演讲稿 | 师德师风演讲稿 | 年会主持词 | 领导致辞 everybody good afternoon:. first of all thank the teacher gave me a story in my own future ideal job. everyone has a dream job. my dream is to bee a boss, own a pany. in order to achieve my dreams, i need to find a good job, to accumulate some experience and wealth, it is the necessary things of course, in the school good achievement and rich knowledge is also very important. good achievement and rich experience can let me work to make the right choice, have more opportunities and achievements. at the same time, munication is very important, because it determines whether my pany has a good future development. so i need to exercise their municative ability. i need to use all of the free time to learn
世界经典哲学类书籍 哲学，是理论化、系统化的世界观，是自然知识、社会知识、思维知识的概括和总结,是世界观和方法论的统一。是社会意识的具体存在和表现形式，是以追求世界的本源、本质、共性或绝对、终极的形而上者为形式，以确立哲学世界观和方法论为内容的社会科学。 《性心理学》 《作为意志和表象的世界》 《理想国》 《西方哲学史》 《自然哲学的数学原理》 《权力意志》 《新工具》 《纯粹理性批判》《文明论概略》 《劝学篇》 《伦理学》 《耶稣传》 《时间简史》 《逻辑哲学论》 《精神现象学》 《物性论》 《感觉的分析》 《精神分析引论》《基督何许人也——基督抹煞论》 《科学的社会功能》《人有人的用处》《科学史》 《人类理智新论》《逻辑学》 《哲学研究》 《新系统及其说明》《道德情操论》 《实践理性批判》《美学》 《判断力批判》 《基督教的本质》《薄伽梵歌》 《伦理学中的形式主义与质料的价值伦理学》《物种起源》 《物理学》《人类的由来》 《人性论》 《人是机器》 《法哲学原理》 《狄德罗哲学选集》 《野性的思维》 《哲学史教程》 《科学与近代世界》 《人类的知识》 《精神分析引论新编》 《自然宗教对话录》 《基督教并不神秘》 《科学中华而不实的 作风》 《一年有半，续一年有 半》 《时间与自由意志》 《哲学辞典》 《历史理性批判文集》 《苏鲁支语录》 《文化科学和自然科 学》 《十六、十七世纪科 学、技术和哲学史》 《科学哲学的兴起》 《灵魂论及其他》 《斯宾诺莎书信集》 《实验心理学史》 《最后的沉思》 《纯粹现象学通论》 《近代心理学历史导 引》 《佛教逻辑》 《神圣人生论》 《逻辑与知识》 《论原因、本原与太 一》 《形而上学导论》 《诗学》 《路标》 《心的概念》 《计算机与人脑》 《十七世纪英格兰的 科学、技术与社会》 《卡布斯教诲录》 《薄伽梵歌论》 《尼各马可伦理学》 《论老年论友谊论责 任》 《实用主义》 《我的哲学的发展》 《拓扑心理学原理》 《在通向语言的途中》 《科学社会学》 《埃克哈特大师文集》 《逻辑大全》 《简论上帝、人及其心 灵健康》 《宗教的本质》 《论灵魂》 《科学的价值》 《内时间意识现象学》 《艺术即经验》 《宗教与科学》 《感觉与可感物》 《行为的结构》 《真理与方法》 《阿维斯塔》 《善的研究》 《人类知识原理》 《伦理学体系》 《科学与方法》 《第一哲学（上下卷）》 《物理学理论的目的 与结构》 《思维方式》 《发生认识论原理》 《爱因斯坦文集》 《伦理学的两个基本问题》 《数理哲学导论 《耶稣传（第一、二卷）》 《美学史》 《原始思维》 《面向思的事情》 《普通认识论》 《莱布尼茨与克拉克论战 书信集》 《对莱布尼茨哲学的批评 性解释》 《物理学和哲学》 《尼采（上下卷）》 《思想录》 《道德原则研究》 《自我的超越性》 《实验医学研究导论》 《巴曼尼得斯篇》 《人类理解论》 《笛卡尔哲学原理》 《人生的亲证》 《认识与谬误》 《哲学史讲演录》 《圣教论》 《哲学作为严格的科学》 《人类知识起源论》 《回忆苏格拉底》 《心的分析》 《任何一种能够作为科学 出现的未来形而上学导论》 《科学与假设》 《宗教经验之种种》 《声音与现象》 《苏格拉底的申辩》 《论个人在历史上的作用 问题》 《论有学识的无知》 《保卫马克思》 《艺术的起源》
小学生个人读书事迹简介怎么写800字 书,是人类进步的阶梯,苏联作家高尔基的一句话道出了书的重要。书可谓是众多名人的“宠儿”。历来,名人说出关于书的名言数不胜数。今天小编在这给大家整理了小学生个人读书事迹,接下来随着小编一起来看看吧! 小学生个人读书事迹1 “万般皆下品,惟有读书高”、“书中自有颜如玉,书中自有黄金屋”,古往今来,读书的好处为人们所重视,有人“学而优则仕”,有人“满腹经纶”走上“传道授业解惑也”的道路……但是,从长远的角度看,笔者认为读书的好处在于增加了我们做事的成功率,改善了生活的质量。 三国时期的大将吕蒙,行伍出身,不重视文化的学习,行文时,常常要他人捉刀。经过主君孙权的劝导,吕蒙懂得了读书的重要性,从此手不释卷,成为了一代儒将,连东吴的智囊鲁肃都对他“刮目相待”。后来的事实证明,荆州之战的胜利,擒获“武圣”关羽,离不开吕蒙的“运筹帷幄,决胜千里”,而他的韬略离不开平时的读书。由此可见,一个人行事的成功率高低,与他的对读书,对知识的重视程度是密切相关的。 的物理学家牛顿曾近说过,“如果我比别人看得更远,那是因为我站在巨人的肩上”,鲜花和掌声面前,一代伟人没有迷失方向,自始至终对读书保持着热枕。牛顿的话语告诉我们,渊博的知识能让我们站在更高、更理性的角度来看问题,从而少犯错误,少走弯路。
