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Mode of Action of Egg Peptides
Published in Claude Gagnon, Controls of Sperm Motility, 2020
Steven E. Domino, David L. Garbers
An additional speract analogue, Gly-Gly-Gly-Gly-Tyr-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly (GGG[Y2]speract), was radiolabeled with 125I and shown to have equal potency to speract in stimulating sperm respiration.25 In the presence of a bifunctional cross-linking agent, disuccinimidyl suberate, the analogue covalently coupled to one protein of apparent molecular weight (Mr) 77,000, as determined on sodium dodecyl sulfate (SDS) polyacry-lamide gels.25 The cross-linking was inhibited by various chemically synthesized analogues of speract that were biologically active in stimulating sperm respiration, but not by inactive chemical analogues. Because high concentrations of GGG[Y2]speract could not be used in cross-linking experiments, a low-affinity receptor may not have been detected. Furthermore, covalent coupling of other associated receptor molecules may not have been possible due to inaccessibility of disuccinimidyl suberate or lack of a properly oriented functional group.25
Structure-Function Relationships of IL-8 and its two Neutrophil Receptors: IL-8-RA and IL-8-RB
Published in Richard Horuk, Chemoattractant Ligands and Their Receptors, 2020
Caroline A. Hébert, Henry B. Lowman
Using disuccinimidyl suberate treatment at 4°C, Besemer et al.25 were able to obtain cross-linked dimers of IL-8 and of the homologous chemokine NAP-2 (Figure 1) at protein concentrations as low as 10 and 130 nM, respectively. Further, these authors were able to show specific cross-linking of IL-8 and NAP-2 to receptors on neutrophils. The four cross-linked bands obtained in these experiments had molecular weights of 55 and 65 kDa for the high-affinity NAP-2 sites (read B-receptor) and molecular weights of 71 and 81 kDa for the low-affinity NAP-2 sites (read A-receptor). Since the molecular weights of each of these pairs of bands differ by ~8 kDa (the molecular weight of an IL-8 monomer), the fact that four, rather than two, bands (for A and B type receptors) were found is consistent with the idea that both monomer and dimer forms of IL-8 might bind to the two receptor types. It was noted that the bands could also result from differential glycosylation of these receptors, or from the existence of unidentified additional neutrophil receptors.25 In subsequent cross-linking experiments,26 these authors estimated the dimerization constant Kdd for IL-8 in solution at -800 nM, far higher than the receptor binding affinity (~2 nM). Nevertheless, a small fraction of the dimeric form could be expected to be present in the nanomolar concentration range; thus the dimer could still not be excluded as the active species of IL-8.
Inhibitory Regulation of Gastrointestinal Organ Growth
Published in Jean Morisset, Travis E. Solomon, Growth of the Gastrointestinal Tract: Gastrointestinal Hormones and Growth Factors, 2017
To evaluate the nature of the somatostatin receptor and determine its molecular size, the following biochemical studies were performed. Ligands were first allowed to bind to the receptor. The complex was then covalently cross-linked and characterized by electrophoresis in SDS-polyacrylamide gels. Unfortunately to date, all such studies were performed using pancreatic acinar cell membranes from rat or guinea pig. Therefore the only information available pertained to pancreatic tissue. The first report85 presents the somatostatin receptor as a 90 kDa protein. This was later confirmed by a different group86 (Table 1). Using a different cross-linking agent, disuccinimidyl suberate (DSS) instead of n-hydroxysuccinimidyl-4-azido-benzoate (HSAB), Srikant and Patel87 identified three proteins of apparent Mr of 200,000, 80,000, and 70,000 under reducing conditions or not. In a more detailed experiment, Zeggari et al.88 evaluated the somatostatin receptor using different cross-linkers on membranes solubilized with Zwittergent 3-14. Of the four cross-linkers used, N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS), N-succinimidyl6-(azido-2’-nitrophenylamine) hexanoate (SANAH), HSAB, and DSS, ANB-NOS was the most efficient and irreversible cross-linker while DSS was virtually ineffective; this last cross-linker was used to identify the three previously described somatostatin receptor proteins.87 Furthermore, for equivalent amounts of initially membrane-bound radioactivity, about twice as much radioactivity was eluted from a gel filtration column after the membranes were prelabeled with 125I-[leu8, dTrp22, Tyr25]S28 than with 125I-Tyr1 ‘-S14. This suggests that the radioligand S28 is more efficiently cross-linked to the receptor by ANB-NOS. However, with both S28 and S14 radioligands, an apparent molecular mass of 93 and 94 kDa was determined, confirming the 90 kDa size of the receptor.88 The glycoprotein nature of the somatostatin receptor was established by its elution from a WGA-agarose column by N-acetylglucosamine or N,N′,N″-triacetylchitotriose which has a higher affinity for WGA.
Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation
Published in Pharmaceutical Biology, 2023
Mei-Yu Shen, Yu-Xi Di, Xiang Wang, Feng-Xiang Tian, Ming-Fei Zhang, Fei-Ya Qian, Bao-Ping Jiang, Xue-Ping Zhou, Ling-Ling Zhou
For standard western blots, cells were collected and lysed in RIPA buffer with protease and phosphate inhibitors (Beyotime, P1045), followed by mixing with SDS loading buffer and heating at 100 °C for 5 min. For detection of dimeric/tetrameric PKM2 expression, cells were collected, washed twice with phosphate-buffered saline (PBS, pH 8.0) and incubated in 2 mM disuccinimidyl suberate (DSS, Sangon Biotech, C100015) which dissolved in PBS (pH 8.0) for 30 min at 37 °C. Cells were then washed, lysed in RIPA buffer, mixed with SDS loading buffer and heated at 100 °C for 5 min. For nuclear extraction, nuclear and cytoplasmic fractions were isolated from cells by nuclear and cytoplasmic protein extraction kit (Beyotime, P0027), following the manufacturer’s instructions. All the protein content was determined using enhanced BCA protein assay kit (Beyotime, P0010S). The protein samples (10 μg per lane) were loaded on 6% or 10% or 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% bovine serum albumin (BSA), incubated with PKM2 primary antibody (Proteintech, 15822-1-AP; 1:1000 dilution) or STAT3 primary antibody (CST, D3Z2G; 1:1000 dilution) or phosphor-STAT3 (Tyr705) (CST, D3A7; 1:2000 dilution) overnight at 4 °C and secondary HRP-conjugated antibodies (proteintech, SA00001-2; 1:3000 dilution) for 1.5 h. Images were obtained and analyzed with the Image Lab software (Bio-Rad). Histone H3 and β-actin were used as nuclear and cytoplasm loading controls, respectively.
Kaempferol alleviates LPS-ATP mediated inflammatory injury in splenic lymphocytes via regulation of the pyroptosis pathway in mice
Published in Immunopharmacology and Immunotoxicology, 2019
Changliang He, Jia Yang, Xiaolin Jiang, Xiaoxia Liang, Lizi Yin, Zhongqiong Yin, Yi Geng, Zhijun Zhong, Xu Song, Yuanfeng Zou, Lixia Li, Wei Zhang, Cheng Lv
We discuss the mechanism of kaempferol effects in further detail. Kaempferol enters the cytoplasm and prevents the TLR4-associated transfer of LPS to the cytoplasm. Our western blot experimental data confirm this. Considering that the NF-κB pathway is involved in NLRP3, proIL-1β, and proIL-18 transcription [35], we suggest that kaempferol may interfere with the NF-κB pathway. This is supported by mRNA expression of NLRP3, proIL-1β, and proIL-18 in our experiments. Kaempferol interfered with the NF-κB pathway, exhibiting an associated anti-inflammatory activity, which was confirmed by Kadioglu et al.’s study [42]. Moreover, we found that kaempferol exerted protective effects and alleviated inflammation via the inhibition of the NLRP3 inflammasome and caspase-1 pathway. Kaempferol treatment enhanced cell viability according to the CCK assay and reduced cell mortality, as shown by the Trypan Blue-positive rate data. The results of the caspase-1 activity assay demonstrated that kaempferol inhibits caspase-1 activity. The level of NLRP3 protein expression, active form p10 of caspase-1 in cell lysate, and supernatant also support this. Furthermore, our data are in agreement with the protective effect of kaempferol shown in cardiac fibroblasts stimulated by LPS-ATP as demonstrated by Tang et al. [43]. However, the protein expression of ASC, a link protein between NLRP3 and caspase-1, showed no significant variation in our experiments. Compared with Liu et al.’s study [23], western blot data of ASC expression in LPS-primed macrophages stimulated by ATP or nigericin also showed no variation. However, unlike the previous studies, we used immunofluorescence detection and a novel immunoblotting technique with disuccinimidyl suberate-cross-linked pellets and anti-ASC antibodies. This immunoblotting was used to show ASC oligomerization specifically.
