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1.
Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with 3H-spiroperidol for common binding sites in rat striatum (IC50 in the range between 10?6 and 10?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites and their potency to evoke “bizarre behavior” in rats . In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites (IC50 greater than 10?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites. 相似文献
2.
The effectiveness of several extraction procedures in solubilizing 3H-spiroperidol receptor sites was examined. Of the solubilizing agents tested, digitonin and lysolecithin were both effective in solubilization of the receptor. Lysolecithin, however, yielded four times as many receptor sites as that obtained with digitonin. The soluble receptor retained the essential characteristics of the membrane bound sites. Butaclamol stereospecificity inhibited the uptake of 2 × 10?9M, 3H-spiroperidol solubilized receptor at an IC50 value similar to that of intact membrane. Stereospecifically of butaclamol antagonism was not maintained, however, when a cerebellum, or heat inactivated caudate preparation was used. The solubilized preparations were sensitive to the effects of the specific dopamine agonist 6,7-dihydroxy-2-aminotetralin (ADTN) which inhibited 3H-spiroperidol binding with low IC50 values similar to those obtained with intact membrane receptor. Displacement of 3H-spiroperidol from 3H-spiroperidol receptor complex was produced by butaclamol stereospecifically, and for other competitive antagonists including haloperidol, spiroperidol and R 1187 in a manner similar to that of the intact membrane receptor. Both microsomes and synaptosomes could be similarly solubilized with digitonin and retained stereospecific reversibility of binding in the presence of butaclamol. Chromatography of solubilized lysolecithin calf caudate, 3H-spiroperidol receptor complex reveals a single peak of radioactivity which was eluted just prior to rabbit gamma globulin, suggesting an estimated molecular weight of 150,000 to 200,000 daltons. 相似文献
3.
L. N. Levanov L. E. Matveev T. E. Yun E. P. Goncharova L. R. Lebedev A. N. Shvalov A. B. Ryzhykov I. K. Baikov V. A. Matveeva V. A. Rikhter N. V. Tikunova 《Russian Journal of Bioorganic Chemistry》2009,35(4):474-481
A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 107 M?1 for the equilibrium state and (2.8 ± 0.3) × 107 M?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC50 value for scl3D6 was 16.7 μg/ml. 相似文献
4.
Monoclonal antibodies (McAbs) against chloramphenicol (CAP) were produced to detect CAP residues, which could be toxic and
possesses a potential threat to human health. The CAP-BSA conjugate was obtained by bovine serum albumin (BSA) coupled with
CAP, and used to immunize the mice. The splenocytes from the immunized mice were fused with mouse myeloma cells SP2/0 to form
hybridoma, which may secrete McAbs against CAP. Hybridomas 1D1 and 3G12 secreting McAbs against CAP were obtained by screening. Ascites containing McAbs were prepared by injecting 1 x 106 cells of hybridoma 1D1 and 3G12 into the abdomen of mice. Protein A affinity chromatography was used to purify McAbs against CAP in a single chromatographic
step with recovery yield above 80% and purity above 95% and full recovery of antibody activity. Experiments showed that McAb
3G12 was highly specific for CAP and had no cross-reactivity with analogues which have a structure similar to CAP. The IC50 value was 50.8 ng/mL. 相似文献
5.
