Experimental studies and potential energy calculations of the blocked tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the third loop of short-chain snake venom neurotoxins

The conformational space of the tetrapeptide Ac-Lys-Pro-Gly-Ile-NMA from the β-bend present in the third loop of short-chain snake venom neurotoxins was investigated with the aid of energy calculations, resulting in the identification of an ensemble of β-turn conformations. These results were compared with the experimentally determined conformations, as observed using NMR and CD spectroscopy. A random coil conformation of the peptide is indicated in polar hydrogen-bonding solvents. In less polar solvents the peptide backbone assumed a more rigid conformation, as reflected by the existence of at least a type II β-turn conformation.     

CD-n.m.r. study of the solution conformation of bradykinin analogs containing α-aminoisobutyric acid

The conformation in aqueous solution of several α-aminoisobutyric acid (AIB)-containing analogs of bradykinin (BK) has been probed by complementary CD and ¹H n.m.r. measurements. The conclusion reached is that substitution of AIB for Pro² and/or Pro³ in BK stabilizes a degree of β-turn conformation in the N-terminal tetrapeptide moiety of the resulting analogs. Changing the solvent from water to DMSO or TFE further enhances the contribution of particular hydrogen bonded structures to the time-averaged conformation of these peptides. Bradykinin and [AIB⁷]-BK adopt similar hydrogen bonded conformations in TFE, apparently with a contribution from a β-turn involving their common Arg¹-Pro²-Pro³-Gly⁴ moiety. The contrasting biological activities of BK and its AIB-analogs are considered in terms of the conformational analogy between the AIB-residue and cis¹ Pro and the propensity for a β-turn at the N-terminus of the peptide.     

嘌呤核苷酸对吗啡依赖大鼠痛阈及急性戒断症状的影响

目的:探讨嘌呤核苷酸对吗啡依赖大鼠痛阈及急性戒断症状的影响。方法:将30只♂ Wistar大鼠随机分为对照组、吗啡组和嘌呤核苷酸+吗啡组,于实验d1、3、5、7用电子压痛仪检测大鼠尾压痛阈值,d2、4、6、8进行热甩尾实验检测大鼠抗热痛觉过敏能力,于d8给药后4h观察纳洛酮急性戒断症状。结果:与对照组相比,d1起吗啡组及嘌呤核苷酸+吗啡组大鼠尾压痛阈值增高(P<0.01),d3起嘌呤核苷酸+吗啡组压痛阈值明显高于吗啡组(P<0.01);d2起吗啡组热甩尾潜伏期延长(P<0.05),d6起嘌呤核苷酸+吗啡组热甩尾潜伏期时间长于吗啡组(P<0.05);嘌呤核苷酸+吗啡组戒断症状评分与吗啡组比有显著的统计学差异(P<0.01)。结论:嘌呤核苷酸能够明显增强吗啡的镇痛作用,并减轻吗啡依赖大鼠的戒断症状。     

Roles of conserved intracellular sequences regions for the proper expression of dopamine D2 and D3 receptors

The dopamine D(2) receptor and D(3) receptor (D(2)R, D(3)R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D(3)R signals are 2 approximately 5 times less efficient than D(2)R. Previous studies have suggested that conformational constraints of D(3)R might be responsible for the poor coupling with the G protein. To this hypothesis, point mutations were introduced into some of the conserved regions between D(2)R and D(3)R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1(st) intracellular loop, SS motif in the 2(nd) intracellular loop, YxxL and TxxS/xS motifs in the 3(rd) intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3(rd) intracellular loop markedly decreased the level of D(3)R expression compared with D(2)R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D(2)R and D(3)R in the 1(st) intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D(2)R and D(3)R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1(st) loop of D(3)R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D(2)R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D(3)R, which might be partly responsible for the quantitative differences in the signaling efficiency between D(2)R and D(3)R.     

Peptide models for β-turns.

