Bovine Serum Albumin Conjugate Research Articles

The mannose receptor is expressed by olfactory ensheathing cells in the rat olfactory bulb

Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.

AN OBLIQUE-INCIDENCE REFLECTIVITY DIFFERENCE STUDY OF THE DEPENDENCE OF PROBE-TARGET REACTION CONSTANTS ON SURFACE TARGET DENSITY USING STREPTAVIDIN-BIOTIN REACTIONS AS A MODEL

A combination of the microarray platform and oblique-incidence reflectivity difference (OI-RD) microscopy was used to study the dependence of kinetic constants of probe-target reactions on the surface density of immobilized targets. Streptavidin-biotin reactions with a very high binding affinity were employed as the study model. Oblique-incidence reflectivity difference microscopy, a label-free and surface-based detection technique, was developed for monitoring real-time binding curves between two interactive biomolecules, enabling the acquisition of kinetic constants such as on-rate, off-rate, and equilibrium dissociation constants. These kinetic constants are important in characterizing biomolecular interactions because in living cells all intercellular and intermolecular reactions are at dynamic rather than at stable equilibrium. The kinetic constant of streptavidin binding to surface-immobilized biotin-bovine serum albumin was demonstrated to be significantly affected by the density of surface bovine serum albumin conjugates, mainly due to mass-transport effects within targets.

Development of an Enzyme Immunoassay Using a Monoclonal Antibody against the Psychoactive Diterpenoid Salvinorin A

Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 μg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

Monoclonal antibody-based enzyme-linked immunosorbent assay for detection of total malachite green and crystal violet residues in fishery products

A rapid easy-to-use trace level direct competitive enzyme-linked immunosorbent assay (dc-ELISA) detection of total residual malachite green (MG), crystal violet (CV) and their corresponding primary metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in fishery products in a single assay was developed. The monoclonal antibodies, anti-MG and anti-CV mAbs, were prepared using carboxyl-malachite green (CMG) and cationized bovine serum albumin (cBSA) conjugates as immunogen. The linear range for the quantitative detection of total MG, CV and their primary metabolites LMG and LCV was between 0.15 to 4.5 ng mL−1 with a half maximal inhibitory concentration (IC50) at 0.56 ± 0.04 ng mL−1 (n = 5). The anti-MG mAbs exhibited 98% cross-reactivity to CV, less than 0.1% cross-reactivity with LMG and LCV, and no cross-reactivity with chloramphenicol, enrofloxacin, sulfadiazine, and tetracycline. Application of the dc-ELISA in fish tissue samples gave a limit of detection (LOD) of 0.37 ng g−1. The improved total detection lead to a recovery of 74.60 ± 8.38% at 0.5 ng g−1 and 87.47 ± 12.83% at 2.0 ng g−1 that was better than existing techniques. The dc-ELISA showed total MG in 7 out of 44 field fish samples that were confirmed with LC-MS/MS. The easy-to-use, inexpensive, and rapid dc-ELISA for the detection of total MG, CV and their corresponding primary metabolites holds promise for field applications.

Detection of Aflatoxin M1 in Milk by Dynamic Light Scattering Coupled with Superparamagnetic Beads and Gold Nanoprobes

This study aimed to develop a rapid and sensitive method for detection of aflatoxin M1 (AFM) by dynamic light scattering (DLS) coupled with superparamagnetic beads and gold nanoprobes. The nanoprobes were synthesized by the conjugate of AFM and bovine serum albumin (AFM-BSA), BSA, and gold nanoparticles. Magnetic beads-based immunosorbent assay (MBISA) was used to measure the concentration of AFM by direct competition between AFM and nanoprobes. DLS was used to determine the concentration of unattached nanoprobes that was positively proportional to the concentration of AFM in the sample. TEM images prove that the as-prepared nanoprobes were able to attach on the magnetic beads through the antibody-antigen reaction. Compared to conventional ELISA, MBISA could effectively reduce the incubation time to 15 min in buffer solution and completely eliminate the color development step, thus simplifying the analysis of AFM. A linear relationship was observed between the inhibition values and the concentrations of AFM in both buffer solution (0-1000 ng·L(-1)) and spiked milk samples (0-400 ng·L(-1)). The limit of detection was found to be 37.7 ng·L(-1) for AFM in buffer solution and 27.5 ng·L(-1) in milk samples. These results demonstrate that DLS coupled with magnetic beads and gold nanoprobes is a rapid and effective method to detect AFM. This method could also be easily extended to rapid detection of other mycotoxins and biological species.

