Cyanogen Bromide-activated Sepharose 4B Research Articles

Purification, Characterization and Antimicrobial Properties of Hemolymph Lectin from the Larva of Red Palm Weevil, Rhynchophorus ferrugineus

Lectins are renowned hemagglutinins and multivalent proteins with a well-known quality for sugar-binding specificity that participate significantly in invertebrate defense functions. Studies on biological activity of lectin from coleopteran insect are very scarce. A lectin with specific affinity for N-acetyl neuraminic acid was purified from the hemolymph of the larva of the red palm weevil, Rhynchophorus ferrugineus by biospecific adsorption using formalinized rabbit erythrocytes and affinity chromatography using PSM-linked cyanogen bromide activated Sepharose 4B. The specific activity of the lectin purified by affinity chromatography was much higher than the lectin purified by biospecific adsorption. The binding specificity of the weevil lectin distinguishes it from other known insect lectins. Like the crude agglutinin, the lectin purified by affinity chromatography also showed the same pattern of specificity towards erythrocytes. However, 4-to-8-fold decrease in HA titer was observed when tested with the purified lectin. In the same way, reduction is also observed in the HAI titer of the purified lectin with most of the inhibitors except PSM where the HAI titer was identical both in the crude agglutinin and purified lectin. Sugars N-acetyl neuraminic acid, N-acetyl mannosamine and N-acetyl-D-galactosamine inhibited the HA titer of the purified lectin with greater efficacy than the crude agglutinin. The sialic acid specificity of the lectin was confirmed by 16-fold reduction in HA titer with asialo rabbit erythrocytes and 32-fold reduction in HAI titer with desialylated PSM. The purified lectin is homogenous on sodium dodecyl sulphate-polyacrylamide electrophorogram with a molecular weight of about 60 kDa. The lectin showed antimicrobial activity against pathogenic bacteria Streptococcus mutans, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeroginosa and fungi Candida albicans and Aspergillus niger.

A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice

BackgroundCryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads.MethodsThirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively.ResultsThe IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues.ConclusionsThe improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.

Continuous monitoring of IgG using immobilized fluorescent reporters.

In the manufacture of therapeutic monoclonal antibodies, the clarified cell culture fluid (CCF) is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of immunoglobulin G (IgG) loading to avoid wastage. Continuous real-time monitoring of IgG flowthrough is of great interest, therefore. We previously developed a fluorescence-based monitoring technology that allows batch mix-and-read mAb detection in the CCF. Here, we report the use of reporters immobilized on cyanogenbromide-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands in a small monitoring column to produce an immediately-detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified CCF emerging from a protein A column, without prior sample preparation. We observed a significant change in fluorescence intensityat 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 18 s at a flow rate of 0.5 ml/min. The current small-scale technology is suitable for use in process development, but the chemistry should be readily adaptable to larger scale applications using fiber-optic sensors, and continuous IgG monitoring could be applicable in a variety of upstream and downstream process settings.

Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818)

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.

Identification of mimotope of Mycoplasma pneumoniae P1 protein and its potential value in serodiagnosis

Traditional diagnostic methods cannot meet the requirements for rapid diagnosis of Mycoplasma pneumoniae. Therefore, developing a serological diagnostic method with high specificity and sensitivity is urgent. In this study, we expressed and purified the recombinant P1’ protein, which comprises amino-acid residues 1160–1498 of the P1 protein. Thereafter, after immunising New Zealand rabbits, we purified the anti-P1’ polyclonal antibody using cyanogen bromide-activated sepharose 4B. By using this purified antibody as target molecule, we then performed a 4-round biopanning for a phage display random 12-mer peptide library. After sequencing analysis, we found that the polypeptide HLQMRLTKLRMP was a 12-mer peptide specifically binding to the anti-P1' polyclonal antibody and had 75% homology with the 1266-1272 amino acid (QVRTKLR) of the M. pneumoniae P1 protein. Besides, we further confirmed that the representative phage 1 which displayed this peptide could specifically bind to the anti-P1’ polyclonal antibody by dot immunobinding assay. Indirect ELISA showed that the synthesized 12-mer peptide could specifically bind to the serum of M. pneumoniae positive patients. The sensitivity and the specificity were 81.87% and 95%, respectively. These results indicated that HLQMRLTKLRMP might be a dominant epitope of the P1 and has the potential for serological diagnosis of M. pneumoniae.