The role of lipids in maintaining ligand binding properties of affinity-purified bovine striatal dopamine D2 receptor was investigated in detail. The receptor, purified on a haloperidol-linked Sepharose CL6B affinity column, exhibited low [3H]spiroperidol binding unless reconstituted with soybean phospholipids. In order to understand the role of individual phospholipids in maintaining the receptor binding activity, the purified preparation was reconstituted separately with individual phospholipids and assayed for [3H]spiroperidol binding. Except for phosphatidylcholine and phosphatidylethanolamine, that respectively restored 30 and 20% binding as compared to that obtained with soybean lipids, reconstitution with other lipids had very little effect. When various combinations of phospholipids were used for reconstitution, a phosphatidylcholine and phosphatidylserine mixture seemed to almost fully restore the receptor binding. A mixture of phosphatidylcholine and phosphatidylethanolamine was as effective as phosphatidylcholine alone in reconstituting ligand binding; however, when phosphatidylserine was also included in the mixture, there was a pronounced increase in binding (about 2-fold compared to the soybean lipids and about 6-fold compared to the phosphatidylcholine-phosphatidylethanolamine mixture). Substitution of other phospholipids or cholesterol for phosphatidylserine in phosphatidylcholine and phosphatidylethanolamine mixture had little effect. Maximal reconstitution of [3H]spiroperidol binding was obtained with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine mixture (2:2:1, w/w) at a concentration of 0.5 mg/ml. The reconstituted receptor exhibited high affinity binding for [3H]spiroperidol which was comparable to that obtained with membrane or solubilized preparations. Various dopaminergic antagonists and agonists showed appropriate order of potency for the reconstituted receptor. The presently described reconstitution data suggest a role of specific phospholipids in preserving the binding properties of dopamine D2 receptor and should prove useful in studies on functional reconstitution of the receptor. 相似文献
6.
M. A. Burkin I. V. Yakovleva V. V. Sviridov A. A. Burkin G. P. Kononenko N. A. Soboleva 《Applied Biochemistry and Microbiology》2000,36(5):496-501
Four hybrid clones (MM-(AB1)-1, MM-(AB1)-2, MM-(AB1)-3, and MM-(AB1)-4) were obtained by hybridoma technology involving the immunization of BALB/c mice with a BSA conjugate of aflatoxin B1 carboxymethyloxime derivative. Antibodies produced by these clones varied in their ability to recognize the aflatoxin B1 analogues. The sensitivity of enzyme immunoassay based on all monoclonal antibodies was higher compared to analysis based
on polyclonal rabbit antibodies (0.1 and 0.4 ng/ml, respectively). 相似文献
7.
利用4-溴丁酸乙酯对小分子半抗原己烯雌酚(DES)进行活化,引入羧基活性基团,应用活泼酯法将其与牛血清白蛋白(BSA)偶联,合成DES-CP-BSA完全抗原,免疫新西兰长耳白兔,制备特异性抗体.结果显示:成功制备了DES完全抗原,且由此获得了特异性的DES抗体,效价达1.28×105,与己烷雌酚、双烯雌酚的交叉反应分别... 相似文献
8.
Sreedhara Sangadala Peggy Wallace Joseph Mendicino 《Molecular and cellular biochemistry》1991,106(1):1-14
Summary RNA was isolated from cultured swine trachea epithelial cells and mucus-secreting tumor cell lines from human pancreas, lung and colon by extraction with guanidine isothiocyanate. Poly(A)+mRNA rich fractions were purified by repeated chromatography on oligo (dT)-cellulose columns and they were translated in a cell-free rabbit reticulocyte system. Translation products labelled with 35S-methionine were isolated by immunoprecipitation with specific antibodies to the polypeptide chains of mucin glycoproteins and they were analyzed by SDS-PAGE and fluorography. A single principal polypeptide band of 67 kDa was found in all cases when the immunoprecipitates were washed with buffer containing bovine serum albumin and unlabeled deglycosylated mucin glycoprotein. The intensity of the 67 kDa band decreased when unlabeled deglycosylated mucin glycoprotein was added to the translation mixture