A simple and rapid optical biosensor for detection of aflatoxin B1 based on competitive dispersion of gold nanorods

This report illustrates a promising one-step and label-free optical biosensor for determination of aflatoxin B1 (AFB1) that is most commonly found in foods and highly dangerous even at very low concentrations. In this research, gold nanorods (GNRs) were employed as a sensing platform, which showed high stability under high ionic strength conditions without addition of any stabilizing agent. GNR-AFB1–BSA (bovine serum albumin) conjugates aggregated after mixing with free antibodies, resulting in significant changes in absorption intensity. At the same time the existence of AFB1 molecules in samples caused dispersion of nanorods, as a result of competitive immune-reaction with antibodies. By taking advantages of the competitive dispersion of GNRs, the developed method could effectively reduce false results caused by undesirable aggregation, which is a big problem for spherical gold nanoparticles. Absorption intensity of UV–vis spectra served as the sensing indicator, with dynamic light scattering (DLS) measurement as another sensing tool. The designed biosensing system could detect AFB1 in a linear range from 0.5 to 20ngmL−1, with a good correlation coefficient of 0.99. And the limit of detection (LOD) was 0.16ngmL−1, indicating an excellent sensitivity with absorbance result. The recoveries of the spiked AFB1 in real peanut samples ranged from 94.2% to 117.3%. Therefore the proposed nano-biosensor was demonstrated to be sensitive, selective, and simple, providing a viable alternative for rapid screening of toxins in agriculture products and foods.

Conjugation of nano and quantum materials with bovine serum albumin (BSA) to study their biological potential

Conjugates of gold nanoparticles (AuNPs) and semiconductor quantum dots (CdS/T) have been synthesized with bovine serum albumin (BSA) using wet chemistry. The optical properties of nano and quantum materials and their BSA conjugate have been studied using UV–Visible and Fluorescence spectroscopy. UV–Visible spectrum of pure BSA showed an absorption maximum at 278nm, which showed blue shift after its conjugation with nano and quantum materials. Increased concentration of AuNPs during conjugation resulted in broadening of BSA peak (278nm), which can be related to the formation of ground state complex formation, caused by the partial adsorption of BSA on the surface of NPs. However, increased concentrations of BSA resulted in decrease in SPR intensity of gold nanoparticles (528nm) and absorbance peak of BSA started diminishing. AuNPs acted as quencher for BSA fluorescence intensity, when excited at 280nm. The binding constant (K) and the number of binding sites (n) between AuNPs and BSA have been found to be 1.97×102 LM−1 and 0.6 respectively. With quantum dots, conjugation resulted in enhancement of fluorescence emission of quantum dots when excited at 300nm, which might be due to the stabilizing effect of BSA on QDs or due to energy transfer from tryptophan moieties of albumin to quantum dots.

Synthesis of Nanogel-Protein Conjugates.

The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics.

Antioxidant activity of conjugation of metalloporphyrin combining with bovine serum albumin