Improved chromatographic method for purification of lactoperoxidase from different milk sources

ABSTRACTOur previous studies showed that sulfanilamide is a new competitive inhibitor of and can be used in the purification of lactoperoxidase (LPO, EC1.11.1.7) from milk. However, this method has some disadvantages like a lower purification factor. The aim of the present study is to improve the purification process of milk LPO from different sources. For this purpose, 16 commercial sulfanilamide derivatives were selected for inhibition studies to determine the best inhibitor of bovine LPO by calculating kinetic parameters. A cyanogen bromide-activated Sepharose 4B affinity matrix was synthesized by coupling with each competitive inhibitor. Among the inhibitors, 5-amino-2-methylbenzenesulfonamide and 2-chloro-4-sulfamoylaniline were used as ligands for the purification of LPO from bovine, buffalo, cow, and goat milks with 1059.37, 509.09, 232.55, and 161.90, and 453.12-, 151.86-, 869.00-, and 447.57-fold, respectively. Our results show that 5-amino-2-methylbenzenesulfonamide, 2-chloro-4-sulfamoylaniline, and 5-amino-1-naphthalenesulfonamide are the best inhibitors for one-step purification of the enzyme.

Combined use of irreversible binding and MRM technology for low- and ultralow copy-number protein detection and quantitation

In this paper, we present a method for the determination of low- and ultralow copy-number proteins in biomaterials based on a combination of concentrating the protein from the sample onto cyanogen bromide-activated Sepharose 4B (via nonspecific binding of free amino groups) and MRM. The detection limit and the dependence of the MRM peak areas on the concentration of protein in the sample were determined using the proteins CYP102 and BSA, as a model system, both in solution and after their addition to human plasma. Nonspecific protein enrichment of proteins from diluted sample volumes of 10-50 mL was found to increase the range of linear dependence of the chromatographic peak area on concentration by more than three orders of magnitude, allowing a lower LOD limit (LLOD) of as low as 10(-18) M. At this LLOD, at least two tryptic peptides of CYP102 and BSA could be detected with S/N of ≥7.0. The results were equally good for samples containing pure protein mixtures and proteins spiked into diluted depleted human blood plasma.

Crataeva tapia bark lectin is an affinity adsorbent and insecticidal agent

Hemagglutinating activity has been associated to presence of lectin, carbohydrate-binding proteins. In this work Crataeva tapia bark lectin (CrataBL) was purified in milligram quantities (28 mg per g of bark) by ion exchange chromatography. The lectin was thermo-stable, ion-independent and N-terminal sequence analysis demonstrated similarity with miraculin and miraculin-like proteins (plant defensive proteins). Glycosylated nature of CrataBL was revealed using glycoprotein staining (periodic acid–Schiff's reagent), positive for polypeptides of apparent molecular masses 21 and 40 kDa on SDS-PAGE. Gel diffusion assay showed that glucose/mannose isolectins from Cratylia mollis recognized CrataBL glycan moiety. CrataBL hemagglutinating activity was inhibited by glycoproteins and CrataBL immobilized on cyanogen bromide-activated sepharose 4B (1 mL) bound 0.54 mg of glycoprotein (casein, fetuin and ovalbumin) per cycle. CrataBL was an insecticide agent against Nasutitermes corniger workers (termite that attack woods) with LC 50 of 0.475 mg mL −1 for 6 days.

