Indirect Binding Assay Research Articles

Overexpression of fucosyltransferase 8 reverses the inhibitory effect of high-dose dexamethasone on osteogenic response of MC3T3-E1 preosteoblasts.

BackgroundCore fucosylation catalyzed by FUT8 is essential for TGF-β binding to TGF-β receptors.MethodsIndirect TGF-β1 binding assay was used to evaluate the ability of TGF-β1 to bind to TGFBRs, Alizarin red and alkaline phosphatase staining were used to detect osteogenic differentiation and mineralization ability , western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Plasmid-mediated gain-of-function study. The scale of core fucosylation modification was detected by Lectin-blot and LCA laser confocal.ResultsOur results showed that compared with vehicle treatment, high-dose (10−6 and 10−5 M) dexamethasone significantly inhibited cell proliferation, osteogenic differentiation, and FUT8 mRNA expression while promoting mRNA expression of adipogenesis-related genes in MC3T3-E1 cells, suggesting that downregulation of FUT8 is involved in the inhibitory effect of high-dose dexamethasone on osteogenesis. Overexpression of FUT8 significantly promoted osteogenic differentiation and activated TGF-β/Smad signaling in MC3T3-E1 cells in the presence of high-dose dexamethasone, suggesting that FUT8 reverses the inhibitory effect of high-dose dexamethasone on osteogenesis. In addition, lectin fluorescent staining and blotting showed that overexpression of FUT8 significantly reversed the inhibitory effects of high-dose dexamethasone on core fucosylation of TGFBR1 and TGFBR2. Furthermore, indirect TGF-β1 binding assay showed that overexpression of FUT8 remarkably promoted TGF-β1 binding to TGFBRs in MC3T3-E1 cells in the presence of high-dose dexamethasone.ConclusionsTaken together, these results suggest that overexpression of FUT8 facilitates counteracting the inhibitory effect of dexamethasone on TGF-β signaling and osteogenesis.

Lab on fiber nano-cavity integrated with charge responsive microgels for biosensing

The development of photonic-based point of care (POC) devices requires the use of compact integrated biosensing platforms enabling reliable detection performances in small volumes. The sensing system should be designed to overcome limitations of benchtop technologies in terms of portability and the easy-of-use. The Lab-on-Fiber (LOF) technology is continuously trying to develop miniaturized, lightweight, and light-coupled optrodes with unprecedented detection performances associated with portable, plug-and-play and user-friendly features. The sensing platforms, yet successfully developed in planar configurations, are being reimagined and suitably scaled down to be directly integrated onto optical fibers. In the wake of this ‘chip-to-fiber translation’ vision, we have realized a micro-sized optical cavity made of smart materials based on synthetic microgels and integrated on the top of an optical fiber. As a specific case study, the optrode has been here tested within a ‘general purpose’ detection assay for biochemical applications. Specifically, we detected nanomolar concentrations of homopolyarginines and positively charged linear polymers that are frequently exploited as surplus in indirect binding assays. The high degree of miniaturization of our probe enables a significant improvement in the minimum detectable concentration (at least 1 nM) compared to those achieved with similar devices having planar configurations. The probe sensitivity and overall performance are strongly influenced by the molecular weight and number of positive charges of the polymer. Overall, our findings represent a step forward in establishing LOF optrodes as sensing units for optical POC, further enlarging their applicability for the detection of a broad range of relevant biomarkers.

SARS-CoV-2 antibody persistence in COVID-19 convalescent plasma donors: Dependency on assay format and applicability to serosurveillance.