In this work, two kinds of water-insoluble metalloporphyrin (MP), meso -tetraphenylporphyrin metal complexes (MTPP, M: Zn Ⅱ , Co Ⅱ ) and meso -tetra( p -hydroxylphenyl)porphyrin metal complexes (MTpHPP, M: Zn Ⅱ , Co Ⅱ ), were combined into bovine serum albumin (BSA), a kind of stable and soluble biopolymer. The obtained water-soluble metalloporphyrin at BSA conjugates (MP@BSA) have been characterized by UV-vis spectra, PAGE gel electrophoresis and circular dichroism spectra. It indicated that the water-solubility and stability of metalloporphyrins were significantly increased after being bound into bovine serum albumin. Their SOD-like activities for scavenging superoxide anion free radical (O 2 · - ) were determined by NBT assay and compared with natural antioxidant. The activities of MP@BSA were more decuple than that of metalloporphyrins. The EC 50 values of MP@BSA were 600-fold and 40-fold higher than that of Vitamin C (VC) and BSA, respectively. The hydroxyl substituted metalloporphyrin showed more active than that of MTPP, and EC 50 of CoTpHPP@BSA got to 1.5 mmol/L. Compared with natural Cu,Zn-SOD, the analog quantity of CoTpHPP@ BSA reached 2.73%.

Enzyme-Linked Immunosorbent Assay for Streptomycin in Animal Feeds

Polyclonal antibody against streptomycin was prepared by using a streptomycin–bovine serum albumin conjugate for the immunization of rabbits. Using this antibody, we developed quantitative assays for streptomycin by means of an indirect competitive enzyme-linked immunosorbent assay (icELISA). Fifty percent inhibition concentration (IC50) for the antibody was 3.6 ng/ml. The detection limit was 0.4 ng/ml. The average of recoveries for all samples was 86.14% and the coefficients of variation of intra- and inter-assays were below 18%. The detection limit using the kit was 15 ng/ml in animal feeds.

A highly sensitive automated flow immunosensor based on kinetic exclusion analysis for determination of the cancer marker 8-hydroxy-2′-deoxyguanosine in urine

A highly sensitive automated flow immunosensor has been developed and validated for the measurement of the cancer marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine. The sensor employed the kinetic-exclusion analytical technology using the KinExA™ 3200 instrument. Various concentrations of 8-OHdG were incubated with a fixed amount of mouse anti-8-OHdG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over a bovine serum albumin conjugate of 8-hydroxyguanosine (8-OHG–BSA) coated onto polymethylmethacrylate beads contained in the observation cell of the KinExA instrument. The free anti-8-OHdG antibody was bound to the immobilized 8-OHG–BSA, however the unbound reagents were removed from the beads bed by flushing the system with phosphate-buffered saline. Fluorescent-labeled secondary antibody was passed rapidly over the beads bed, and the fluorescence was recorded during the flow of the secondary antibody through the beads. A calibration curve was generated by plotting the fluorescence signals that were retained on the beads as a function of 8-OHdG concentrations. The assay limit of detection was 0.05 ng ml−1 and the working range of the assay was 0.05–5 ng ml−1. The analytical recovery of urine-spiked 8-OHdG was 96.05–106.55 ± 3.00–6.40%. The precision of the sensor was satisfactory; relative standard deviation values were 3.28–4.89 and 4.40–6.89% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was found to be superior to the conventional enzyme-linked immunosorbent assay for 8-OHdG. The proposed sensor is anticipated to have a great value in measurement of 8-OHdG where a more confident result is needed.