before immunoprecipitation. Affinity purified monospecific antibodies elicited against chemically deglycosylated polypeptide chains of purified mucin glycoproteins from human and swine trachea and Cowper's gland were all equally effective in immunoprecipitating the 67 kDa translation product. Monospecific antibodies directed against the glycosylated and unglycosylated regions of the polypeptide chain yielded single bands with a molecular size of 67 kDa in each case. Peptide profiles obtained by digestion of the 67 kDa translation product with S. aureus V-8 protease were identical to those obtained with deglycosylated human and swine trachea mucin glycoproteins.These stydies clearly demonstrate that the translation product of swine trachea and human lung, colon and pancreatic mucin glycoprotein gene is a single polypeptide chain of 67 kDa. The relative size and properties of the translation products synthesized with poly (A)+RNA isolated from mucus-secreting cells derived from three different tissues are similar to those of mucin glycoproteins purified directly from mucus secretions of human and swine trachea epithelium.Abbreviations TFMS
Trifluoromethanesulfonic acid
- SDS
Sodium Dodecyl Sulfate
- PAGE
Polyacrylamide Gel Electrophoresis
- GalNAc
N-Acetylgalactosamine
- HTMG
Human Trachea Mucin Glycoprotein
- deHTMG
deglycosylated Human Trachea Mucin Glycoprotein
- STMG
Swine Trachea Mucin Glycoprotein
- deSTMG
deglycosylated Swine Trachea Mucin Glycoprotein
- CCMG
Cowper's Gland Mucin Glycoprotein
- deCGMG
deglycosylated Cowper's Gland Mucin Glycoprotein
- HPMG
Pancreatic Mucin Glycoprotein from BxPC-3 cells
- HCMG
Colon Mucin Glycoprotein from SW 403 cells
- HLMG
Human Lung Mucin Glycoprotein from A-549 cells
- STMG+deSTMG–
antibodies which bind to immobilized STMG but do not bind to immobilized deSTMG
- deSTMG+STMG–
antibodies which bind to immobilized deSTMG but do not bind to immobilized STMG
- STMG+deSTMG+
antibodies which bind to both STMG and deSTMG
- HTMG+deHTMG–
antibodies which bind to immobilized HTMG but do not bind to immobilized deHTMG
- deHTMG+HTMG–
antibodies which bind to immobilized deHTMG but do not bind to immobilized HTMG
- HTMG+deHTMG+
Antibodies which bind to both HTMG and deHTMG 相似文献
9.
D-Ribitol, a five–carbon sugar alcohol, is an important metabolite in the pentose phosphate pathway; it is an integral part of riboflavin (vitamin B2) and cell wall polysaccharides in most Gram-positive and a few Gram-negative bacteria. Antibodies specific to D-ribitol were generated in New Zealand white rabbits by using reductively aminated D-ribose-BSA conjugate as the immunogen. MALDI-TOF and amino group analyses of ribitol-BSA conjugate following 120 h reaction showed ~27–30 mol of ribitol conjugated per mole BSA. The presence of sugar alcohol in the conjugates was also confirmed by an increase in molecular mass and a positive periodic acid–Schiff staining in SDS-PAGE. Caprylic acid precipitation of rabbit serum followed by hapten affinity chromatography on ribitol–KLH–Sepharose CL-6B resulted in pure ribitol–specific antibodies (~45–50 μg/mL). The affinity constant of ribitol antibodies was found to be 2.9?×?107 M?1 by non-competitive ELISA. Ribitol antibodies showed 100 % specificity towards ribitol, ~800 % cross–reactivity towards riboflavin, 10–15 % cross–reactivity with sorbitol, xylitol and mannitol, and 5–7 % cross–reactivity with L-arabinitol and meso-erythritol. The specificity of antibody to ribitol was further confirmed by its low cross-reactivity (0.4 %) with lumichrome. Antibodies to D-ribitol recognized the purified capsular polysaccharide of Haemophilus influenzae type b, which could be specifically inhibited by ribitol. In conclusion, antibodies specific to D-ribitol have been generated and characterized, which have potential applications in the detection of free riboflavin and ribitol in biological samples, as well as identification of cell-surface macromolecules containing ribitol. 相似文献
10.