BackgroundAntibody response duration following severe acute respiratory syndrome coronavirus 2 infection tends to be variable and depends on severity of disease and method of detection.Study design and methodsCOVID‐19 convalescent plasma from 18 donors was collected longitudinally for a maximum of 63–129 days following resolution of symptoms. All the samples were initially screened by the Ortho total Ig test to confirm positivity and subsequently tested with seven additional direct sandwich or indirect binding assays (Ortho, Roche, Abbott, Broad Institute) directed against a variety of antigen targets (S1, receptor binding domain, and nucleocapsid [NC]), along with two neutralization assays (Broad Institute live virus PRNT and Vitalant Research Institute [VRI] Pseudovirus reporter viral particle neutralization [RVPN]).ResultsThe direct detection assays (Ortho total Ig total and Roche total Ig) showed increasing levels of antibodies over the time period, in contrast to the indirect IgG assays that showed a decline. Neutralization assays also demonstrated declining responses; the VRI RVPN pseudovirus had a greater rate of decline than the Broad PRNT live virus assay.DiscussionThese data show that in addition to variable individual responses and associations with disease severity, the detection assay chosen contributes to the heterogeneous results in antibody stability over time. Depending on the scope of the research, one assay may be preferable over another. For serosurveillance studies, direct, double Ag‐sandwich assays appear to be the best choice due to their stability; in particular, algorithms that include both S1‐ and NC‐based assays can help reduce the rate of false‐positivity and discriminate between natural infection and vaccine‐derived seroreactivity.

The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling

Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.

SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that infects swine and humans with high mortality and morbidity. Although a number of virulence-associated factors have been reported, the understanding of the molecular mechanism underlying SS2 pathogenicity remains limited. Our previous studies revealed that srtBCD-associated protein 2’ (SBP2′) contributed to the pathogenesis of SS2, but the function of another member in the srtBCD cluster, srtBCD-associated protein 1 (SBP1) was still unknown. Here, we found that sbp1 was widely distributed among high virulent SS2 strains, suggesting that sbp1 may be involved in the pathogenesis of SS2. To investigate the function of SBP1, we firstly conducted Western blotting analyses to confirm that SBP1 was expressed in the high virulent SS2 strain ZY05719 both in vivo and in vitro, then constructed the deletion mutant of sbp1 by homologous recombination. Bacterial adhesion assay, indirect immunofluorescence assay and protein binding assay all demonstrated that SBP1 was associated with adhesion of SS2 to HEp-2 cells. However, SBP1 did not influence the invasion, phagocytosis or intracellular survival of SS2. Furthermore, infection assays in vivo showed that inactivation of sbp1 failed to impair the ability of SS2 to cause zebrafish and mouse mortality. Overall, these results indicate that SBP1 is an adhesion-associated factor without the involvement of virulence in Streptococcus suis serotype 2.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

BackgroundSperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay.MethodsIndirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay.ResultsMonoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding.ConclusionGAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm/oocyte binding.

Abstract 2086: Dual inhibitors of Flt3 and c-Fest tyrosine kinases potently inhibit proliferation of AML cells expressing an active Flt3 mutant.

Abstract Acute myelogenous leukemia (AML) is a hematologic cancer associated with advancing age and relatively poor prognosis. Although diverse genetic changes are associated with AML, about one-third of cases involve activating mutations in the receptor tyrosine kinase, Flt3. Clinical trials of most Flt3 tyrosine kinase inhibitors have been disappointing, indicating that additional signaling pathways contribute to the transformed phenotype. Previous studies have linked the c-Fes protein tyrosine kinase to Flt3 in AML, and RNAi-knockdown studies suggest that c-Fes may be required for Flt3 function. Recently, our laboratory reported several classes of inhibitors with favorable selectivity profiles for c-Fes both in vitro and in cell-based assays. In this study, we tested c-Fes inhibitors from four different chemical classes for their growth-suppressive activity against myeloid leukemia cells lines with an activating mutation in Flt3 (MV4-11 cells) vs. wild-type Flt3 (THP-1). All c-Fes inhibitors tested potently inhibited the growth of MV4-11 AML cells, but were far less active against THP-1 cells. The IC50 values for inhibition of MV4-11 cell proliferation by c-Fes inhibitors of the diaminopyrimidine, phenoxyquinazoline and pyrrolopyrimidine classes ranged from 25-140 nM, rivaling the potency of the clinical Flt3 inhibitors lestaurtinib and tandutinib. In contrast, two pyrazolopyrimidine c-Fes inhibitors were less potent in MV4-11 cells, with IC50 values for growth arrest in the low micromolar range. KINOMEscan analysis, an indirect binding assay which provides a relative measure of inhibitor selectivity across the kinome, suggested that the more potent compounds may have dual specificity for both Flt3 and c-Fes while the less potent compounds may be more selective for c-Fes alone. To test this idea, we performed in vitro kinase assays with recombinant purified c-Fes and Flt3 proteins. We observed that the most potent inhibitors of MV4-11 AML cell proliferation inhibited both c-Fes and Flt3 kinase activity, with IC50 values in the 0.2-1.5 μM range for c-Fes and in the 0.2-4.0 μM range for Flt3. For example, the phenoxyquinazoline analog TL02-59 showed similar potency against both c-Fes (IC50 of 1.0-1.5 μM) and Flt3 (IC50 of 0.6-4.0 μM) in vitro, and potently blocked MV4-11 cell proliferation (IC50 of 26.5 nM) and induced apoptosis. In contrast, the pyrazolopyrimidine inhibitor WZ4-49-8, which exhibited 20-fold greater selectivity for c-Fes over wild-type Flt3 in vitro, was less active against MV4-11 cell proliferation (IC50 of 1.86 μM) and produced a lower apoptotic response than TL02-59. Interestingly, the established Flt3 inhibitor lestaurtinib was also a relatively potent inhibitor of c-Fes, yielding IC50 values in the 0.4-0.8 μM range. These results suggest that dual inhibition of both Flt3 and c-Fes may provide a therapeutic advantage for the treatment of AML. Citation Format: Mark Weir, Sabine Hellwig, Nathanael S. Gray, Thomas E. Smithgall. Dual inhibitors of Flt3 and c-Fest tyrosine kinases potently inhibit proliferation of AML cells expressing an active Flt3 mutant. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2086. doi:10.1158/1538-7445.AM2013-2086