读书的好处是显而易见的,但是,在社会发展日新月异的今天,依然不乏对读书,对知识缺乏认知的人,《今日说法》中我们反复看到农民工没有和用人单位签订劳动合同,最终讨薪无果;屠户不知道往牛肉里掺“巴西疯牛肉”是犯法的;某父母坚持“棍棒底下出孝子”,结果伤害了孩子的身心,也将自己送进了班房……对书本,对知识的零解读让他们付出了惨痛的代价,当他们奔波在讨薪的路上,当他们面对高墙电网时,幸福,从何谈起?高质量的生活,从何谈起? 读书,让我们体会到“锄禾日当午,汗滴禾下土”的艰辛;读书,让我们感知到“四海无闲田,农夫犹饿死”的无奈;读书,让我们感悟到“为报倾城随太守,西北望射天狼”的豪情壮志。 读书的好处在于提高了生活的质量,它填补了我们人生中的空白,让我们不至于在大好的年华里无所事事,从书本中,我们学会提炼出有用的信息,汲取成长所需的营养。所以,我们要认真读书,充分认识到读书对改善生活的重要意义,只有这样,才是一种负责任的生活态度。 小学生个人读书事迹2 所谓读一本好书就是交一个良师益友,但我认为读一本好书就是一次大冒险,大探究。一次体会书的过程,真的很有意思,咯咯的笑声,总是从书香里散发;沉思的目光也总是从书本里透露。是书给了我启示,是书填补了我无聊的夜空,也是书带我遨游整个古今中外。所以人活着就不能没有书,只要爱书你就是一个爱生活的人,只要爱书你就是一个大写的人,只要爱书你就是一个懂得珍惜与否的人。可真所谓
关于坚持的英语演讲稿 Results are not important, but they can persist for many years as a commemoration of. Many years ago, as a result of habits and overeating formed one of obesity, as well as indicators of overall physical disorders, so that affects my work and life. In friends to encourage and supervise, the participated in the team Now considered to have been more than three years, neither the fine rain, regardless of winter heat, a day out with 5:00 time. The beginning, have been discouraged, suffering, and disappointment, but in the end of the urging of friends, to re-get up, stand on the playground. 成绩并不重要，但可以作为坚持多年晨跑的一个纪念。多年前，由于庸懒习惯和暴饮暴食，形成了一身的肥胖，以及体检指标的全盘失常，以致于影响到了我的工作和生活。在好友的鼓励和督促下，参加了晨跑队伍。现在算来，已经三年多了，无论天晴下雨，不管寒冬酷暑，每天五点准时起来出门晨跑。开始时，也曾气馁过、痛苦过、失望过，但最后都在好友们的催促下，重新爬起来，站到了操场上。 In fact, I did not build big, nor strong muscles, not a sport-born people. Over the past few years to adhere to it, because I have a team behind, the strength of a strongteam here, very grateful to our team, for a long time, we encourage each other, and with sweat, enjoying common health happy. For example, Friends of the several run in order to maintain order and unable to attend the 10，000 meters race, and they are always concerned about the brothers and promptly inform the place and time, gives us confidence and courage. At the same time, also came on their own inner desire and pursuit for a good health, who wrote many of their own log in order to refuel for their own, and inspiring. 其实我没有高大身材，也没健壮肌肉，天生不属于运动型的人。几年来能够坚持下来，因为我的背后有一个团队，有着强大团队的力量，在这里，非常感谢我们的晨跑队，长期以来，我们相互鼓励着，一起流汗，共同享受着健康带来的快
