胰岛素诱导低血糖大鼠心房肌细胞特殊颗粒的形态计量和心房利钠肽免疫组织化学研究

通过形态计量学和免疫组织化学方法发现胰岛素诱导低血糖大鼠心房肌细胞核周区特殊颗粒（ＡＳＧ）的体密度、面数密度和数密度及平均直径均高于对照组（Ｐ＜０．０５）,但高尔基复合体各参数与对照组比较没有差别（Ｐ＞０．０５）。实验组的心房利钠肽（ＡＮＰ）的免疫反应强度比对照组强（Ｐ＜０．００１）。提示胰岛素诱导低血糖对心房利钠肽的释放具有抑制作用,表明ＡＮＰ作为生理和病理调节递质与代谢刺激相拮抗。     

戊肝病毒活性肽的选择,合成与应用

对戊型肝炎病毒（ＨＥＶ）编码蛋白序列进行了亲水性分析及二级结构预测,选择亲水性强、具有β－转角与β－折叠的区段,采用多肽固相合成法合成了ＨＥＶ基因组３个开读框架（ＯＲＦ１,ＯＲＦ２和ＯＲＦ３）中可能的抗原表位,以免疫学方法进行鉴定并选出了分别来自ＨＥＶ３个ＯＲＦ的、具有重要生物活性与应用前景的３段肽（ＥＨ１７４、ＥＨ２６５、ＥＨ３６２）。在此基础上,进行了抗ＨＥＶＥＬＩＳＡ新型检测试剂盒的实验室研究及临床试用。结果表明,所研究的戊肝抗体检测试剂盒特异性高、临床符合性好、具有可重复性,在戊肝辅助诊断及流行病学调查中具有重要意义。     

Synthesis and lodination of human (phenylalanine13, tyrosine19) melanin-concentrating hormone for radioreceptor assay

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr¹³ and Val¹⁹ of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe¹³, Tyr¹⁹] -MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [¹²⁵I]-[Phe¹³, Tyr¹⁹]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD ) was 1.18 × 10^?10 M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as α-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.     

Anchor-linked intermediates in peptide amide synthesis are caused by dimeric anchors on the solid supports

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4′-aminomethyl-3′,5′-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH₂, which was then degraded to H-Tyr-NH₂, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH₂ which could be isolated in preparative amounts. Cleavage reactions with ¹⁵N-labelled H-Ala-4-ADPV-[¹⁵N]-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH₂, which contained no ¹⁵N as demonstrated by ¹H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.     

Use of a gel-forming dipeptide derivative as a carrier for antigen presentation

A dipeptide of the formula Fmoc-Leu-Asp and some other related dipeptides were synthesized in solution by standard methods. When such peptides are dissolved in water at concentrations below 1% at 100 °C and cooled below 60 °C they form turbid solutions and eventaully visocelastic gels at lower temperatures. Such gels are thermoreversible and can also be disrupted by mechanical agitation. At a concentration of 2 mg/ml the peptide Fmoc-Leu-Asp forms an aqueous gel at 60 °C with a shear modulus of 80 Pa measured at a frequency of 1 rad/s. Peptide solutions undergo an abrupt increase in light scattering between 1 and 1.5 mg/ml at both 23 and 60 °C. By analogy with previous observations of other systems, this increse appears to be due to the formation of filamentous micelles and the aggregation of filamaents into a three-dimensional network. When low molecular weight adamantanamine derivatives, which are inherently non-antigenci antiviral drugs, were incorporated into the Fmoc-Leu-Asp gel and injected into rabbits, high titre specific antibodies were efficiently produced without the need for additional adjuvant. Both the physical properties of the gel and its effect on the antigenicity of low molecular weight compounds suggest a number of practical applications.     