The circular dichroism spectra of four β-turn model peptides, Z-Aib-Pro-Aib-Pro-OMe (1), Piv-Pro-Aib-NHMe (2), Piv-Pro-D-Ala-NHMe (3) and Piv-Pro-Val-NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type II β-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type II β-turn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents, suggesting a variant of a Type I (III) structure. The Type II β-turn is characterized by a CD spectrum having two positive CD bands at ? 230 nm and ? 202 nm, a feature observed in Piv-Pro-D-Ala-NHMe in cyclohexane and methanol and for Piv-Pro-Aib-NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type II, III and V reverse turn structures. Piv-Pro-Val-NHMe adopts non-β-turn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I β-turns.     

A novel alpha conotoxin (α-PIB) isolated from C. purpurascens is selective for skeletal muscle nicotinic acetylcholine receptors

The alpha-conotoxin family is comprised of peptides that share the following arrangement of cysteine residues in the primary amino acid sequence: -CC-C-C-, where each dash represents a variable number of amino acids. The number of amino acids between cysteine residues has been used to group the alpha-conotoxins into distinct subfamilies. These subfamilies include the alpha 4/7-, alpha 4/3- and alpha 3/5-conotoxins, so named for the number of amino acids between 2nd/3rd and 3rd/4th cysteine residues, respectively. The alpha 3/5-conotoxins antagonize vertebrate-muscle nicotinic acetylcholine receptors (nAChRs), while the alpha 4/7- and alpha 4/3-conotoxins primarily inhibit vertebrate neuronal nAChRs. To date, these three subfamilies are the most extensively characterized of the alpha-conotoxin family. Here we report the purification and characterization of an unusual alpha 4/4-conotoxin, alpha-conotoxin PIB (alpha-PIB), from the venom of Conus purpurascens, with the following amino-acid sequence: ZSOGCCWNPACVKNRC (Z=pyroglutamate, O=hydroxyproline). This peptide demonstrates high affinity inhibition of vertebrate-muscle nAChRs, and paralytic effects when injected in vivo. Testing of alpha-PIB against other receptors indicated that the inhibitory effect is specific for skeletal muscle nAChRs. alpha-PIB shares the key biochemical and pharmacological characteristics of the alpha-conotoxin family.     

Conformation of model,alanine and proline containing tetrapeptides in water

CD spectra of model alanine and prolyl-alanine tetrapeptides were measured at different _pH values. An analysis of the spectra shows that proline in position 2 or 4 of a tetrapeptide favours folding of the peptide chain, and unfolding when it is in position 3. Changes in CD spectra evidence growing amounts of the β-turn conformation upon increasing ^pH, independent of proline position in the peptide chain.     

A study of derivative spectrophotometry for the determination of carboxyhemoglobin in blood

An application of derivative spectrophotometry to the determination of carboxyhemoglobin (HbCO) in blood was examined. The 1st, 2nd, 3rd, and 4th derivative spectra were recorded in the Soret region for HbCO, reduced hemoglobin (Hb), and their mixed solution. The 4th derivative spectrum had the highest sensitivity to the change of HbCO-Hb ratio in the solution. A maximum point of the 4th derivative curve was employed for the HbCO determination. Blood samples with varied HbCO concentrations were analyzed using this technique and the results were compared with those obtained by conventional spectrophotometric and gas chromatographic procedures. The derivative method was in satisfactory agreement with both procedures.     

Comments on the use of a dichromophoric circular dichroism assay for the identification of β-turns in peptides

Use of the dichromophoric CD assay for β-turn formation in peptide sequences has been investigated. The assay involves the observation of Cotton effects in CD spectra, originating from the approach of N- and C-terminal aromatic chromophores in tetrapeptides. The approach of the chromophores was believed to be brought about by a beta;-turn in the peptide structure. Our investigations were paralleled by NMR studies which revealed the presence of a previously unreported hydrogen bond in the β-turn conformers, which appears to play a role in the generation of the observed Cotton effects. This suggests caution in the use of the CD technique alone as an assay for β-turn conformers in peptides.     

