The antibody binding site. Labelling of a specific antibody against the photo-precursor of an aryl nitrene

The isolation of specific rabbit antibodies for the haptenic group 4-azido-2-nitrophenyl, is described. These antibodies bind 1.8-2.0mol of hapten [in-(4-azido-2-nitrophenyl)-l-lysine]/mol with an association constant of nearly 10(7)m(-1) at 4 degrees C. On photolysis of the antibody-hapten complex, resulting in the formation of an aryl nitrene at the binding site, hapten was covalently bound to the antibody, and the antibody binding site was blocked. The ratio of labelling of heavy- and light-chains was 2.5:1. Two small peptides were isolated from digests of labelled heavy-chain, indicating that some 13% of the label in the antibody was attached to cysteine-92 and to alanine-93. These residues are adjacent to the major hypervariable region in rabbit heavy-chain (residues 95-105).     

The utility of photo-affinity labels as `mapping' reagents. A study of sub-populations of a specific rabbit antibody by using structurally related photo-affinity reagents

A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.     

Separation and concentration of delta 17-6-keto-PGF1 alpha using monoclonal antibody to omega 3-olefin structure of trienoic prostanoids.

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-prostaglandin F1 alpha (PGF1 alpha) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal antibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for omega 3-olefin structure. The 4G9-12B antibody became more specific for delta 17-6-keto-PGF1 alpha than 6-keto-PGF1 alpha by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, omega 3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating delta 17-6-keto-PGF1 alpha in the human blood or urine.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

Distinguishing mammalian sialidases by inhibition kinetics with novel derivatives of 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid, an unsaturated derivative of N-acetylneuraminic acid.

Kinetic analysis of mammalian sialidases was carried out using analogs of the potent sialidase inhibitor, 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic+ ++ acid (1). Substitutents at C-9 in place of the terminal hydroxyl group included a, 4-azido-2-nitrophenylthio group to give 5-acetamido-2,6-anhydro-9-S-(4-azido-2-nitrophenyl)-3,5, 9-trideoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (2), and an azide group to give 5-acetamido-2,6-anhydro-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-non-2 -enonic acid (3). Competitive inhibition kinetics were observed when 1,2, and 3 were tested with the lysosomal sialidase (cultured fibroblasts) and the plasma membrane sialidase (adenovirus DNA-transformed, human embryonic kidney cells), giving a Ki of about 10 microM for both enzymes with all three compounds. In contrast, only 1 was a potent inhibitor of the microsomal sialidase (rat muscle).     

Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis

Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.     

Synthesis of 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D binding protein

Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.     

Structural modeling of the complex between an acetylcholine receptor-mimicking antibody and its snake toxin antigen

The antibody Mα2-3 neutralizes the functional, acetylcholine receptor binding activity of its antigen, neurotoxin α, and exhibits several other properties in common with the receptor itself. We present here the results of calculations examining the three-dimensional structure of the toxin α:Mα2-3 complex. The antigen structure, determined by nuclear magnetic resonance spectroscopy,¹ was docked to models of the variable fragment of the antibody combining site² by using a method based on surface complementarity and maximization of buried surface area^3,4 while taking into account the possibility of conformational change on complexation. Extensive experimental information on the location of the functional epitope was incorporated into the analysis and used to screen candidate geometries of the complex resulting from the modeling. Eight plausible structures that are in accord with the experimental data were derived. Common structural features of the models are discussed, and residues of the antibody-combining site that are expected to play important roles in complexation are identified. In particular, three epitope residues that, according to mutagenesis experiments, make particularly strong contributions to the binding, interact excentrically and do not make contact with the central loops of the combining site, L3 and H3. Proteins 30:249–263, 1998. © 1998 Wiley-Liss, Inc.     

Photo-affinity labelling of carbohydrate-binding sites in proteins: synthesis of potential inhibitors

The synthesis and properties of 4-azido-2-nitrophenyl α-D-mannopyranoside, N-diazoacetyl-β-D-glucopyranosylamine, and 4-diazoacetamidophenyl α-D-mannopyranoside are described. These compounds are potential reagents for photo-affinity labelling of carbohydrate-binding sites in proteins.     