E.P. Smorodin O.A. Kurtenkov B.L. Sergeyev G.V. Pazynina N.V. Bovin 《Glycoconjugate journal》2003,20(2):83-89
The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were
found in human serum. A set of polyacrylamide (PAA)—based glycoconjugates was applied to the direct and competitive enzyme-linked
immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were
affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and
-Tn antibodies were shown to be specific. The anti-TF IgG bound both Galβ1-3GalNAcα- and Galβ1-3GalNAcβ-PAA, the latter was
three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcα-PAA. The anti-SiaTn
IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 × 10−8 M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown
for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody
specificity to GalNAcβ and GalNAcβ1-3GalNAcβ ligands was found in human serum. Published in 2004.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
Sheep or guinea pig antisera against the purified Ca++ transport ATPase of sarcoplasmic reticulum inhibit Ca++ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca++. The Ca++-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca++ transport. Antibodies prepared against the Ca++ binding protein (C1 protein) have no influence upon either ATPase activity or Ca++ transport and ferritin-labeled anti-C1 antibodies do not bind to microsomes. 相似文献
12.
Kazem Karami Zohreh Mehri Lighvan Hossein Farrokhpour Maryam Dehdashti Jahromi Amir Abbas Momtazi-borojeni 《Journal of biomolecular structure & dynamics》2013,31(13):3324-3340
[Pd{(C,N)–C6H4CH2NH(Et) (Qu)] (2) and [Pd{(C,N)–C6H4CH2NH(Et) (Nar)] (3) (Qu = Quercetin, Nar = Naringin) mononuclear palladium (II) complexes have been synthesized and characterized using elemental analysis, IR and electronic spectroscopy. The interaction of the prepared complexes with calf thymus DNA and bovine serum albumin (BSA), monitored by UV–visible and fluorescence titrations, respectively, have been carried out to better understand the mode of their action under biological conditions. Intercalative binding mode between the complexes and DNA is suggested by the binding constant (Kb) values of 2.5 × 106 and 3.2 × 106 for complexes 2 and 3, respectively. In particular, the in vitro cytotoxicity of the complexes on two cancer cells lines (bladder carcinoma TCC and breast cancer MCF7) showed that the compounds had broad spectrum, anti-cancer activity with low IC50 values and the order of in vitro anticancer activities is consistent with the DNA-binding affinities. In the meantime, the quenching of tryptophan emission with the addition of complexes using BSA as a model protein indicated the protein binding ability. The quenching mechanisms of BSA by the complexes were static processes, according to the results obtained. The competitive binding using Warfarin, Digoxin and Ibuprofen site markers, which contain definite biding sites, demonstrated that the complexes bind to site I on BSA. Ultimately, the binding sites of DNA and BSA with the complexes have been determined by molecular modelling studies. 相似文献
13.
A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles 下载免费PDF全文
A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe3O4@SiO2 magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe3O4@SiO2 and carboxylic acid‐functionalized Fe3O4@SiO2). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC50) values of the two methods were about 0.024 ng ml?1 and 0.046 ng ml?1 respectively and LODs were approximately 0.0002 ng ml?1 and 0.001 ng ml?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs. 相似文献
14.
Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO–CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100–150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC50 = 0.13–0.2 ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03 ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC50 = 1.6 ng/ml and LOD = 0.14 ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory. 相似文献
15.
本研究利用中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV) Erns蛋白,并分析其免疫原性。以BVDV-1 NADL标准毒株基因序列为基础,构建BVDV Erns蛋白重组真核表达质粒pcDNA3.1-BVDV-Erns,转染悬浮培养的CHO细胞,进行上清分泌表达。SDS-PAGE分析Erns蛋白的表达和纯化,并用抗His单克隆抗体和BVDV阳性血清进行Western blotting鉴定纯化蛋白;进一步使用纯化的Erns蛋白免疫新西兰大白兔,通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和细胞间接免疫荧光(indirect immunofluorescence,IFA)实验检测血清抗体水平及其免疫反应活性,用病毒中和实验测定免疫兔血清的中和抗体滴度。BCA蛋白定量试剂盒检测纯化的Erns 相似文献
16.
C. Sivapathasekaran Palashpriya Das Soumen Mukherjee J. Saravanakumar Mahitosh Mandal Ramkrishna Sen 《International journal of peptide research and therapeutics》2010,16(4):215-222
A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension
up to 27 mN m−1. The microorganism produced 1.64 ± 0.1 g l−1 of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays
using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify
the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products
were found to be 90 and 40 mg l−1 respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of
flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human
colon cancer cell lines HCT-15 (IC50 80 μg ml−1) and HT-29 (IC50 120 μg ml−1). 相似文献
17.