Application of the multiple antigenic peptides (MAP) strategy to the production of prophormone convertases antibodies: Synthesis,characterization and use of 8-branched immunogenic peptides

Antiserum against an N-terminal sequence of murine prohormone convertase-1 (mPC1) incorporating the sequence immediatley following the junction between the putative pro-region and the active enzyme was obtained. This was accomplished using the multiple antigenic peptide (MAP) approach whereupon an 8-branched polylysine core to which are grafted multiple copies of a 16 amino acid peptide representing the N-terminal sequence of mPC1 (positions 84–99) was synthesized by solid-phase Fmoc chemistry. The ensuing peptide was purified and fully characterized by RP-HPLC, ¹H-NMR, amino acid composition, peptide sequencing and ion-spray mass spectrometry. The immunological properties of the resulting antibodies in detecting recombinant PC1 in both crude and purified preparations were compared with antibodies raised against a similar N-terminal segment of PC1 but using the conventioanl method of peptide–carrier protein conjugation and also developed against a C-terminal fusion protein of PC1. Our data indicate that the MAP antibody was as efficient as both the amino and carboxy-terminal antibodies in qualitative as well as quantitative analysis of PC1 encoded protein by radioimmunoassay. Following an identical approach, antibodies against other prohormone convertases like furin, PC5/6 and PACE4 were also developed and subsequently applied to a number of biochemical and immunological studies. In each case, the ease of preparation and high immunogenicity of the MAP approach were confirmed and reside in the simplicity and rapidity with which a potent and useful antiserum is obtained.     

Chemical synthesis of a 7 kDa insect gonadotropic neurohormone

An original insect neurohormone of 65 residues was synthesized by the solid-phase methodology using t-Boc strategy and Boc-Val-PAM–resin. The purification, conducted by several steps of liquid chromatography having mass, polarity or charge as separative criteria, yielded the product with the correct molecular weight of 6922 Da determined by mass spectrometry. The synthetic peptide had both the same affinity for the antinative neurohormone serum and the same biological activity as the native neurohormone.     

Multiple peptide synthesis

The synthesis of large numbers of peptides can be very labor intensive and, if a conventional peptide synthesizer is used, only small numbers of peptides can be produced within a reasonable time. The techniques described below can make large numbers of different peptides simultaneously with varying degrees of mechanization, ranging from the wholly manual methods, to those involving complete mechanization of the whole synthesis process. Most of the multiple synthesis methods are primarily intended for small scale production ranging from microgram amounts up to a few tens of milligrams. All of the systems are economical in use of solvents and reagents, enabling cost-effective synthesis. The techniques described can also be used to prepare peptide libraries, containing several millions of peptide sequences, to enable the rapid screening of all possible permutations of amino acids within short peptides. However, it is considered that multiple synthesis methods are not particularly suited where extreme high purity or very long peptides are required.     

Colocalization of NADPH-diaphorase activity and certain neuropeptides in the esophagus of opossum (Didelphis virginiana)

Nitric oxide and various neuropeptides in the myenteric plexus regulate esophageal motility. We sought colocalization of nitric oxide synthase and neuropeptides in frozen sections of mid-portion of smoothmuscled opossum esophagus using NADPH-diaphorase activity to mark the synthase and immunoreactivity to detect peptides. The peptides, all with demonstrated physiological activity in this organ, were calcitonin generelated peptide, galanin, neuropeptide Y, substance P, and vasoactive intestinal polypeptide. The ExtrAvidin Peroxidase immunostain for each peptide was carried up to the final peroxidase reaction with 3-amino-9-ethylcarbazole. The NADPH-diaphorase reaction was applied with short incubation to provide light staining just before the peroxidase reaction was performed. We examined sections for the proportions of singly and dually labeled nerve cells in the myenteric plexus. NADPH-diaphorase activity was highly colocalized with calcitonin gene-related peptide (59%), galanin (54%), and vasoactive intestinal polypeptide (53%). It showed little colocalization with neuropeptide Y (10%) and substance P (8%). The proportions of all nerve cells containing each of the substances were: NADPH-diaphorase-33%, calcitonin gene-related peptide-30%, galanin-55%, neuropeptide Y-16%, substance P-35%, and vasoactive intestinal polypeptide-58%. We conclude that the nerves responsible for peristalsis in the esophagus may act by releasing nitric oxide along with other inhibitory substances, calcitonin gene-related peptide, galanin, and vasoactive intestinal polypeptide, but not excitatory substances, neuropeptide Y and substance P.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.