Identification of the NADH-NAD+ transhydrogenase peptide of the mitochondrial NADH-CoQ reductase (Complex I). A photodependent labeling study utilizing arylazido-beta-alanyl NAD+

Incubation of Complex I (NADH-CoQ reductase) of ox heart mitochondria at 4 degrees C in the presence of 0.5 M NaClO4 followed by ammonium sulfate fractionation of the solubilized proteins results in the isolation of a resolved preparation still capable of catalyzing NADH-NAD+ transhydrogenation but having only low levels of NADH dehydrogenase activity. A number of NAD(H) analogues, including the photoaffinity probes, arylazido-beta-alanyl NAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]NAD+ and arylazido-beta-alanyl AcPyAD+ (A3'-O-[3-[N-(4-azido-2-nitrophenyl)amino]propionyl]AcPyAD+ can be utilized as substrates for transhydrogenation in this preparation. A further incubation (10 min) of the resolved NADH-NAD+ transhydrogenase in the presence of 0.5 M NaClO4, but now at 30 degrees C, results in the complete loss of this transhydrogenase activity. Photoaffinity labeling experiments utilizing arylazido-[3-3H]beta-alanyl NAD+ and arylazido-[3-3H]beta-alanyl AcPyAD+ with the resolved NADH-NAD+ transhydrogenase preparation prior to and following NaClO4 (30 degrees C) treatment indicates that the 42,000 molecular weight component of Complex I is the pyridine nucleotide binding site responsible for the major NADH-NAD+ (DD) transhydrogenase activity of Complex I.     

A photoreactive competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts

A photoreactive, potent, competitive inhibitor of the human lysosomal neuraminidase in cultured skin fibroblasts has been prepared. The starting material, 2,3 dehydro-N-acetyl neuraminic acid methyl ester, was selectively tosylated at the C-9 position with tosyl chloride and subsequently peracetylated with acetic anhydride. The tosyl group was displaced with potassium thio acetate in dimethylformamide at 60 degrees C for 80 min. 4-fluoro-3-nitrophenylazide was incorporated by reaction with the thio acetate product and equimolar sodium methoxide in methanol followed by reacetylation. Base hydrolysis gave the final product, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6 anhydro-2,3,5,9-tetradeoxy-9-thio-D-glycero-D-galacto-non-2-enonic acid (W5). The yields at each step were 50-70%. Competitive inhibition kinetics were observed when W5 was tested with the fibroblast neuraminidase using 4-methylumbelliferyl-N-acetyl-neuraminic acid as substrate giving an apparent Ki of about 10 microM. These results suggest that the terminal hydroxyl group at C-9 may not be important in the recognition and binding of the substrate by the enzyme. Also, the compounds prepared here may be useful as photoaffinity probes or ligands for affinity chromatography for purification.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 [Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-[(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.     

Nucleotide interactions with membrane-bound transporter associated with antigen processing proteins

The transporter associated with antigen processing (TAP) contains two nucleotide-binding domains (NBD) in the TAP1 and TAP2 subunits. When expressed as individual subunits or domains, TAP1 and TAP2 NBD differ markedly in their nucleotide binding properties. We investigated whether the two nucleotide-binding sites of TAP1/TAP2 complexes also differed in their nucleotide binding properties. To facilitate electrophoretic separation of the subunits when in complex, we used TAP complexes in which one of the subunits was expressed as a fluorescent protein fusion construct. In binding experiments at 4 degrees C using the photo-cross-linkable nucleotide analogs 8-azido-[gamma-(32)P]ATP and 8-azido-[alpha-(32)P]ADP, TAP2 was found to have reduced affinity for nucleotides compared with TAP1, when the two proteins were separately expressed. Complex formation with TAP1 enhanced the binding affinity of the TAP2 nucleotide-binding site for both nucleotides. Binding analyses with mutant TAP complexes that are deficient in nucleotide binding at one or both sites provided evidence for the existence of two ATP-binding sites with relatively similar affinities in TAP1/TAP2 complexes. TAP1/TAP2 NBD interactions appear to contribute at least in part to enhanced nucleotide binding at the TAP2 site upon TAP1/TAP2 complex formation. Binding analyses with mutant TAP complexes also demonstrate that the extent of TAP1 labeling is dependent upon the presence of a functional TAP2 nucleotide-binding site.     