Studies on conformational consequences of i to i+ 3 side-chain cyclization in model cyclic tetrapeptides

In an effort to explore the effect of ring size on the biologically active conformation of cyclic analogs of the mating pheromone α-factor (WHWLQLKPGQPMY) from Saccharomyces cerevisiae, eight cyclic tetrapeptides corresponding to the KPGQ portion of α-factor were synthesized. These N-α-acetyl/carboxyl amide terminal cyclic tetrapeptides were prepared on a 4-methylbenzhydrylamine resin using orthogonal Boc, Frnoc, OFm and OtBut protecting groups and HOBt-DIPC accelerated active esters or urethane-protected N-carboxyanhydrides. On-resin cyclization of the side-chain amino and carboxyl groups of the first and fourth residues, respectively, was performed with the BOP reagent to generate lactams containing 14–18 atoms. HF cleavage resulted in two products, the desired cyclic tetrapeptide and a major side product. All peptides were purified to near homogeneity (>99%) by using reversed-phase HPLC and were characterized by FABMS and ¹H NMR. Certain constrained cyclic tetrapeptides appear to be a mixture of isomers at room temperature as evidenced by HPLC and NMR. The major side product has been identified as a cyclo dimer, obtained as a consequence of interchain cyclization on the resin. CD analysis in several solvents gives evidence that some of the cyclic tetrapeptides exist in β-turn conformations. © Munksgaard 1995.     

药物联合神经阻滞治疗带状疱疹后神经痛的临床观察

目的观察药物联合神经阻滞治疗带状疱疹后神经痛的临床疗效。方法带状疱疹后神经痛患者37例,根据受损部位不同给予相应的神经阻滞,并联合药物进行综合治疗。于治疗后的第1、2、3、4周进行视觉模拟(VAS)和睡眠质量(QS)评分。结果治疗后1、2、3和4周VAS评分分别为(4.68±1.69)分、(3.13±0.53)分、(2.06±0.41)分和(1.10±0.58)分,均明显低于治疗前的(7.92±1.32)分(P<0.05);治疗后1、2、3和4周的QS评分分别为(1.50±0.52)分、(0.89±0.45)分、(0.74±0.37)分和(0.55±0.24)分,均明显低于治疗前的(3.15±0.68)分(P<0.05)。结论药物联合神经阻滞治疗带状疱疹后神经痛疗效好,能迅速解除疼痛,缓解临床症状。     

N-Methyl peptides

The natural occurrence of N-methyl peptides in various plant metabolites has made N-methylation a subtle and attractive possible modification for structure-activity relationship studies of endogeneous peptides. However, little is known about the conformational specificity induced by the N-methylation of a given peptide, and particularly concerning the β-turn conformation. A spectroscopic investigation (i.r., n.m.r., CD) and X-ray diffraction experiments have been carried out on tBuCO-X-Me-Y-NHMe blocked dipeptides (X = Gly, L-Ala, L-Pro, and Y = Gly, and L- or D-Ala, Leu, Phe) with reference to the homologous desmethylated species. The influence of the N-methylation on conformation depends to a large extent on the chirality of the X and Y residues. Homochiral sequences are the most affected, with a strong preference for the βVI-folded conformation containing a middle cis amide bond. Heterochiral sequences are essentially unaffected and retain the βII-folded conformation with a trans middle amide bond. Glycine-containing sequences undergo a more complex perturbation according to the X or Y position of the Gly residue. The available data for larger N-methyl peptides are consistent with our observations, suggesting that these simple dipeptides well reflect the conformational perturbations induced by N-methylation on the β-turn conformation.     

Simulations of conformers of tuftsin and a cyclic tuftsin analog

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr-Lys-Pro-Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β-turn. Our results indicate that water-peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β- nor a γ-turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr-Lys-Pro-Arg-Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side-chain orientations of the Lys and Arg residues. © Munksgaard 1995.     