Application of Affinity Selection-Mass Spectrometry Assays to Purification and Affinity-Based Screening of the Chemokine Receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.

Development of a label-free competitive ligand binding assay with human serum albumin on a molecularly engineered surface plasmon resonance sensor chip

The binding interactions between ligands and receptors play a vital role in their biological functions. In this work, a label-free indirect competitive binding assay based on surface plasmon resonance (SPR) detection was developed, using fatty acid–human serum albumin (HSA) as a model system. In the assay, a fatty acid analog was immobilized on the surface of an SPR gold chip by self-assembly, and was allowed to bind with HSA in solution. Addition of a ligand under investigation into the solution set up a competition with the analog for the protein, causing a reduction in the SPR signal. Due to the asymmetric structure of the ligand binding channel, HSA exhibited remarkable selectivity for the orientation of the fatty acid analog on a gold surface. When the carboxylic acid group of the analog was exposed on the sensor surface, the amount of bound HSA was very low. However, it increased by more than 7 fold when the alkyl chain was exposed. Dilution of the surface coverage of the fatty acid analog also enhanced HSA binding by reducing steric hindrance on the surface. Using a 1 : 4 mercaptoundecanoic acid–mercaptohexanol modified surface derivatized with octylamine, competitive binding assays for octanoic acid and perfluorooctane sulfonate with HSA were conducted. Using a derived kinetic equilibrium model, the equilibrium association constant was determined to be 4.57 × 105 M−1 and 2.11 × 105 M−1, respectively, which are consistent with the values measured by other established methods. The engineered sensor chip surface can be used to study the binding interactions of other chemicals with this important serum transport protein.

Diagnosis and discrimination of autoimmune Graves' disease and Hashimoto's disease using thyroid-stimulating hormone receptor-containing recombinant proteoliposomes

Graves' disease (GD) is an autoimmune disease of the thyroid gland caused by autoantibodies against thyroid-stimulating hormone receptor (TSHR). Currently, the diagnostic test for TSHR autoantibodies is based on an indirect competitive binding assay that measures the ability of TSHR autoantibodies to inhibit the binding of thyroid-stimulating hormone (TSH) to TSHR. Here, we have developed a specific and direct diagnostic method for autoantibodies in GD that incorporates immobilized TSHR-containing recombinant proteoliposomes into an enzyme-linked immunosorbent assay (ELISA). To reduce non-specific binding of autoantibodies to recombinant proteoliposomes, we investigated the effect of polyethylene glycol (PEG)-lipid on the binding of commercially available anti-TSHR antibodies (aTSHRAb). The incorporation of PEG-lipids into liposomes decreased non-specific binding, as compared to liposomes that did not contain PEG-lipids, and the addition of blocking reagents further decreased non-specific reactivity. aTSHRAb exhibited higher reactivity towards PEG-modified TSHR recombinant proteoliposomes than PEG-modified liposomes without TSHR (bare liposomes). Importantly, serum autoantibodies from patients with GD, which is associated with hyperthyroidism, exhibited remarkably specific binding to TSHR recombinant proteoliposomes. Serum autoantibodies from patients with Hashimoto's disease (HD), which is associated with hypothyroidism, also reacted specifically with proteoliposomal TSHR. These results suggest that immobilized TSHR recombinant proteoliposomes can serve as a direct diagnostic test for GD and HD. Furthermore, given that there is no competition test currently available for detecting autoantibodies in HD, the combination of TSHR recombinant proteoliposome ELISA and indirect competitive TSHR binding assay might be an effective way to discriminate between GD and HD.