1.How to build a business that lasts100years 0:11Imagine that you are a product designer.And you've designed a product,a new type of product,called the human immune system.You're pitching this product to a skeptical,strictly no-nonsense manager.Let's call him Bob.I think we all know at least one Bob,right?How would that go? 0:34Bob,I've got this incredible idea for a completely new type of personal health product.It's called the human immune system.I can see from your face that you're having some problems with this.Don't worry.I know it's very complicated.I don't want to take you through the gory details,I just want to tell you about some of the amazing features of this product.First of all,it cleverly uses redundancy by having millions of copies of each component--leukocytes,white blood cells--before they're actually needed,to create a massive buffer against the unexpected.And it cleverly leverages diversity by having not just leukocytes but B cells,T cells,natural killer cells,antibodies.The components don't really matter.The point is that together,this diversity of different approaches can cope with more or less anything that evolution has been able to throw up.And the design is completely modular.You have the surface barrier of the human skin,you have the very rapidly reacting innate immune system and then you have the highly targeted adaptive immune system.The point is,that if one system fails,another can take over,creating a virtually foolproof system. 1:54I can see I'm losing you,Bob,but stay with me,because here is the really killer feature.The product is completely adaptive.It's able to actually develop targeted antibodies to threats that it's never even met before.It actually also does this with incredible prudence,detecting and reacting to every tiny threat,and furthermore, remembering every previous threat,in case they are ever encountered again.What I'm pitching you today is actually not a stand-alone product.The product is embedded in the larger system of the human body,and it works in complete harmony with that system,to create this unprecedented level of biological protection.So Bob,just tell me honestly,what do you think of my product? 2:47And Bob may say something like,I sincerely appreciate the effort and passion that have gone into your presentation,blah blah blah-- 2:56(Laughter) 2:58But honestly,it's total nonsense.You seem to be saying that the key selling points of your product are that it is inefficient and complex.Didn't they teach you 80-20?And furthermore,you're saying that this product is siloed.It overreacts, makes things up as it goes along and is actually designed for somebody else's benefit. I'm sorry to break it to you,but I don't think this one is a winner.