Amino acid sequences of eight phospholipases A2 from the venom of Australian king brown snake, Pseudechis australis

The amino acid sequences of eight phospholipases A2 (Pa-1G, Pa-3, Pa-5, Pa-9C, Pa-10A, Pa-12A, Pa-12C and Pa-15) which had been isolated from the venom of Australian king brown snake (Pseudechis australis) were elucidated. Pa-1G, Pa-3 and Pa-15 showed micro-heterogeneity at the 103rd position and Pa-5 was separated into two components, Pa-5a ([Pro-18 and Tyr-61]Pa-5) and Pa-5b ([ Ser-18 and Phe-61]Pa-5). All the phospholipase A2 molecules except Pa-1Ga and Pa-1Gb which lack the 118th residue, consisted of a single chain of 118 amino acid residues including 14 half-cystine residues and all the common residues among phospholipases A2 from other sources. From comparison studies, Asp-50, Lys-58 and Asp-90 seem to be important for the toxicity, and we propose that the domain for the presynaptic toxicity consists of seven hydrophilic residues, i.e. Arg-43, Lys-46, Asp-50, Glu-54, Lys-58, Asp-90 and Glu-94.     

Conformational analysis of NKB and potent and selective NK-3 agonists

The three-dimensional structures of NKB, [Arg⁰] NKB, [Cys^2.5] NKB and a potent, water-soluble, selective NK-3 agonist, [Pro⁷] NKB have been studied by ¹H-n.m.r. spectroscopy in dimethylsulfoxide and methanol. From this study, the postulated common conformational features of potent NK-3 agonists are: 1) either an α-helical structure from residues 2 to 6 or a β-turn orientation for residues 3 to 6 and 2) a C₇ orientation for the common phenylalanine, Phe⁶. The C-terminal tetrapeptide should adopt an extended structure.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH

Abstract: The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-Val9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide ( 1 ), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide–receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1–Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible β-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first β-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second β-turn. Furthermore, the presence of a β-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH–NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I β-turn structure is also supported by CD spectral analysis. The cIBR peptide ( 1 ) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

比较 EST 与开腹取石对胆总管结石患者机体炎症反应和免疫功能的影响

目的：探讨内镜下乳头括约肌切开取石术（ EST）与开腹取石对胆总管结石患者机体炎症反应和免疫功能的影响。方法选择该院2009年4月至2013年10月胆总管结石患者160例,其中行EST患者80例,行开腹手术患者80例,比较两组患者围手术期炎性反应指标：降钙素原（PCT）、C反应蛋白（CRP）、IL-6及免疫功能指标：免疫球蛋白（IgA、IgG、IgM）及T淋巴细胞亚群（CD4、CD8、CD4／CD8）的变化。结果开腹组和EST组的IL-6、PCT和CRP术前水平差异无统计学意义（P＞0．05）,术后1、3 d EST组的IL-6、CRP、PCT水平均高于开腹组（ P＜0．05）,术后5 d两组的IL-6、CRP、PCT水平无统计学差异（ P＞0．05）。开腹组和EST组的IgG、IgA、IgM术前水平差异无统计学意义（P＞0．05）,术后1、3、5 d EST组的IgG、IgA、IgM水平明显高于开腹组（ P＜0．05）。两组患者术前CD3＋、CD4＋、CD4＋／CD8＋,差异无统计学意义（ P＞0．05）;术后1 d传统开腹组术后的CD4＋、CD4＋／CD8＋水平低于EST组（ P＜0．05）,CD3＋水平差异无统计学意义（ P＞0．05）;术后3、5 d传统开腹组的CD3＋、CD4＋、CD4＋／CD8＋水平明显低于EST组（P＜0．05）。结论与开腹手术治疗胆总管结石相比,EST对机体组织损伤小、免疫抑制轻,虽短时间内出现炎症反应增强,但经过对症治疗,对于患者生理和免疫功能恢复无明显影响。同时EST操作简便、创伤小、手术时间短、并发症少且疼痛轻微,可广泛应用于临床。     

