Antibody labeling of cholesterol/ceramide ordered domains in cell membranes

A monoclonal antibody raised against mixed monolayers of 60:40 mol % cholesterol/C16-ceramide of known structure was used to label cholesterol/ceramide-rich domains in cell membranes. The antibody, Cer-Chol 405F specifically recognizes the mixed crystalline and homogeneous phase in monolayers, but it does not interact with either of the components separately. It interacts differently with mixed monolayers that contain ceramides of different acyl chain length. When used on cells, the antibody labeling is sensitive to changes in cholesterol and ceramide levels, as well as to over-expression of specific ceramides; this is in agreement with the results that were obtained on lipid monolayers. This represents a proof of concept of the applicability of a new approach to the structural characterization of lipid microdomains in cell membranes. The approach consists of raising antibodies that recognize specific structural organizations of lipids in artificial mixtures, characterizing the antibody/ordered domain complexes in vitro, and subsequently using them to detect the presence of the same (or similar) domains in cell membranes.     

Chiral recognition of D-kyotorphin by lipidic membranes: relevance toward improved analgesic efficiency

D-Kyotorphin (D-KTP), the most potent isomer of the endorphin-like dipeptide kyotorphin (KTP), is a good drug candidate for the treatment of chronic pain and is thought to be involved in receptor-mediated processes. According to the "membrane catalysis" model, ligands interact with membrane lipids to attain high local concentrations in the receptor vicinity and to adopt the necessary conformation for docking. Therefore, the interaction and recognition of D-KTP by membranes is potentially important to its increased analgesic effect. In spite of the neutral net charge of D-KTP at pH 7.4, fluorescence spectroscopy reveals that the interaction with large unilamellar vesicles is more extensive than was observed for KTP. The tyrosine residue interacts extensively with rigid membranes, with a location and well-defined orientation in the bilayer. This suggests not only that D-KTP meets the structural constraints needed for receptor-ligand interaction in a manner similar to that of KTP, but also that the stronger membrane interaction and ability to discriminate rigid membrane domains might contribute to its improved analgesic effect.     

The effect of specific growth rate and death rate on monoclonal antibody production in hybridoma chemostat cultures

Experimental data collected from bench-scale chemostat cultures of mouse-mouse hybridoma cells have been used for the development of a kinetic model of monoclonal antibody production. The specific antibody productivity was found to be proportional to the specific death rate, indicating a stimulating effect of stress on the ability of the cells to produce antibody. The death rate was found to be an exponential function of the average cell age in the culture. Furthermore, a Generalized Maintenance Energy (GME) model is proposed, to take account of the effects of culture stress on the nutrient uptake rates. The proposed relationships agreed very well with other mouse-mouse hybridoma chemostat culture data in the literature.     

Stereoselective recognition of monolayers of cholesterol, ent-cholesterol, and epicholesterol by an antibody

The interaction between a monoclonal antibody and four distinct monolayers with varying degrees of structural, chemical, and stereochemical similarity were studied and quantified. The antibody, raised and selected against cholesterol monohydrate crystals, interacts with cholesterol monolayers stereospecifically, but not enantiospecifically. Monolayers of ent-cholesterol molecules, which are chemically identical to cholesterol and whose structure is the exact mirror image of the cholesterol monolayer, interact with the antibody to the same extent as the cholesterol monolayers. The affinity of the antibody for both enantiomeric monolayers is extremely high. However, the antibody does not interact with monolayers of epicholesterol, which is an epimer of cholesterol: The hydroxy group in epicholesterol is in the 3alpha position rather than in the 3beta position, imposing a different angle between the hydroxy group and the rigid steroid backbone, and a different packing of the molecules. Monolayers of triacontanol, a long-chain primary aliphatic alcohol, interact with the antibody to a lesser extent than the cholesterol and ent-cholesterol monolayers, presumably due to the structural flexibility of the triacontanol molecule. The lack of chiral discrimination by the antibody is thus correlated to the level at which the chirality is exposed at the surface of the monolayers.     