METHODS FOR SANITARY SURVEILLANCE OF LIVESTOCK PRODUCTION. VII. ENZYMOIMMUNOASSAY OF STREPTOMYCIN

Summary The polyclonal rabbit antibodies against streptomycin conjugated with bovine serum albumin in reaction with diglycidyl ether of 1,4-butanediol presents high specificity to streptomycin and its similar structural analog — dihydrostreptomycin. In the con- ditions of indirect competitive enzymoimmunoassay with immobilized antigens, heterologous to immunogene on protein carrier and methods of synthesis, the limit of streptomycin detection is 0.1 ng/ml. The authors consider the use of developed immunoenzyme test- systems for control of antibiotic contamination in milk and eggs with the limit of detection of 10 mkg/kg and in meat with the limit of 20 mkg/kg. Streptomycin (SM) is aminoglycoside antibiotic widely used for many years in our country and abroad for the treatment of acute infectious diseases of animals, despite its dangerous side effects such as allergic reactions, disorders of neuromuscular conduction, and ototoxicity (1 2). In modern practice, the control of SM residues in animal products is often based on enzyme-linked immunosorbent assay (ELISA). This method was implemented in several countries in the form of test systems of various types including biosensor technology (3-12). Russian researchers also have proposed a test system based on direct ELISA and the evaluation test using immunochromatography for the control of SM in milk and dairy products (13, 14). The purpose of this work was obtaining specific immunoreagents based on streptomycin, optimization of conditions of the indirect competitive solid-phase immunoassay, and assessing the applicability of this test system for the control of SM residues in milk, eggs and meat. Technique. The experiment was performed using the following chemicals: streptomycin sulfate, diglycidyl ether of 1,4- butanediol, adipic acid dihydrazide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), human transferrin (HT) (Sigma, USA ), sodium borohydride (Serva, Germany), dihydro-streptomycin (G.F. Gauze Research Institute of New Antibiotics, Moscow), dimethylformamide (Fluka, Germany), bovine serum albumin (BSA), gelatin (Gel) and antispecies enzyme conjugate prepared according to the description (15) from horseradish peroxidase (EC 1.11.1.7) and donkey antiserum to rabbit immunoglobulin, sodium periodate of domestic production. ELISA was conducted on high-binding polystyrene plates (Costar, USA) using a photometer Dynatech MR 5000 (Dynatech, Germany). The reaction with diglycidyl ether of 1,4-butanediol for the synthesis of conjugates of SM with BSA and Gel - BSA- SM(100)e, Gel-SM(10)e, Gel-SM(30)e, and Gel-SM(100)e: 6,8 mg SM sulfate (10 umol) in 1 ml 1% NaHCO3 was added with 14 ul (10 umol) diglycidyl ether of 1,4-butanediol (10% solution in dimethylformamide) and stirred at room temperature for 24 hours. Then, 4 mg BSA (0,06 umol) in 0,5 ml of 0,05 M carbonate-bicarbonate buffer (CBB, pH 9,5) was added with 600 ul of the abovementioned mixture (100-fold molar excess of hapten), and three portions of 4 mg Gel (0,025 umol) in 0,5 ml CBB - with, respectively, 25; 75, and 250 ul (10-, 30-, and 100-fold molar excesses), then stirred for 3 hours and dialyzed . The reaction with adipic acid dihydrazide for the synthesis of SM conjugates with BSA and Gel- BSA-SM(100)h, Gel- SM(10)h, Gel-SM(30)h, and Gel-SM(100)h: firstly, 4 mg BSA (0,06 umol) and 12 mg Gel (0,075 umol) (resp., in 0,5 and 1,5 ml water) were added with EDCI (resp., 15 and 30 mg), stirred for 30 minutes, and then the mixture with Gel was divided to 3 equal portions. Then, an aqueous solution of 6,8 mg SM sulfate (10 umol) was added with 1,8 mg of adipic acid dihydrazide and stirred at room temperature overnight. After that, the portion with BSA was added with 600 ul of the mixture containing the reaction product of SM and adipic acid dihydrazide (100-fold molar excess), while the portions with Gel - with, respectively, 25; 75, and 250 ul (10-, 30-, and 100-fold molar excesses), then stirred for 2 hours at room temperature and dialyzed.

Emissive Behavior, Cytotoxic Activity, Cellular Uptake, and PEGylation Properties of New Luminescent Rhenium(I) Polypyridine Poly(ethylene glycol) Complexes