The Hemolymph of the Ascidian Styela plicata (Chordata-Tunicata) Contains Heparin inside Basophil-like Cells and a Unique Sulfated Galactoglucan in the Plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

A Study of Soluble HLA-G 1 Protecting Porcine Endothelial Cells Against Human Natural Killer Cell-Mediated Cytotoxicity

Human natural killer (NK) cells, which can mediate direct lysis of porcine endothelial cells, play an important role in xenograft rejection. HLA-G, which is a critical molecule in maintaining maternal immune tolerance of semi-allogenic fetus, is able to protect susceptible target cells from lysis induced by NK cells. In this study, we investigated whether soluble HLA-G 1 (sHLA-G 1) protected porcine xenogeneic cells against human NK cell-mediated lysis. Methods The human sHLA-G 1 genomic DNA (pcDNA3-sHLA-G 1) was transfected into a B lymphoblastoid cell line 721.221 (LCL721.221) by nucleofector. The sHLA-G 1 expression of the transfected LCL721.221 cells was identified by RT-PCR and Dot-ELISA. The sHLA-G 1 protein was purified by affinity chromatography on anti–HLA-ImAb W6/32 coupled to cyanogen-bromide–activated Sepharose 4B from culture supernates of transfectants. Various concentrations of sHLA-G 1 protein (0, 2, 4, 6, or 8 μg/mL) were added to a NK cell-mediated xenogenic cell lysis system with either NK92 cells or fresh human peripheral blood mononuclear cells (PBMCs) cocultured with the porcine endothelial cells line. A LDH release assay was used to evaluate NK cell-mediated cytotoxicity. Results sHLA-G 1 provided significant protection of porcine endothelial cells against human NK-mediated cytotoxicity in a dose-dependent manner. The rates of NK92 cell-mediated cytotoxicity were reduced to 83.4 ± 5.7% (2 μg/mL), 56.6 ± 9.3% (4 μg/mL), 39.3 ± 10.2% (6 μg/mL), and 31.2 ± 4.9% (8 μg/mL) versus 96.9 ± 3.0% in the control group ( P < .01). Similarly, adding 6 μg/mL sHLA-G1 reduced the mean rate of PBMC-mediated cytotoxicity ( n = 4) to 5.8 ± 1.6% from 23.9 ± 1.3% in the control group ( P < .01). Conclusions These results indicated that sHLA-G 1 protected xenogeneic porcine endothelial cells against attack by human NK cells, thus providing a new approach to overcome NK-mediated immunity to xenografts.

Cyanogen bromide-activated coupling: DNA catalytic chromatography purification of EcoRI endonuclease

A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4-6 d.

Characterization of Alternative Cytosolic Forms and Cellular Targets of Mouse Mitochondrial Thioredoxin Reductase

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.

Two Point Mutations of RPE65 from Patients with Retinal Dystrophies Decrease the Stability of RPE65 Protein and Abolish Its Isomerohydrolase Activity

RPE65 is the isomerohydrolase in the retinoid visual cycle essential for recycling of 11-cis retinal, the chromophore for visual pigments in both rod and cone photoreceptors. Mutations in the RPE65 gene are associated with inherited retinal dystrophies with unknown mechanisms. Here we show that two point mutations of RPE65, R91W and Y368H, identified in patients with retinal dystrophies both abolished the isomerohydrolase activity of RPE65 after a subretinal injection into the Rpe65-/- mice and in the in vitro isomerohydrolase activity assay, independent of their protein levels. Further, the R91W and Y368H mutants showed significantly decreased protein levels but unchanged mRNA levels when compared with the wild-type RPE65 (wtRPE65). Protein stability analysis showed that wtRPE65 is a fairly stable protein, with an apparent half-life longer than 10 h, when expressed in 293A cells. Under the same conditions, mutants R91W and Y368H both showed substantially decreased protein stabilities, with half-lives less than 2 and 6 h, respectively. Subcellular fractionation and Western blot analysis demonstrated that wtRPE65 predominantly exists in the membrane fraction, while both of the mutants are primarily distributed in the cytosolic fraction, suggesting that these mutations disrupt the membrane association of RPE65. However, palmitoylation assay showed that wtRPE65 and both of the mutants were palmitoylated. These results suggest that these mutations may result in critical structural alterations of RPE65 protein, disrupt its membrane association, and consequently impair its isomerohydrolase activity, leading to retinal degeneration.