Synthesis of 4-cyanophenyl and 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-arabino- and -beta-D-lyxopyranosides possessing antithrombotic activity

Tri-O-acetyl-5-thio-D-ribopyranosyl bromide was converted into 3,4-di-O-benzoyl-1,5-anhydro-5-thio-D-erythro-pent-1-enitol (3,4-di-O-benzoyl-5-thio-D-ribal), the azidonitration of which afforded an unstable mixture of 2-azido-3,4-di-O-benzoyl-2-deoxy-1-O-nitro-5-thio-D-pentopyranoside++ + isomers. This was converted without separation into the corresponding 1-O-acetyl derivatives from which an alpha,beta anomeric mixture of the 1-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-5-thio-D-arabinopyranose+ ++ isomers could be isolated in high yield. Glycosidation of this mixture with 4-cyano- or 4-nitrobenzenethiol, using trimethylsilyl triflate or boron trifluoride etherate, respectively, as promoters gave the corresponding D anomers exclusively. Zemplén debenzoylation afforded 4-cyanophenyl as well as 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-arabinopyranoside, respectively. When 1-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-5-thio-D-lyxopyranose was used as glycosyl donor only the corresponding 1 anomers, i.e., 4-cyanophenyl as well as 4-nitrophenyl 2-azido-2-deoxy-1,5-dithio-beta-D-lyxopyranosides, could be isolated after Zemplén debenzoylation in high yield. All four 1,5-dithioglycosides possess significant oral antithrombotic activity.     

Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles

A new competitive immunostrip assay was developed to detect human serum albumin (HSA) in urine sample with use of conjugated monoclonal antibody gold nanoparticles (mAb–AuNPs) and mobile crystalline material (MCM)-41–HSA bioconjugate. To prepare the immunostrip, the colloidal AuNPs with an average particle diameter of 20 nm, was synthesized, labeled with antibody and applied on the conjugate pad as the detection reagent. Then, HSA was attached to the MCM-41 mesoporous nanoparticles and immobilized to a nitrocellulose membrane as the test line. In the optimized investigational conditions, the immunostrip could detect HSA in a high linear range (from 1 to 200 μg/ml) and low detection limit (ng/ml). The reliability of the testing procedure was examined by performing the immunostrip test with 30 urine samples and comparing the results with those obtained via immunoturbidimetry. The immunostrip was adequately sensitive and accurate for a rapid screening of HSA in the urine. This new strategy for competitive immunostrip design can be used and developed for other antigen based immunostrip assay.     

Different immune response of mice immunized with conjugates containing multiple copies of either consensus or mixotope versions of the V3 loop peptide from human immunodeficiency virus type 1

The critical role that antibody responses to the V3 loop epitope play in human immunodeficiency virus type 1 (HIV-1) neutralization has caused this peptide to be used in many HIV-1 vaccine candidates. To enhance cross-reactivity toward several V3 sequences, a database of 50 peptides of the V3 region from HIV-1 subtype A was used to design both a consensus peptide and a combinatorial peptide (mixotope) library representative of these sequences. The two immunogens (consensus and mixotope) were incorporated into multiple antigen peptide (MAP) constructions, conjugated to a recombinant surface antigen from hepatitis B virus (HbsAg) carrier protein, and inoculated to mice in combination with a C4 (CD4-binding) peptide MAP construction, also conjugated to HBsAg. The respective responses and cross-reactivity to several V3 loop sequences of both types of immunogens were compared. Mice inoculated with the V3 consensus-MAP-HBsAg + C4-MAP-HBsAg mixture elicited higher antibody responses than those given the V3 mixotope-MAP-HBsAg + C4-MAP-HBsAg mixture. In addition, pooled serum from the first group of immunogens analyzed at dilution 1:100 had higher cross-reactivity against V3 peptides on cellulose membranes than those from mice given the combinatorial immunogen. Fine epitope mapping of both consensus and C4 peptide by the spot synthesis technique showed that sera of the first group strongly recognized both sequences in their entirety, whereas mice immunized with the mixotope library recognized only the N-terminal region of V3. These results seem to suggest that the V3 consensus peptide is superior to the combinatorial strategy in inducing potent and cross-reactive responses to HIV.     

Fluorescent and photoactive probes for the study of protein kinase C

A fluorescent diacylglycerol, 2-(12-N-dansylaminododecanoyl)-1-myristoyl-sn-glycerol (dansyl-DAG) and a photoactive diacylglycerol, 2-(12-[N-(4-azido-2-nitrophenyl)] aminododecanoyl)-1-myristoyl-sn-glycerol (azido-DAG) have been synthesized. Both have been shown to bind to protein kinase C by inhibition of phorbol dibutyrate binding. Dansyl-DAG was able to activate protein kinase C at low calcium concentrations. Stimulation of neutrophils with dansyl-DAG resulted in a large release of superoxide radicals from the cells. The physicochemical properties of dansyl-DAG and azido-DAG may allow one to label and follow specifically changes in the location of protein kinase C and to understand some aspects of its function and regulation.     

Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.