Comparative production of a monoclonal antibody specific for enrofloxacin in a stirred-tank bioreactor

A hybridoma producing monoclonal antibody (MAb) specific for enrofloxacin was cultivated in a 2-L stirred-tank bioreactor in various modes and the performances of each mode were compared. In batch mode, a maximum viable cell and MAb concentration of 9.21 × 10⁵ cells mL^?1 and 67.3 mg L^?1, respectively, were obtained. When the hybridoma was cultivated in a fed-batch culture with the addition of specific nutrients, no improvement in either the viable cell number or MAb concentration was observed. On the other hand, an increase in the production of toxic metabolites, i.e. ammonia and lactate, was observed with growth inhibition of the hybridoma cells occurring at ammonia and lactate concentrations of 2.0 mM and 2.0 g L^?1, respectively. However, the best performance of hybridoma cultivation was achieved in a perfusion culture mode using a spin filter, which was installed in the stirred-tank reactor as a cell retention device with a perfusion rate of 0.80 vvd. Under these conditions a steady viable cell concentration of 1.57 × 10⁶ cells mL^?1 was obtained within five days with an overall productivity and yield of 73.7 mg L^?1 d^?1 and 61.4 mg d^?1, respectively, which was a significant increase over that attained with the batch process.     

Synthesis and biological activity of a novel inhibitor of dihydroceramide desaturase

A novel mechanism-based dihydroceramide desaturase inhibitor (XM462) in which the substrate C5 methylene group is replaced by a sulfur atom is reported. Dihydroceramide desaturase inhibition occurred both in vitro and in cultured cells with IC(50) values of 8.2 and 0.78 microM, respectively, at a substrate concentration of 10 microM. In vitro experiments showed that XM462 produced a mixed-type inhibition (K(i)=2 microM, alpha=0.83). LC-MS analyses showed that accumulation of endogenous dihydroceramides occurred in cells upon treatment with XM462 in serum-free medium, whereas ceramides built up in controls. In addition, XM462 was found to be metabolised to its 1-glucosyl and 1-phosphocholine derivatives, and to the products of N-deacylation and reacylation with palmitoyl and stearoyl groups. In Jurkat A3 cells cultured in serum-free medium, viability, as the percentage of trypan blue unstained cells in total cells, was reduced upon XM462 treatment (5 microM, 24 h), but not in controls. The interest of this compound is discussed.     

Expression and characterisation of recombinant human CD48 and isolation of a human anti‐CD48 monoclonal antibody by phage display

Human CD48, a membrane‐bound, glycosylphosphatidylinositol (GPI)‐linked glycoprotein, is a potential tumour target for the treatment of leukaemias and lymphomas. CD48 is expressed on T‐ and B‐cells, however <5% of CD34⁺ progenitor cells express CD48. A truncated, 45 kDa soluble form of the full length CD48 was expressed in Chinese hamster ovary (CHO) cells, and was shown to consist of a broad range of charge isoforms, with the most abundant isoforms between pI 4.5 and 5.0. The truncated form of CD48 was shown to bind to antibodies raised against native, GPI‐linked CD48 by surface plasmon resonance analysis. A synthetic, human, scFv immunoglobulin gene library was screened against recombinant CD48 by phage display, and an scFv antibody fragment, (designated N2A) was isolated after four rounds of biopanning. N2A was reassembled as a human IgG1 human monoclonal antibody, expressed in CHO cells and the binding of IgG1‐N2A to recombinant CD48 was confirmed by surface plasmon resonance. Flow cytometry studies of IgG1‐N2A binding to Raji cells showed the specificity of N2A for GPI‐linked CD48 was conserved, and presents the potential for IgG1‐N2A as a lead antibody candidate for the treatment of white blood cell malignancies. Copyright © 2005 Society of Chemical Industry     

Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody

A molecular model of the binding site of an anti-carbohydrateantibody (YsT9.1) has been developed using computer-assistedmodeling techniques and molecular dynamics calculations. Sequencehomologies among YsT9.1 and the Fv regions of McPC603, J539and human Bence-Jones protein REI, all of which have solvedcrystal structures, provided the basis for the modeling. Thegroove-type combining site model had a topography which wascomplementary to low energy confonners of the polysaccharide,a Brucella O-antigen, and the site could be almost completelyfilled by a pentasaccharide epitope in either of two dockingmodes. Putative interactions between this epitope and the antibodyare consistent with the known structural requirements for bindingand lead to the design of oligosaccharide inhibitors that probethe veracity of the modeled docked complex. Ultimately boththe Fv model and the docked complex will be compared with independentcrystal structures of YsT9.1 Fab with and without pentasaccharideinhibitor, currently at the stage of refinement.     