PDE7A1, a cAMP-specific Phosphodiesterase, Inhibits cAMP-dependent Protein Kinase by a Direct Interaction with C

The N-terminal regulatory region of the high affinity cAMP-specific phosphodiesterase, PDE7A1, contains two copies of the cAMP-dependent kinase (PKA) pseudosubstrate site RRGAI. In betaTC3 insulinoma cells, PDE7A1 co-localizes with PKA II in the Golgi-centrosome region. The roles PDE7A1 and its regulatory region play in cAMP signaling were examined by studying interactions with PKA subunits. PDE7A1 associates with the dissociated C subunit of PKA (C), but does not bind tetrameric PKA holoenzyme. High affinity binding of C by PDE7A1 inhibits kinase activity in vitro (IC50 = 0.5 nm). The domain containing PKA pseudosubstrate sites at the N terminus of PDE7A1 mediates complex formation with C. The PDE7A1 N-terminal repeat region inhibits C activity in CHO-K1 cells and also suppresses C dependent, cAMP-independent, physiological responses in yeast. Thus, PDE7A1 possesses a non-catalytic activity that can contribute to the termination of cAMP signals via direct inhibition of C. This study identifies a novel inhibitor of PKA and a non-catalytic affect of a cyclic nucleotide phosphodiesterase.

Determination of Chloramphenicol Residue in Chicken Tissues by Immunoaffinity Chromatography Cleanup and Gas Chromatography with aMicrocell Electron Capture Detector

A rapid and sensitive gas chromatography (GC) method was developed to detect chloramphenicol in chicken tissues. The extracted samples were cleaned up using the immunoaffinity column prepared by coupling antichloramphenicol monoclonal antibody with cyanogen bromide-activated Sepharose 4B. The dynamic column capacity of chloramphenicol was 3265 ng/mL gel. The eluate was evaporated to dryness, and residues were derivatized and determined by GC with a microcell electron capture detector. Average recoveries were 86.6 to 96.9% for chicken muscle and 74.3 to 96.1% for chicken liver. The limit of quantitation of the method was 0.05 ng/g for chicken muscle and 0.1 ng/g for chicken liver.

Isolation of protein subpopulations undergoing protein-protein interactions.

A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.

Differential Effects of Circulating IgA Isolated from Patients with IgA Nephropathy on Superoxide and Fibronectin Production of Mesangial Cells

Background: IgA nephropathy (IgAN) is characterized by predominant deposition of IgA in the glomerular mesangium. Serum IgA is often elevated in patients with IgAN, and it has been postulated that it is responsible for the mesangial lesions. However, the direct effect of circulating IgA on mesangial cells is not clear. Methods: We investigated the effects of sera and IgA which were isolated from patients with IgAN on thymidine uptake, superoxide and fibronectin production and fibronectin mRNA expression of cultured rat mesangial cells, and we compared the findings to the effects of IgA isolated from patients with non-IgA mesangial proliferative glomerulonephritis (MsPGN) and normal controls. IgA was isolated with affinity chromatography using cyanogen bromide activated Sepharose 4B coupled to sheep antihuman IgA antiserum. Results: Our results demonstrated that both sera and IgA from patients with IgAN dose-dependently increased mitogenesis of mesangial cells as measured by ³H-labeled thymidine uptake. The thymidine uptake by sera and IgA isolated from patients with IgAN was significantly higher than that of sera and IgA isolated from patients with MsPGN and normal controls. Sera and IgA from patients with IgAN significantly enhanced superoxide and fibronectin production and fibronectin mRNA expression of mesangial cells. The superoxide and fibronectin production was also significantly higher as compared with patients with MsPGN and normal controls. Conclusions: Our results indicate that circulating IgA isolated from patients with IgAN is different from that of patients with MsPGN and normal controls and may potentially induce oxidative injury and production of extracellular matrix of glomerular mesangial cells in IgAN.