Eun-Seong Seo Jin Kang Jin-Ha Lee Go-Eun Kim Ghahyun J. Kim Doman Kim 《Enzyme and microbial technology》2009,44(5):355-357
We synthesized a hydroquinone glucoside (HG) as a potential skin-whitening agent using Leuconostoc mesenteroides (B-1299CB BF563) dextransucrase with hydroquinone (HQ) as an acceptor and sucrose as a donor. The product was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HG was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295 (C12, H16, O7 Na)+. HG was identified as 4-hydroxyphenyl-α-d-glucopyranoside. The optimum condition of HG synthesis, determined using a response surface methodology, was 450 mM HQ, 215 mM sucrose, and 0.55 U/mL dextransucrase; the final HG produced was 544 mg/L. The IC50 of diphenylpicryl-hydrazyl scavenging activity was 3.85 mM indicating a higher antioxidant activity compared to β-arbutin (IC50 = 6.04 mM). HG-mediated inhibition of lipid peroxidation was 3.51% that of HQ (100%) and much higher than that of β-arbutin (0.81% of HQ). In addition the IC50 value of nitrite-scavenging activity was 14.76 mM showing a superior scavenging activity to that of β-arbutin (IC50 = 27.09 mM). 相似文献
18.
Marie-Christine Birling Guy Roussel Françoise Nussbaum Jean-Louis Nussbaum 《Neurochemical research》1993,18(8):937-945
Bovine myelin/oligodendrocyte glycoprotein (MOG) was purified from a Wolfgram protein fraction of brain myelin by molecular sieving and preparative gel electrophoresis. The N-terminal sequence of this wheat germ agglutinin reacting glycoprotein was determined. Antibodies against purified MOG and synthetic N-terminal octapeptide of MOG were produced in rabbits. Respective affinity purified antibody preparations gave identical results on Western blots. Treatment with specific glycosidases indicated that the oligosaccharide chains of MOG are only of N-chain type. This glycoprotein seems to be restricted to mammalian species since it was not detected in other animal species, ranging from fish up to reptiles. Immunohistochemical investigations on rat brain sections revealed that MOG is restricted to myelin sheaths and oligodendrocytes, thus corroborating previous results obtained with the MOG 8-18C5 monoclonal antibody. Decreased staining pattern in Jimpy brain further attested its specific localization in myelin-related structures. The octapeptide site-specific antibodies were not reactive on brain sections which may be attributed to the burying of this N-terminal sequence in the membrane. These MOG polyclonal antibodies appear to be valuable tools for further studies concerning this minor glycoprotein.Abbreviations BSA
bovine serum albumin
- CNS
central nervous system
- DM-20
minor myelin proteolipid protein
- MAG
Myelin-associated glycoprotein
- MBP
myelin basic proteins
- MOG
Myelin/oligodendrocyte glycoprotein
- OMgp
Oligodendrocyte/Myelin glycoprotein
- PAGE
polyacrylamide gel electrophoresis
- PBS
phosphate buffered saline
- PeptMOG
n-terminal octapeptide of MOG
- PLP
major myelin proteolipid protein
- PMSF
phenylmethylsulfonylfluoride
- SDS
sodium dodecylsulphate
- TBS
Tris buffered saline
- WPF
Wolfgram protein fraction
- WGA
Wheat germ agglutinin 相似文献
19.