Contrasting Roles for Domain 4 of VCAM-1 in the Regulation of Cell Adhesion and Soluble VCAM-1 Binding to Integrin α4β1

Cell adhesion mediated by the interaction between integrin alpha4beta1 and VCAM-1 is important in normal physiologic processes and in inflammatory and autoimmune disease. Numerous studies have mapped the alpha4beta1 binding sites in VCAM-1 that mediate cell adhesion; however, little is known about the regions in VCAM-1 important for regulating soluble binding. In the present study, we demonstrate that 6D VCAM-1 (an alternatively spliced isoform of VCAM-1 lacking Ig-like domain 4) binds alpha4beta1 with a higher relative affinity than does the full-length form of VCAM-1 containing 7 Ig-like extracellular domains (7D VCAM-1). In indirect binding assays, the EC50 of soluble 6D VCAM-1 binding to alpha4beta1 on Jurkat cells (in 1 mM MnCl2) was 2 x 10(-9) M, compared with 7D VCAM-1 at 11 x 10(-9) M. When used in solution to inhibit alpha4beta1 mediated cell adhesion, the IC50 of 6D VCAM-1 was 13 x 10(-9) M, compared with 7D VCAM-1 measured at 150 x 10(-9) M. Removal of Ig-like domains 4, 5, or 6, or simply substituting Asp328 in domain 4 of 7D VCAM-1 with alanine, caused increased binding of soluble 7D VCAM-1 to alpha4beta1. In contrast, cells adhered more avidly to 7D VCAM-1 under shear force, as it induced cell spreading at lower concentrations than did 6D VCAM-1. Finally, soluble 6D VCAM-1 acts as an agonist through alpha4beta1 by augmenting cell migration and inducing cell aggregation. These results indicate that the domain 4 of VCAM-1 plays a contrasting role when VCAM-1 is presented in solution or as a cell surface-expressed adhesive substrate.

Non-opioid actions of opioid peptides

Beside the well known actions of opioid peptides on μ-, δ- and κ-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include β-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta (ζ) receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to κ 2 and δ-opioid receptors, MERF can act also through σ 2- or probably via FMRF-NH 2 receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.

A new enzymatic variant of Leishmania (Leishmania) forattinii isolated from Proechimys iheringi (Rodentia, Echimydae) in Espírito Santo, Brazil.