Immunological significance of metal induced conformational changes in the mitogenic achatininH binding to carbohydrate ligands.

9-O-Acetyl neuraminic acid specific lectin (AchatininH) was isolated from the hemolymph of the land snail Achatina fulica by affinity chromatography on sheep submaxillary mucin (SSM) coupled cyanogen bromide activated Sepharose 4B. The molecular weight of the native protein was 2.42 kDa. UV-Vis absorption, fluorescence and circular dichroism spectroscopic studies on AchatininH revealed the importance of divalent metal ions (Ca2 +, Mg2+ and Mn2+) on lectin conformational change associated with activity of lectins. The binding of these cations changes lambdamax to shorter wavelength in the far UV region (blue shift) and longer wavelength in UV region (red shift), indicating substantial contribution of aromatic side chain in the far UV region on binding with metal ions. The results infer that divalent cations cause conformational changes in lectin which may be responsible for affinity with their carbohydrate moiety.

Simultaneous analysis of delta9-THC and its major metabolites in urine, plasma, and meconium by GC-MS using an immunoaffinity extraction procedure.

A simple extraction procedure for delta9-tetrahydrocannabinol (delta9-THC) and its metabolites from various biological specimens was developed based on immunoaffinity chromatography. Using the affinity resin prepared by immobilization of THC antibody onto cyanogen bromide-activated Sepharose 4B, delta9-THC and its major metabolites including 11-nor-delta9-THC-9-carboxylic acid (delta9-THCCOOH), 11-hydroxy-delta9-THC (11-OH-delta9-THC), and 8beta,11-dihydroxy-delta9-THC (8beta,11-diOH-delta9-THC) were extracted simultaneously from plasma or urine after enzyme hydrolysis. The samples were derivatized as TMS derivatives and analyzed by gas chromatography-mass spectrometry in EI mode with SIM monitoring. Greater than 87% extraction recovery of the four analytes was obtained from both plasma and urine at 5 and 50 ng/mL concentration levels. The method was also used for meconium analysis with some modification. The extraction recovery from meconium, however, was lower than that of plasma and urine, ranging from 52 to 72% at the 10-ng/g level. All compounds showed good linearity within the tested ranges up to 100 ng/mL (g). The limits of detection ranged from 0.5 to 2.5 ng/mL in plasma and urine, and from 1.0 to 2.5 ng/g in meconium. Analysis of 24 meconium specimens showed that 11-OH-delta9-THC is indeed an important metabolite in meconium.

Evaluation of alpha-glucosidase inhibition by using an immobilized assay system.

The inhibitory effects of natural and synthetic inhibitors on the intestinal membrane-bound hydrolase, alpha-glucosidase (AGH), were evaluated by using an immobilized cyanogen bromide-activated Sepharose 4B support. Immobilized AGH (iAGH) inhibition study by synthetic inhibitors (acarbose and voglibose) revealed that the magnitude of inhibition differed from that in the free AGH (fAGH) study: IC50 value of acarbose in iAGH-maltase assay system, 340-430 nM; fAGH, 11 nM. iAGH-maltase inhibition by both inhibitors was influenced by blocking reagents with different functional groups (COOH, OH, CH3, and NH2 groups). On the other hand, significant iAGH-sucrase inhibitory activity was observed only when using the negatively charged support induced by 0.1 M beta-alanine. The Km values obtained in the iAGH assay system were similar to those from the fAGH method. With natural inhibitors, the iAGH-sucrase inhibitory activity of D-Xylose, with in vivo glucose suppression, increased twice compared to that in fAGH. Green tea extract gave almost the same inhibition for both AGH assay systems.