We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N(^)N)(CO)(3)(py-PEG-NH(2))](PF(6)) (py-PEG-NH(2) = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MW(PEG) = 5000 Da, PDI(PEG) < 1.08; N(^)N = 1,10-phenanthroline (phen) (1-PEG-NH(2)), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2-PEG-NH(2)), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3-PEG-NH(2))) and [Re(bpy-PEG)(CO)(3)(py-NH(2))](PF(6)) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine; py-NH(2) = 3-aminopyridine) (4-PEG-NH(2)) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N(^)N)(CO)(3)(py-Et-NH(2))](PF(6)) (py-Et-NH(2) = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-Et-NH(2)), Me(4)-phen (2-Et-NH(2)), Ph(2)-phen (3-Et-NH(2))) and [Re(bpy-Et)(CO)(3)(py-NH(2))](PF(6)) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4-Et-NH(2)). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC(50) = 6.6 to 1152 μM) than their PEG-free counterparts (IC(50) = 3.6 to 159 μM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH(2)-4-PEG-NH(2) have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N(^)N)(CO)(3)(py-PEG-NCS)](PF(6)) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-NCS), Me(4)-phen (2-PEG-NCS), Ph(2)-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)(3)(py-NCS)](PF(6)) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N(^)N)(CO)(3)(py-PEG-Bu)](PF(6)) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-Bu), Me(4)-phen (2-PEG-Bu), Ph(2)-phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO)(3)(py-Bu)](PF(6)) (py-Bu = 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA-4-PEG-BSA and 1-PEG-PEI-4-PEG-PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH(2) and its PEG-free counterpart 3-Et-NH(2) has been investigated using zebrafish embryos as an animal model. Embryos treated with the PEG complex at high concentrations revealed delayed hatching, which has been ascribed to hypoxia as a result of adhering of the complex to the external surface of the chorion.

Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies

Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5' or 8-8' linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA-BSA and negatively reacted with pAHA-BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA-BSA conjugates containing 8-O-4' linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4', 8-8', or 5-5' linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4', 8-5', or 5-5' linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5' and 8-8' linked structure of lignin in the cell walls.

Preparation, structural analysis and bioactivity of ribonuclease A-albumin conjugate: Tetra-conjugation or PEG as the linker

Ribonuclease A (RNase A) is a therapeutic enzyme with cytotoxic action against tumor cells. Its clinical application is limited by the short half-life and insufficient stability. Conjugation of albumin can overcome the limitation, whereas dramatically decrease the enzymatic activity of RNase A. Here, three strategies were proposed to prepare the RNase A–bovine serum albumin (BSA) conjugates. R-SMCC-B (a conjugate of four RNase A attached with one BSA) and R-PEG-B (a mono-conjugate) were prepared using Sulfo-SMCC (a short bifunctional linker) and mal-PEG-NHS (a bifunctional PEG), respectively. Mal-PEG-NHS and hexadecylamine (HDA) were used to prepare the mono-conjugate, R-HDA-B, where HDA was adopted to bind BSA. The PEG linker can elongate the proximity between RNase A and BSA. In contrast, four RNase A were closely located on BSA in R-SMCC-B. R-SMCC-B showed the lowest Km and the highest relative enzymatic activity and kcat/Km in the three conjugates. Presumably, the tetravalent interaction of RNase A in R-SMCC-B can increase the binding affinity to its substrate. In addition, the slow release of BSA from R-HDA-B may increase the enzymatic activity of R-HDA-B. Our study is expected to provide strategies to develop protein–albumin conjugate with high therapeutic potential.

Comparison of Immune Responses to the O-Specific Polysaccharide and Lipopolysaccharide of Vibrio cholerae O1 in Bangladeshi Adult Patients with Cholera

Immunity against Vibrio cholerae O1 is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) part of lipopolysaccharide (LPS). Despite this, human immune responses to V. cholerae OSP have not previously been characterized. We assessed immune responses against V. cholerae OSP in adults with cholera caused by V. cholerae O1 El Tor serotype Inaba or Ogawa in Dhaka, Bangladesh, using O1 OSP-core-bovine serum albumin (OSPc:BSA) conjugates; responses targeted OSP in these conjugates. Responses of Inaba-infected patients to Inaba OSP and LPS increased significantly in IgG, IgM, and IgA isotypes from the acute to convalescent phases of illness, and the responses correlated well between OSP and LPS (R = 0.86, 0.73, and 0.91, respectively; P < 0.01). Plasma IgG, IgM, and IgA responses to Ogawa OSP and LPS in Ogawa-infected patients also correlated well with each other (R = 0.60, 0.60, and 0.92, respectively; P < 0.01). Plasma IgM responses to Inaba OSP and Ogawa OSP correlated with the respective serogroup-specific vibriocidal antibodies (R = 0.80 and 0.66, respectively; P < 0.001). Addition of either OSPc:BSA or LPS, but not BSA, to vibriocidal assays inhibited vibriocidal responses in a comparable and concentration-dependent manner. Mucosal IgA immune responses to OSP and LPS were also similar. Our study is the first to characterize anti-OSP immune responses in patients with cholera and suggests that responses targeting V. cholerae LPS, including vibriocidal responses that correlate with protection against cholera, predominantly target OSP. Induction of anti-OSP responses may be associated with protection against cholera, and our results may support the development of a vaccine targeting V. cholerae OSP.