Synthesis of nanomedicines by nanohybrids conjugating ginsenosides with auto-targeting and enhanced MRI contrast for liver cancer therapy
Published in Drug Development and Industrial Pharmacy, 2018
Xiaoxiong Zhao, Junmei Wang, Yujun Song, Xinhua Chen
Hepatocellular carcinoma (HCC) is the fifth most common fatal human malignancy worldwide. HCC is highly resistant to chemotherapeutic drugs, and there is no single effective medicine against it [1–5]. Two or three medicines are often combined to enhance the efficacy of therapy. Nevertheless, chemotherapeutics, even in combination, often causes serious toxic side effects. Thus, there is an urgent need to develop novel treatment modalities [6]. The successful and explosive development of nanomaterials inevitably promotes their coupling with biology and medicine, which produces the blooming area of nanobiotechnology [7–16]. These nanomaterials themselves can be used as drugs without serious toxic effects, both for cancer therapy and for lesion imaging, after careful modification of their surfaces and/or conjugated with certain medicines or organic components [9–11,13–19]. Surface modification of NPs with desired surface active groups and/or coupling NPs with medicine or organics are two key routes in the development of these kinds of nanomedicines in addition to their reasonable microstructure for the enhanced interaction among different components in the drugs [7,11,19–21]. There are several conventional surface modification processes for conjugating desired biomolecules (e.g. peptide, DNA, RNA, or proteins) or medicines (e.g. folic acid and paclitaxel) with NPs based on carbodiimide [e.g. 1-thyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)] coupling reactions (i.e. for NPs with surface –COOH ligands) [22–24] or carbazole derivative [e.g. disuccinimidyl suberate (DSS)] coupling reactions (e.g. for NPs with surface –NH2 ligands) [25–27], and/or metal-sulfide and metal-nitride bonds (e.g. Au-S and Au-N bonding) [8,28]. Recently, superparamagnetic Fe3O4 NPs have attracted considerable attention for a local hyperthermia agent in nanomedicine due to its officially approved high biocompatibility. Superparamagnetic Fe3O4 NPs not only have a pure superparamagnetic phase for easy transportation, good circulation, no agglomeration in blood vessels, and a small particle size for effective injection, but also have been used as contrast agents for fluorescence imaging, magnetic resonance imaging (MRI), computed tomography (CT), photoacoustic tomography (PAT), and surface-enhanced Raman scattering [29]. Furthermore, the magnetic NPs with the medicines are easily retained and absorbed by the target tissue of endothelial cells and target sites and thus have a better biocompatibility effect. It is well-known that many natural products that are isolated from medicinal herbs (e.g. Chinese ginseng and paclitaxel) have anti-cancer effects, such as chemotherapeutic agents with few toxic side effects and a wide spectrum of anti-tumor activities when used as monotherapy or in combination with chemotherapy regimens, a wide spectrum of in vitro cell experiments and animal experiments in vivo and/or even in human clinical tests [30–32]. Certain of the NPs conjugated with biomolecules have been approved as chemotherapeutic anti-cancer drugs by the Food and Drug Administration (FDA) after several years of study and clinical tests (e.g. Abraxane®, paclitaxel albumin-stabilized nanoparticle formulation) [33].