一种单克隆抗体的电荷异质体分析

目的对抗程序性死亡受体-1(programmed death-1,PD-1)单克隆抗体的电荷异质体进行结构鉴别及生物学功能分析。方法通过检测抗PD-1单克隆抗体经专一性蛋白酶(唾液酸酶及羧肽酶)酶切前后的变化,初步确认电荷异质体的种类。采用阳离子交换色谱对电荷异质体进行分离和收集,经联用液相色谱-质谱(LC-MS)法分析电荷异质体各组分的组成;通过抑制抗原与配体相互作用影响NFAT荧光素酶报告基因表达的方法确定电荷异质体的生物学功能。结果碱性异质体含量较少,主要变化为重链N-末端谷氨酰胺未环化;酸性异质体主要变化为重链N383脱氨化和糖链末端唾液酸化。各异质体的生物学活性为89%~102%。结论抗PD-1单克隆抗体电荷异质体大部分修饰位点均远离互补决定区(complementary determining region,CDR),且在体外生物学活性方面差异较小。     

Inadvertent introduction of squalene,cholesterol, and other skin products into a sample

Recent developments in analytical techniques permit the chemical ecologist to achieve identification of naturally occurring compounds with relatively small amounts of the products of interest. However, the microanalytical techniques employed frequently require the handling of sample vials and other transferral instruments such as syringes and micropipets, where the analyst's hands come into close contact with the sample. Here we show how inadvertent contamination of a sample with skin lipids can occur simply by catching a 1-ml sample vial by the neck rather than the base or by activating a syringe by holding the plunger extension between the fingers rather than taking it by the head. Squalene, cholesterol, and, to a lesser extent, hydrocarbons and fatty acids from fingers are easily introduced into the sample in this manner. These findings are particularly relevant for a parasitology laboratory such as ours, investigating the function of vertebrate-derived products in hematophagous arthropods.     

Regulation of exocytosis in chromaffin cells by trans-insertion of lysophosphatidylcholine and arachidonic acid into the outer leaflet of the cell membrane

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans-insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans-insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.     

Molecular recognition of the Lewis Y antigen by monoclonal antibodies

The murine monoclonal antibody BR55-2 is directed against thetumor-associated antigen Lewis Y oligosaccharide. The LewisY core antigen is a difucosylated structure consisting of fourhexose units. Analysis of binding profiles of lactoseries isomericstructures by BR55–2 suggest that the binding epitopeincludes the OH-4 and OH-3 groups of the ß-D-galactoseunit, the 6-CH₃ groups of the two fucose units and the N-acetylgroup of the subterminal ß-D-N-acetylglucosamine (ßDGlcNAc).To elucidate the molecular recognition properties of BR55–2for the Y antigen, BR55–2 was cloned, sequenced and itsthree-dimensional structure was examined by molecular modeling.The crystal structure of BR96, another anti-Lewis Y antibody,solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide,and the lectin IV protein in complex with a Lewis b tetrasaccharidecore were used as a guide to probe the molecular basis for BR55–2antigen recognition and specificity. Our modeling study showsthat BR55–2 shares similar recognition features for thedifucosylated type 2 lactoseries Lewis Y structure observedin the BR96-sugar complex. We observe that a major source ofspecificity for the Lewis Y structure by anti-Y antibodies emanatesfrom interaction with the ß-D-N-acetylglucosamineresidue and the nature of the structures extended at the reducingsite of the fucosylated lactosoamine.     