Taxonomic studies of leishmanial isolates fromthe New World indicate tremendous diversitywithin this genus (E Cupolillo et al. 1994 Am JTrop Med Hyg 50: 296-311). A number of newLeishmania species from sylvan areas of theNeotropics have been described recently. Some ofthose parasites are associated with disease in hu-mans; others appear to be restricted to lower or-ders of mammals, such as rodents and edentates(G Grimaldi et al. 1989 Am J Trop Med Hyg 41:687-725). However, some of the latter parasitesmay yet be shown capable of causing human dis-ease, particularly in persons with altered cellularimmune responses.During a survey of potential reservoir hosts ofLeishmania done in the State of Espirito Santo, Bra-zil (1982-1985), three leishmanial parasites (des-ignated with stock codes MPRO/BR/82/RV203,MPRO/BR/82/RV228, and MPRO/BR/85/RV260)were isolated from skin lesion samples taken fromspiny-rats, Proechimys iheringi (Rodentia,Echimydae), captured in a secondary forest locatedin the municipality of Viana (A Falqueto et al. 1985Mem Inst Oswaldo Cruz 80: 497). In studies us-ing an indirect radioimmune binding assay (RIA)and a large panel of monoclonal antibodies (Mabs)derived for selected species of Leishmania , the iso-lates were characterized as members of the L.mexicana complex. These strains were differentfrom those species infective to humans, in that theyreacted with either the L.(L.) amazonensis or L.(L.) mexicana specific MAbs. Furthermore, thiswas also the case with cloned organisms from twostocks showing that they were not mixed popula-tions of Leishmania (G Grimaldi et al. 1987 Am JTrop Med Hyg 36: 270-287). This group of dis-tinct parasites presented, however, similarserodeme patterns in relation to the new recentlydescribed species of the Leishmania subgenus, L.(L.) forattinii (E Yoshida et al. 1993 Mem InstOswaldo Cruz 88: 397-406).The latter parasite was isolated from sylvaticreservoir hosts (Didelphis marsupialis aurita andP. iheringi denigratus ) captured on regions endemicfor American cutaneous leishmaniasis in Brazil,respectively in the municipalities of Conchas, SaoPaulo (E Yoshida et al. 1979 Rev Inst Med Trop SPaulo 21: 110-113) and Tres Bracos, Bahia (ABarreto et al. 1985 Rev Soc Bras Med Trop 18:243-246). The general morphology and the growthcharacteristics in vitro of the new species were simi-lar to those of other L. mexicana complex para-sites. Here we have further studied the Espirito Santoisolates using additional techniques for the char-acterization of Leishmania . The molecular proce-dures used for typing the strains (isoenzyme elec-trophoresis, blot enzyme binding assay usingMAbs, restriction endonuclease fragment patternsof k-DNA, and molecular karyotype analysis) havebeen described in detail in previous publications(Yoshida et al. 1979, 1983 loc. cit. , Cupolillo et al.1994 loc. cit., A Franco et al. 1997 Mem InstOswaldo Cruz 92: 63-68).Results of electrophoretic analysis were basedon the following enzymatic loci, namely: glucose-6-phosphate dehydrogenase (G6PDH), malate de-hydrogenase (MDH), isocitrate dehydrogenase(IDHNADP), 6-phosphogluconate dehydrogenase(6PGDH), nucleotidase (NH1 and NH2), glucosephosphate isomerase (GPI), phosphoglucomutase(PGM), proline dipeptidase (PEPD), leucine pep-tidase (PEP2), malic enzyme (ME), mannose phos-phate isomerase (MPI), and aconitate hydratase(ACON). The enzyme profiles of the selected iso-lated signature MPRO/BR/82/RV228, which rep-resents the poorly defined group of parasites from

Human primary Sjögren's syndrome autoantibodies as mediators of nitric oxide release coupled to lacrimal gland muscarinic acetylcholine receptors

IgG obtained from sera of primary Sjögren's syndrome (pSS-IgG) patients and its interaction with M 3 muscarinic cholinoceptors of rat exorbital lacrimal glands were studied by indirect immunofluorescence (IFI) and binding assay. Primary Sjögren's syndrome IgG stained epithelial cells with a continuous fluorescence pattern. The IFI imagen was attenuated by incubating the pSS-IgG with a synthetic peptide corresponding to the second extracellular loop of M3 muscarinic cholinoceptor. Primary SS-IgG was also able to bound irreversibly to muscarinic acetylcholine receptors (mAChRs) displacing the specific cholinergic antagonist QNB. Moreover, these antibodies triggered intracellular signals coupled to M 3 muscaric cholinoceptors such as nitric oxide synthase (NOS) activation and cGMP production. Both primary Sjögren's syndrome IgG effects mimicked carbachol action and were abrogated by specific muscarinic antagonist 4-DAMP. The nitric oxide pathway through muscarinic cholinoceptors activation by pSS-IgG on rat exorbital lacrimal gland is also described. We proposed that chronic interaction of these autoantibodies on lacrimal gland muscarinic acetylcholine receptors could lead to tissue damage through nitric oxide release after immunological stimulation.