关于工作的优秀英语演讲稿 Different people have various ambitions. Some want to be engineers or doctors in the future. Some want to be scientists or businessmen. Still some wish to be teachers or lawers when they grow up in the days to come. Unlike other people, I prefer to be a farmer. However, it is not easy to be a farmer for Iwill be looked upon by others. Anyway,what I am trying to do is to make great contributions to agriculture. It is well known that farming is the basic of the country. Above all, farming is not only a challenge but also a good opportunity for the young. We can also make a big profit by growing vegetables and food in a scientific way. Besides we can apply what we have learned in school to farming. Thus our countryside will become more and more properous. I believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. All the working position can provide him with a good chance to become a talent. 1 ————来源网络整理，仅供供参考
个人先进事迹简介 01 在思想政治方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.利用课余时间和党课机会认真学习政治理论,积极向党组织靠拢. 在学习上,xxxx同学认为只有把学习成绩确实提高才能为将来的实践打下扎实的基础,成为社会有用人才.学习努力、成绩优良. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意识和良好的心理素质和从容、坦诚、乐观、快乐的生活态度,乐于帮助身边的同学,受到师生的好评. 02 xxx同学认真学习政治理论,积极上进,在校期间获得原院级三好生,和校级三好生,优秀团员称号,并获得三等奖学金. 在学习上遇到不理解的地方也常常向老师请教,还勇于向老师提出质疑.在完成自己学业的同时,能主动帮助其他同学解决学习上的难题,和其他同学共同探讨,共同进步. 在社会实践方面,xxxx同学参与了中国儿童文学精品“悦”读书系,插画绘制工作,xxxx同学在班中担任宣传委员,工作积极主动,认真负责,有较强的组织能力.能够在老师、班主任的指导下独立完成学院、班级布置的各项工作. 03 xxx同学在政治思想方面积极进取,严格要求自己.在学习方面刻苦努力,不断钻研,学习成绩优异,连续两年荣获国家励志奖学金；作
为一名学生干部,她总是充满激情的迎接并完成各项工作,荣获优秀团干部称号.在社会实践和志愿者活动中起到模范带头作用. 04 xxxx同学在思想方面,积极要求进步,为人诚实,尊敬师长.严格 要求自己.在大一期间就积极参加了党课初、高级班的学习,拥护中国共产党的领导,并积极向党组织靠拢. 在工作上,作为班中的学习委员,对待工作兢兢业业、尽职尽责 的完成班集体的各项工作任务.并在班级和系里能够起骨干带头作用.热心为同学服务,工作责任心强. 在学习上,学习目的明确、态度端正、刻苦努力,连续两学年在 班级的综合测评排名中获得第1.并荣获院级二等奖学金、三好生、优秀班干部、优秀团员等奖项. 在社会实践方面,积极参加学校和班级组织的各项政治活动,并 在志愿者活动中起到模范带头作用.积极锻炼身体.能够处理好学习与工作的关系,乐于助人,团结班中每一位同学,谦虚好学,受到师生的好评. 05 在思想方面,xxxx同学积极向上,热爱祖国、热爱中国共产党,拥护中国共产党的领导.作为一名共产党员时刻起到积极的带头作用,利用课余时间和党课机会认真学习政治理论. 在工作上,作为班中的团支部书记,xxxx同学积极策划组织各类 团活动,具有良好的组织能力. 在学习上,xxxx同学学习努力、成绩优良、并热心帮助在学习上有困难的同学,连续两年获得二等奖学金. 在生活中,善于与人沟通,乐观向上,乐于助人.有健全的人格意 识和良好的心理素质.