Daniela Tavella Carl B. Watt Ying-Yet Thomas Su Kwen-Jen Chang Sarah Handlin Valita Gaskie Dominic Man-Kit Lam 《Neurochemistry international》1985,7(3):455-466
The hybridoma technology of Kohler and Milstein (1975) was utilized to produce monoclonal antibodies against the enkephalins. Two hybridomas, AD4 and DB4, produced monoclonal antibodies of the IgG type 1 class against Leu5-enkephalin that were highly specific for Leu5- and Met5-enkephalin. AD4 exhibited almost equal reactivity with either Leu5- or Met5-enkephalin, whereas DB4 exhibited only a 20% cross-reactivity with Met5-enkephalin. The IC50 of these monoclonal antibodies were approximately two orders of magnitude greater than the IC50 a polyclonal antiserum against enkephalins (A206; Miller et al 1978) used routinely in many immunochemical and immunocytochemical studies.The monoclonal antibodies, AD4 and DB4, exhibited specific sequence and size requirements for binding enkephalin-related peptides. The amino acid sequence Gly-Gly-Phe-Leu or Gly-Gly-Phe-Met was essential for recognition by AD4 and DB4. However, Tyr-Gly-Gly-Phe which lacks Leu or Met in the fifth position did not react with our monoclonal antibodies. Moreover, enkephalin-related peptides in which the enkephalin sequence was situated at the amino terminus and which contained six or more amino acids did not react significantly with AD4 or DB4. In particular, unlike the polyclonal antiserum A206, our monoclonal antibodies do not react with dynorphins 1–6 or 1–13. However, when the monoclonal antibody (AD4) was used to localize immunohistochemically the population of enkephalinergic amacrine cells in the chicken retina, it provided a staining pattern quite comparable to that observed in previous studies (Watt et al., 1983) using the polyclonal enkephalin antiserum A206. This finding therefore demonstrates that the immunoreactive products visualized in the enkephalin-immunoreactive amacrine cells of the chicken retina with the polyclonal antiserum correspond to authentic enkephalin or peptides very closely related to the enkephalins. 相似文献
20.
Yun Li Nana Wang Dong Lin Xiaohui Liu Yong Yang 《Journal of biomolecular structure & dynamics》2020,38(17):4977-4996
AbstractTwo new nickel (II) triphenylphosphine complexes derived from tridentate aroylhydrazone ligands [H2L1 = 2-hydroxy-3-methoxybenzylidene)benzohydrazone and H2L2 = N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone] and triphenylphosphine were prepared and their molecular structures were determined by single crystal X-ray diffraction analysis. Both nickel(II) complexes showed slightly distorted square planar geometry with one tridentate aroylhydrazone ligand coordinated through ONO donor atoms and one triphenylphosphine ligand coordinated to the nickel center through the phosphorus atom. DNA interaction studies indicated that both complexes possessed higher affinity to herring sperm DNA (HS-DNA) than the corresponding free aroylhydrazone ligand. Molecular docking investigations showed that both complexes could bind to DNA through intercalation of the phenyl rings between adjacent base pairs in the double helix. Meanwhile, bovine serum albumin (BSA) binding studies revealed the complexes could effectively interact with BSA and change the secondary structure of BSA. Further pharmacological evaluations of the synthesized complexes by in vitro antioxidant assays demonstrated high antioxidant activity against NO· and O2˙? radicals. The anticancer activity of each complex was assessed through in vitro cytotoxicity assays (CCK-8 kit) toward A549 and MCF-7 cancer cell and normal L-02 cell lines. Significantly, the Ni(II) complex derived from H2L1 ligand was found to be more effective cytotoxic toward MCF-7cancerous cell with the IC50 value equaled 9.7?μM, which showed potent cytotoxic activity over standard drug cisplatin. Abbreviations A549 human lung carcinoma cell BSA bovine serum albumin CCK-8 Cell Counting Kit-8 DFT density functional theory DNA deoxyribonucleic acid DPPH˙ 2,2-diphenyl-1-picrylhydrazyl H2L1 2-hydroxy-3-methoxybenzylidene)benzohydrazone N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone H2L2 N′-(2-hydroxy-3-methoxybenzylidene)-2-hydroxybenzoylhydrazone HOMO highest occupied molecular orbital IC50 the 50% activity L-02 human normal liver cell LOMO lowest unoccupied molecular orbital (LUMO) MCF-7 human breast carcinoma cell NO˙ nitric oxide O2˙? superoxide anion SOD superoxide dismutase Communicated by Ramaswamy H. Sarma 相似文献