Uncoupling of Ligand-binding Affinity of the Bacterial Serine Chemoreceptor from Methylation- and Temperature-modulated Signaling States

The Escherichia coli chemoreceptor Tsr mediates tactic responses to serine, repellents, and changes in temperature. We have previously shown that the serine-sensing ability of Tsr-T156C, which has a unique cysteine in place of threonine at residue 156, is specifically inactivated by thiol-modifying reagents and that L-serine protects the receptor from modification. In this study, we demonstrated the correlation between protective effects of various attractants and their potencies to elicit attractant responses. This indirect binding assay was used to monitor the affinity of the receptor for L-serine under various conditions. It has been demonstrated by in vitro assays that the ligand-binding affinities of Tsr and the related chemoreceptor Tar are unaffected by changes in the methylation state of the receptor. Using the serine protection assay, we re-examined this issue both in vitro and in vivo. The methylation levels of Tsr-T156C did not affect its ligand-binding affinity. We also showed both in vitro and in vivo that the ligand-binding affinity was unaffected by temperature. These results suggest that the structure of the periplasmic domain of the receptor is uncoupled from the signaling states of the cytoplasmic domain. This ligand-binding assay system should be applicable to other receptors.

A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent.

P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets to leukocytes and thus further promote thrombolysis. The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPAIR) together with two TPAIR/P-selectin fusion constructs (P280IR and P121IR) were expressed with the use of baculovirus vectors. After infection of Sf21 cells with the recombinant baculovirus, recombinant TPAIR and P-selectin/TPAIR fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPAIR and the ligand binding capability of P-selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPAIR, P280IR, and P121IR (P > .5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant (P < .05) and similar (P > .5) among TPAIR, P280IR, and P121IR. No significant bleeding was noted among treated animals. Chimeric proteins P280IR and P121IR have clot lysis activities that are similar to TPAIR both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.

Cytomegalovirus glycoprotein B-specific antibody analysis using electrochemiluminescence detection-based techniques.

An electrochemiluminescence technique was used to develop versatile and sensitive assay strategies for determination of seroreactivities against biologically important cytomegalovirus neutralization epitopes expressed on glycoprotein B. Indirect binding assays showed wide linear assay ranges and revealed that serum samples diluted in parallel with a monoclonal antibody-based standard, simplifying quantitative analytical assessments.

Immunological similarities among trichocyst secretory proteins of Paramecium caudatum, Paramecium tetraurelia and Pseudomicrothorax dubius

Secretory vesicles called trichocysts structurally show a body and a tip in Paramecium and a shaft and four arms in Pseudomicrothorax. Biochemical and immunological relationships between both trichocyst types were examined. Three polyclonal antibodies were produced against 3 groups of protein bands of isolated Pseudomicrothorax dubius trichocysts separated by one-dimensional gel electrophoresis: G 1 (30-31 kDa), G 2 (26-27 kDa) and G 3 (15-20 kDa). By indirect immunofluorescent binding assay, the three antisera label extended (i.e. discharged) and condensed (i.e. non-discharged) trichocysts of both Ps. dubius and Paramecium caudatum. By immunogold labeling on ultrathin sections, the three antisera label the trichocyst shaft of Ps. dubius and the trichocyst body of Pa. caudatum on both condensed and extended secretory vesicles. On two-dimensional immunoblots, remarkable antigenic similarities are shown by trichocysts of Ps. dubius, Paramecium tetraurelia nd6 and Pa. caudatum. Anti-G 1 serum detects proteins at 28-32 kDa, pI 5.0-5.7, in the three cells. Anti-G 3 serum labels one protein group at ~ 15-22 kDa and a second protein group at ~ 35-50 kDa in Ps. dubius, Pa. tetraurelia nd6 and Pa. caudatum. Anti-G 2 serum labels proteins at ~ 15-22 kDa, pI 4.7-5.2, in all three species. Anti-G 2 serum also detects bands at 24-28 kDa and 30-31 kDa, pI 4.7-6.1, in Ps. dubius. No equivalent M(r) proteins are observed in either Paramecium spp. Comparisons of immunoblots of proteins of entire cells with and without ejectable trichocysts allowed identification of non-ejectable trichocyst proteins, some of which may be precursors localized within pretrichocysts. Such proteins are at 41-47 kDa in Ps. dubius, and at ~ 45 kDa in Paramecium.