Localization of cholecystokinin-like and calcitonin-like peptides in infant carotid bodies: a light- and electron-microscopic immunohistochemical study

Previous immunohistochemical studies have identified several regulatory peptides in the carotid body chief cells in both humans and animals. These peptides, together with amines, may be important in the modulation of the chemoreflex by the carotid body. We report the localization and distribution of calcitonin and cholecystokinin-like (CCK) immunoreactivity in chief cells of human infant carotid body by light- and electron-microscopic immunohistochemical techniques. Consecutive sections immunostained with calcitonin and/or CCK antibodies revealed positively stained chief cells, both alone and in clusters, scattered throughout the carotid body lobule. Generally more chief cells were positive for calcitonin than for CCK. This was confirmed by quantiative analysis showing that the ratio of calcitonin to CCK immunoreactive cells was consistently >2:1 in all cases studied. There was no apparent correlation between the immunoreactivity for the two peptides and the age, sex, or postmortem interval. Calcitonin-like and CCK-like immunoreactivities were localized electron-microscopically over the dense core granules of the chief cells. Calcitonin and CCK-like peptides in carotid body chief cells may act as neutransmitters or neuromodulators involved in chemoreception.     

Synthetic glycosylation of peptides using unprotected saccharide β-glycosylamines

Glycopeptides can be valuable tools in determining the influence of carbohydrate moieties on the intrinsic properties of glycoproteins. However, glycopeptides of sufficient quantity and purity are as yet not readily available from biological sources. The chemical coupling of a -glycosylamino group of an unprotected carbohydrate with an activated aspartic acid residue of an unprotected peptide is a simple method for synthesizing asparagine-linked glycopeptides. In this report we demonstrate that the use of this method is not restricted to -glycosylamines of simple monosaccharides or short aspartic acid-containing pentapeptides. This is illustrated by the syntheses of several glycopentapeptides containingN,N-diacetylchitobiose, a glutamine-linked glycopentapeptide containing a biantennary complex oligosaccharide, and glycosylated variants of two analogs of a polypeptide hormone, atriopeptin, containingN,N-diacetylchitobiose.Abbreviations Ac acetyl - Bzl benzyl - DMF dimethylformamide - Fmoc 9-fluorenylmethoxycarbonyl - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - HBTU O-benzotriazol-1-yl-N,N,N,N-tetramethyluroniumhexa-fluorophosphate - HOBt 1-hydroxybenzotriazole - Man mannose - m/z mass/charge - NMR nuclear magnetic resonance - Xyl xylose - Z benzyloxycarbonyl; unless otherwise specified, amino acids are abbreviated using their one-letter codes.     

乙型肝炎病毒表面抗原preS1在大肠杆菌中的表达与鉴定

本文报告利用pWR590质粒为载体,构建了含1ac启动子、β-半乳糖苷酶(1—590)基因、Xa因子的四肽识别位点和HBV preS1、preS2编码序列的表达质粒,并成功地在大肠杆菌中获得稳定表达。融合蛋白经Xa因子消化和高效液相层析,得到了preS1(1—91)纯肽。此肽特异性地与人肝细胞质膜结合,从而为肝细胞上存在preS1受体提供了直接的实验依据,也为分离和鉴定肝细胞上preS1受体打下了良好的基础。     

利用饵料微藻表达抗菌肽及其初步应用

为了解抗菌肽在饵料微藻中表达后的抗菌特性,构建海洋微拟球藻(Nannochloropsis oceanica)、湖泊微拟球藻(N.limnetica)和三角褐指藻(Phaeodactylum tricornutum)的抗菌肽(源自虹鳟,Cath-1a)表达质粒,分别转化相应的微藻,检测转化子中抗菌肽的表达量和体外抑菌效果,将藻株作为鱼饲料添加剂喂食斑马鱼,初步分析了抗菌肽及藻体自身的岩藻黄素和多不饱和脂肪酸对鱼免疫系统的影响。结果表明,外源抗菌肽在3种微藻中均可以成功表达,体外抑菌试验表明,仅三角褐指藻对水产领域常见致病菌爱德华氏菌(Edwardsiella tarda)有一定的抑菌效果,然而抗菌肽的表达并未使3种藻株的体外抑菌性增加。添加藻粉对斑马鱼的生长无明显影响,通过检测鱼体肝脏中与抗氧化和免疫相关基因的表达水平及丙二醛的含量,表明添加藻粉可增强斑马鱼的抗氧化和抗炎症能力,表达抗菌肽(PtC组)能进一步提高斑马鱼的免疫力。另外,添加Pt6(富含岩藻黄素)藻粉组比添加PtC的抗炎效果更显著,表明三角褐指藻中的岩藻黄素和二十碳五烯酸对增强鱼的抗病能力具有潜在作用。     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).     

Synthesis and characterization of two potential impurities (des-ethyl-Ganirelix) generated in the Ganirelix manufacturing process

Controlling certain diseases using peptide drugs has remarkably increased in the past two decades. In this regard, a generic formulation is an upfront solution to fulfill market demands. Ganirelix, a leading peptide active pharmaceutical ingredient (API) primarily used as a gonadotropin-releasing hormone antagonist (GnRH), has established a potential market value worldwide. But its generic formulation mandates detailed impurity profiles from a synthetic source and contemplates the sameness of a reference-listed drug (RLD). Post-chemical synthesis and processing of Ganirelix, some commercial sources have revealed two new potential impurities among many known, which show the deletion of an ethyl group from the hArg(Et)₂ residue at the sixth and eighth positions, named des-ethyl-Ganirelix. These impurities are unprecedented in traditional peptide chemistry, and such monoethylated-hArg building blocks are not easily accessible commercially to synthesize these two impurities. Here, we have outlined the synthesis, purification, and enantiomeric purity characterization of the amino acids and their incorporation in the Ganirelix peptide sequence to synthesize these potential peptide impurities. This methodology will enable the convenient synthesis of side-chain substituted Arg and hArg derivatives in peptide drug discovery platforms.     

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases

The conjugates of an adenosine mimetic and oligo-l -arginine or oligo-d -arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l -methionine (SAM) and S-adenosyl-l -homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.     

New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.