Synthesis of putrescine-imprinted double-layer nanofiber membrane by electrospinning for the selective recognition of putrescine

In this work, we have developed a double-layer nanofiber membrane (NFM) with high selectivity based on molecular imprinting technique by electrospinning methods and applied it in recognizing putrescine. Poly(vinyl alcohol) (PVA) was used as spinning solution, 1,4-butylene glycol as the imitate template, and ninhydrin as chromogenic agent. The adsorption of the putrescine is corresponded to purple-color stains on the NFM, which is digitizing intuitively by the ImageJ software. By quantifying the gray value of the stains on the NFM, its imprinting efficiency (IE) is estimated. The adsorption performance of the double-layer molecular imprinted NFM was studied in detail, showing higher adsorption capacity (71.912 mg mL⁻¹), shorter adsorption equilibrium time of 40 min and higher selectivity to putrescine than our previous works. Furthermore, the material showed remarkable selectivity for putrescine with respect to other three kinds of aliphatic amine in similar molecular structure. The selectivity coefficient (α) of putrescine against cadaverine, spermine, and spermidine are calculated to be 1.734, 2.224, and 2.576, respectively. In addition, since PVA is a water-soluble material, the NFM is not reusable. © 2020 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48932.     

The Chemistry and Biology of 6‐Hydroxyceramide,the Youngest Member of the Human Sphingolipid Family

Sphingolipids are crucial for the life of the cell. In land‐dwelling mammals, they are equally important outside the cell—in the extracellular space of the skin barrier—because they prevent loss of water. Although a large body of research has elucidated many of the functions of sphingolipids, their extensive structural diversity remains intriguing. A new class of sphingolipids based on 6‐hydroxylated sphingosine has recently been identified in human skin. Abnormal levels of these 6‐hydroxylated ceramides have repeatedly been observed in atopic dermatitis; however, neither the biosynthesis nor the roles of these unique ceramide subclasses have been established in the human body. In this Minireview, we summarize the current knowledge of 6‐hydroxyceramides, including their discovery, structure, stereochemistry, occurrence in healthy and diseased human epidermis, and synthetic approaches to 6‐hydroxysphingosine and related ceramides.     

Purification of monoclonal antibodies from cell culture supernatants by Gradiflow™ electrophoresis technology

Monoclonal antibodies (mAbs) are used extensively for analytical, diagnostic and therapeutic applications. The purification of mAbs from cell culture supernatants typically consists of protein A, G or L affinity chromatography, often in association with other conventional chromatographic techniques such as ion exchange and gel filtration. We report the application of Gradiflow^? preparative electrophoresis technology, for the separation of mouse and mouse/human chimeric mAbs from cell culture supernatants in their native state. The one‐step purification of murine mAb HuLym3 shows that mAbs can be purified from hybridoma cell culture supernatants to high purity, and is thus an alternative to other purification methods based on conventional and affinity chromatography for the production of mAbs for analytical and diagnostic applications. A mouse/human IgG1 chimeric mAb produced by Chinese hamster ovary cells was also purified from cell culture supernatant, and the purity achieved suggests that Gradiflow^? electrophoresis could replace affinity chromatography in the downstream processing of mAbs for therapeutic use. Gradiflow^? electrophoresis technology is scaleable and thus is applicable to industrial‐scale purification of mAbs. Copyright © 2005 Society of Chemical Industry     

The Lipids of the Early Endosomes: Making Multimodality Work

Early endosomes are dynamic intracellular compartments that fuse with incoming endocytic carrier vesicles and associated cargoes from the plasma membrane. It has been long known that the chemical structures of lipids confer striking properties and rich biochemistry on bilayers. Although the organisational principles of the plasma membrane are relatively better understood, understanding endosomal membranes has been challenging. It has become increasingly apparent that endosomal membranes, because of their lipid compositions and interactions, use distinct lipid chemistries. We discuss the biochemical and biophysical phenomena in play at the early endosomal membrane. We focus on cholesterol, phosphoinositides, and phosphatidylserine and their clear roles in endosome functions. We discuss the various principles and mechanisms underpinning how these lipids are implicated at the functional level in the working of endosomes, and we summarise early endosomes as a multimodal organelle employing distinct lipid‐specific mechanisms.