尊敬的领导，老师，亲爱的同学们， 大家好！我是5班的梁浩东。今天早上我坐车来学校的路上，我仔细观察了路上形形色色的人，有开着小车衣着精致的叔叔阿姨，有市场带着倦容的卖各种早点的阿姨，还有偶尔穿梭于人群中衣衫褴褛的乞丐。于是我问自己，十几年后我会成为怎样的自己，想成为社会成功人士还是碌碌无为的人呢，答案肯定是前者。那么十几年后我怎样才能如愿以偿呢，成为一个受人尊重，有价值的人呢？正如我今天演讲的题目是：自主管理。 大家都知道爱玩是我们孩子的天性，学习也是我们的责任和义务。要怎样处理好这些矛盾，提高自主管理呢？ 首先，我们要有小主人翁思想，自己做自己的主人，要认识到我们学习，生活这一切都是我们自己走自己的人生路，并不是为了报答父母，更不是为了敷衍老师。 我认为自主管理又可以理解为自我管理，在学习和生活中无处不在，比如通过老师，小组长来管理约束行为和同学们对自身行为的管理都属于自我管理。比如我们到一个旅游景点，看到一块大石头，有的同学特别兴奋，会想在上面刻上：某某某到此一游话。这时你就需要自我管理，你需要提醒自己，这样做会破坏景点，而且是一种素质低下的表现。你设想一下，如果别人家小孩去你家墙上乱涂乱画，你是何种感受。同样我们把自主管理放到学习上，在我们想偷懒，想逃避，想放弃的时候，我们可以通过自主管理来避免这些，通过他人或者自己的力量来完成。例如我会制定作息时间计划表，里面包括学习，运动，玩耍等内容的完成时间。那些学校学习尖子，他们学习好是智商高于我们吗，其实不然，在我所了解的哪些优秀的学霸传授经验里，就提到要能够自我管理，规范好学习时间的分分秒秒，只有辛勤的付出，才能取得优异成绩。 在现实生活中，无数成功人士告诉我们自主管理的重要性。十几年后我想成为一位优秀的，为国家多做贡献的人。亲爱的同学们，你们们？让我们从现在开始重视和执行自主管理，十几年后成为那个你想成为的人。 谢谢大家！
关于工作的英语演讲稿 【篇一：关于工作的英语演讲稿】 关于工作的英语演讲稿 different people have various ambitions. some want to be engineers or doctors in the future. some want to be scientists or businessmen. still some wish to be teachers or lawers when they grow up in the days to come. unlike other people, i prefer to be a farmer. however, it is not easy to be a farmer for iwill be looked upon by others. anyway,what i am trying to do is to make great contributions to agriculture. it is well known that farming is the basic of the country. above all, farming is not only a challenge but also a good opportunity for the young. we can also make a big profit by growing vegetables and food in a scientific way. besides we can apply what we have learned in school to farming. thus our countryside will become more and more properous. i believe that any man with knowledge can do whatever they can so long as this job can meet his or her interest. all the working position can provide him with a good chance to become a talent. 【篇二：关于责任感的英语演讲稿】 im grateful that ive been given this opportunity to stand here as a spokesman. facing all of you on the stage, i have the exciting feeling of participating in this speech competition. the topic today is what we cannot afford to lose. if you ask me this question, i must tell you that i think the answer is a word---- responsibility. in my elementary years, there was a little girl in the class who worked very hard, however she could never do satisfactorily in her lessons. the teacher asked me to help her, and it was obvious that she expected a lot from me. but as a young boy, i was so restless and thoughtless, i always tried to get more time to play and enjoy myself. so she was always slighted over by me. one day before the final exam, she came up to me and said, could you please explain this to me? i can not understand it. i