Synthesis of chitosan networks: Swelling, drug release, and magnetically assisted BSA separation using Fe3O4 nanoparticles

Chitosan (CS) nanohydrogel networks were prepared by reaction with glyceroldiglycidylether (GDE) and poly(dimethylsiloxane) (PDMS), as crosslinking agents in an emulsion system. The nanogel content increased with increasing the amount of crosslinkers and reached to a maximum of 90% with GDE. The nanogels structure was characterized by FT-IR, AFM, DSC, and TGA. The average size for CS-GDE and CS-PDMS particles were 59nm and 180nm, respectively. The swelling behavior of nanohydrogels was observed to be dependent on pH, temperature, degree of crosslinking, and on the chemical structure of crosslinker. The equilibrium water content of CS-GDE nanohydrogels reached to a maximum of 600% at neutral pH, and decreased at high and low pH and low temperature. These nanohydrogels were tested for sodium diclofenac (SDF) loading and releasing efficiency. The covalent conjugation of bovine serum albumin (BSA) and magnetic Fe3O4 nanoparticles on the hydrogels were found to hold a potential application in magnetically assisted bioseparation.

Membrane-initiated estrogen signaling: oncogenic or impotent?

A recent study by Kampa et al. provided further evidence of nongenomic, membrane-initiated estrogen signaling. Using a cell-impermeable estradiol–bovine serum albumin conjugate, the authors identified genes and kinases that were modulated by a membrane-associated estrogen receptor, which they have termed ‘ERx.’ They describe how estradiol-induced ERx function is insensitive to antiestrogens tamoxifen and fulvestrant. The ERx components upregulated kinase signaling pathways and genes implicated in cancer progression, suggesting that ERx may promote antiestrogen resistance. However, the existence of membrane-initiated estrogen signaling and its contribution to antiestrogen resistance in vivo await confirmation.

Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens.

Escherichia coli O148 is a nonencapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit [3)-α-L-Rhap-(1→2)-α-D-Glcp-(1→3)-α-D-GlcNAcp-(1→3)-α-L-Rhap-(1→] differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a D-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SPs of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti-O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPSs of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-SPs that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SPs of both bacteria.

Binding affinity of anti-xylitol antibodies to canine hepatic vessels

Xylitol is used as a sugar substitute in food products. Dogs have been reported to experience lethal liver injury after accidental ingestion of xylitol. Because liver injury may be a serious consequence of canine immune-mediated reactions, antibodies produced against xylitol may attack the liver. Therefore, in the present study, we evaluated whether binding sites for xylitol antibodies are located at the liver or not. Anti-xylitol antibodies were generated by immunization of rabbits with a xylose–bovine serum albumin conjugate. Immunohistological examination showed that binding sites for the anti-xylitol antibodies were located in the hepatic arteries and the portal veins. Western blotting analyses by using a canine liver homogenate showed 4 protein bands with different molecular weights which reacted with anti-xylitol antibodies. Therefore, binding of anti-xylitol antibodies to the vessels may be the first step in an immune-mediated pathogenic response in xylitol toxicity. Further studies are necessary to determine the effects of anti-xylitol antibodies on the liver in the pathogenesis of xylitol toxicity.