高压氧对围生期缺氧缺血事件新生儿脑血流的影响

目的通过检测围生期缺氧缺血事件新生儿高压氧（HBO）治疗前后的脑血流,了解高压氧治疗对有围生期缺氧缺血事件新生儿脑灌注的影响。方法于2008年12月-2010年12月在我院住院的有围生期缺氧缺血病史的新生儿中,随机选取30例,用彩色超声多普勒检测每次HBO治疗前后通过大脑中动脉（McA）脑血流情况,连续检测其大脑前动脉的收缩期峰值（VS）、舒张末期速度（Vd）、阻力指数（RI）。所得数据采用SPSS13．0统计软件进行统计分析。结果脑血流速度的改变：HBO治疗的第1、2天,治疗前后脑血流速度无明显改变;治疗第3天,Vs、Vd较前降低,其中Vs差异有显著性（P〈0．05）,Vd差异有极显著性（P〈0．01）;治疗第4天,Vs、Vd较前降低,Vs、Vd差异有极显著性（P〈0．01）。血管阻力指数的变化：HBO治疗的第1、2天,无明显改变;治疗第3天,RI较前升高,RI差异有极显著性垆〈0．01）治疗第4天,RI较前升高,RI差异有极显著性（P〈0．01）。结论对围生期缺氧缺血事件新生儿进行HBO治疗,初期脑灌注影响不明显,治疗后期产生明显影响,可使脑血管收缩并致血流速度减慢,从而影响脑灌注。     

Circular dichroism studies on synthetic peptides corresponding to the cleavage site region of precursor proteins

The conformations of synthetic peptides which span the region in which the precursor part of proteins (signal sequences) destined for export are cleaved by signal peptidases, were investigated by circular dichroism spectroscopy. Pentapeptides comprising amino acids only from the carboxy-terminus of signal sequences or the amino terminus of the mature protein do not have any preferred conformation in a variety of solvents. Octa- and nonapeptides containing amino acids from the carboxy-terminal protion of signal sequences and the amino-terminus of the mature portions of precursor proteins tend to adopt β-turn conformations in trifluoroethanol and micelles of sodium dodecylsulphate. Hence, in addition to the distribution of amino acids with small side chains at the carboxy terminus of signal sequences, it is conceivable that signal peptidases also recognize a β-turn conformation in the cleavage site region of precursor proteins.     

Interaction between enrofloxacin and monensin in broiler chickens

Enrofloxacin, a fluoroquinolone, and its interaction with monensin, an ionophore drug, was studied to explore the influence of enrofloxacin on drug metabolizing enzymes that can lead to physiological and toxicological consequences upon coadministration with monensin in broiler chickens. Group I, treated with 100 mg monesin/kg feed from 1 d old to 41st d of age, did not show any influence on aniline hydroxylase and cytochrome b5 levels. Group II, treated with 10 mg enrofloxacin/kg body weight per os for three consecutive days on 33rd, 34th, 35th d of age, had a highly significant decrease in aniline hydroxylase on 38th d (ie on 3rd d post-treatment with enrofloxacin); a reversal effect was noticed on the 41st day (ie on 6th d post-treatment with enrofloxacin). There was no alteration in cytochrome b5 level. Group III with monensin and enrofloxacin coadministration 100 mg monensin/kg feed from 1 d old to the 41st day + 10 mg enrofloxacin/kg body weight, per os for 3 consecutive days on the 33rd, 34th, 35th d of age) had a significant decrease in aniline hydroxylase level on the 3rd d post-treatment with enrofloxacin, but an elevation tending to reach normal on the 6th d post-treatment with enrofloxacin. Monensin + enrofloxacin coadministration did not produce any alteration in cytochrome b5 level. Creatine kinase (CK) and alanine amino transferase (ALT) levels significantly increased on the 3rd d post-treatment with enrofloxacin, but on the 6th d post-treatment with enrofloxacin the increase declined. Aspartate amino transferase (AST) significantly increased on the 6th d post enrofloxacin treatment. This study demonstrated the reversible competitive type of inhibition of enrofloxacin on CYP450 enzymes, and with coadministration with monensin produced increased CK, AST and ALT serum enzymes suggesting heart and liver injury. Simultaneous administration of enrofloxacin and monensin even at recommended levels could result in adverse interactions.