第五章老年社会工作 第一节老年社会工作概述 第二节老年社会工作的主要内容 第三节老年社会工作的主要方法 第一节老年社会工作概述 一、老年人与老年期 （一）年龄界定 生理年龄：生理指标与功能确定 心理年龄：心理活动程度确定 社会年龄：与他人交往的角色确定 （二）老年期的划分 低龄老年人：60-69岁 中龄老年人：70-79 高龄老年人：80岁以上 （三）划分的意义 1.三种年龄：不能仅凭日历年龄判断服务需求，而要关注不同个体在生理、心理与社会方面的差异。 2.三个时期：关注老年同期群的共性需要。 二、老年期的特点 （一）生理变化 1.生理变化的特点：九大生理系统老化 2.对开展老年社会工作的影响 （1）要特别关注老年人的身体健康状况； （2）处理好隐私的健康问题，如大小便； （3）帮助机构和家庭策划环境的调整。 （二）心理变化 1.智力、人格、记忆力的变化 智力：结晶智力强，但处理问题速度下降 人格：总结自己生命的意义 记忆力：记忆速度下降，动机决定是否学习 2.对开展老年社工的影响 （1）提供机会但尊重选择 （2）所有事放慢节奏 （3）关注身体健康对心理功能的重要性 （三）社会生活方面的变化 1.对老年社会生活变化的理论解释 （1）角色理论：丧失象征中年的社会角色 （2）活动理论：生活满足感与活动有积极联系 （3）撤离理论：接受减少与社会的交往 （4）延续理论：不能割裂看待老年阶段 （5）社会建构理论：老年是一个独特的个人过程 （6）现代化理论：现代化使老年人地位下降
2.理论在社会工作中的应用 （1）注意角色转变的重大生活事件 （2）注意老年个体的差异性，尊重其对生活意义的不同理解 （3）注意社会隔离可能对老年人造成的伤害 （4）改变总有可能 （5）关注社会变迁对老年人的影响，推动社会政策的调整 三、老年人的需要及问题 （一）老年人的需要 1.健康维护 2.经济保障 3.就业休闲 4.社会参与 5.婚姻家庭 6.居家安全 7.后事安排 8.一条龙照顾 （二）老年人的问题 1.慢性病问题与医疗问题 2.家庭照顾问题 3.宜居环境问题 4.代际隔阂问题 5.社会隔离问题 四、老年社会工作 （一）老年社会工作的对象 1.老年人自身：空巢、残疾、高龄老人，也包括健康老人 2.老年人周围的人：家庭成员、亲属、朋友、邻居等 3.宏观系统：单位与服务组织 （二）老年社会工作的目的 根本目标：老有所养、老有所医、老有所教、老有所学、老有所为、老有所乐 （三）老年社会工作的作用 1.个体层面：维持日常生活、获得社会支持 2.宏观层面：参与制定有关老年人的服务方案与政策 五、老年社会工作的特点 （一）老年歧视等社会价值观会影响社会工作者的态度与行为 （二）反移情是社会工作者的重要课题 （三）社会工作者要善于运用督导机制 （四）需要多学科合作 第二节老年社会工作的主要内容 一、身体健康方面的服务 二、认知与情绪问题的处理 三、精神问题的解决 四、社会支持网络的建立 五、老年人特殊问题的处理 一、身体健康方面的服务
Dear teacher and colleagues: my topic is on “spare time”. It is a huge blessing that we can work 996. Jack Ma said at an Ali's internal communication activity, That means we should work at 9am to 9pm, 6 days a week .I question the entire premise of this piece. but I'm always interested in hearing what successful and especially rich people come up with time .So I finally found out Jack Ma also had said :”i f you don’t put out more time and energy than others ,how can you achieve the success you want? If you do not do 996 when you are young ,when will you ?”I quite agree with the idea that young people should fight for success .But there are a lot of survival activities to do in a day ,I want to focus on how much time they take from us and what can we do with the rest of the time. As all we known ,There are 168 hours in a week .We sleep roughly seven-and-a-half and eight hours a day .so around 56 hours a week . maybe it is slightly different for someone . We do our personal things like eating and bathing and maybe looking after kids -about three hours a day .so around 21 hours a week .And if you are working a full time job ,so 40 hours a week , Oh! Maybe it is impossible for us at