Differential Binding Patterns Research Articles

Subpopulations of human dendritic cells display a distinct phenotype and bind differentially to proteins of the extracellular matrix

CD1a pos dendritic cells (DCs) and Langerhans cells (LCs) are highly specialized antigen-presenting cells mainly localized in the skin. Various cells have been identified as precursors of cutaneous DCs, but the definitive precursor subpopulations remain to be defined and characterized in detail. In this study, DCs were generated in vitro from monocytes (monocyte-derived DCs, MoDCs) and from CD34 pos stem cells (CD34 pos cell-derived DCs, CD34DCs). By virtue of their CD14 and CD1a expression, four CD34DC subpopulations were characterized while MoDCs contain three different subpopulations. Of these, CD14-expressing cells are considered to be precursors of fully differentiated DCs, which themselves are CD14 negCD1a pos. Both, MoDCs and CD34DCs expressed the α integrins LFA-1, Mac-1, CR4, VLA-4, VLA-5 and the β2 integrin CD18. CD34DCs and MoDCs were negative for VLA-3, whereas MoDCs, but not CD34DCs expressed VLA-6. Phenotypic and functional characterization of the cells generated herein at earlier time points revealed that DCs at day 3 of culture may reflect the in vivo situation more closely than at day 7. Adhesion of DC precursors to endothelial cells and to components of the extracellular matrix is a prerequisite for their migration towards the epidermis. To this end, we investigated adhesion of CD34DCs and MoDCs to components of the cutaneous extracellular matrix. Distinct DC subsets showed a differential binding pattern to proteins of the extracellular matrix. MoDCs and CD34DCs bound preferentially to laminin 332 via CD49f and to fibronectin via CD49e, but only weakly to laminin 111 or to collagens. While CD14 pos cells preferentially bound to laminin 332, CD1a pos cells adhered to fibronectin. In summary, subpopulations of CD34DCs and MoDCs are phenotypically related to each other, but not identical and display differential binding to components of the extracellular matrix.

Association Between a Polymorphism in the Lymphotoxin−a Promoter Region and Migraine

The aim of this study was to determine whether polymorphisms in the lymphotoxin (LTA)-tumor necrosis factor (TNF) region are associated with the risk of migraine. Previous studies concerning the role of TNFalpha in migraine have provided conflicting results. It has been reported that LTA could be a susceptibility gene in migraine. It is possible that the TNFalpha polymorphism associated with migraine is in linkage disequilibrium with other functional polymorphisms that influence migraine risk. Moreover, there are significant differences among the allele frequencies of TNF gene variants among populations from different ethnic groups. In a case-control study, including 439 migraine patients and 382 controls, we examined the association between 15 single nucleotide polymorphisms in the coding and promoter regions of LTA and TNFalpha genes, which are located within the 22 kb around TNF and the risk of migraine. We performed a chromatin immunoprecipitation (ChIP) assay and an electrophoretic mobility shift assay (EMSA) to identify differential protein-DNA binding of LTA-294. Homozygosity for the LTA-294C allele was significantly associated with an increased risk of migraine compared with CT/TT carriers (corrected P= .005, odds ratio [OR]= 1.7, 95% confidence interval [CI] 1.3-2.3). Haplotype TGAAC was found to be significantly associated with a protective effect against migraine (P= .0005, Bonferroni corrected P= .003, OR = 0.7, 95% CI 0.5-0.8). There was no differential protein-DNA binding pattern in both EMSA and ChIP assays. We found that the LTA haplotypes were associated with migraine among Koreans and that the best marker for this is the LTA-294 T/C polymorphism. Our results indicate that these associations should be defined in the context of the involvement of other genetically linked region, such as human leukocyte antigen (HLA) loci.

Alteration in acetylcholinesterase glycosylation of rat brain in memory disorder

Acetylcholinesterase in CSF has been observed to be post-translationally modified by abnormal glycosylation in Alzheimer’s disease. Present study was conducted to study the glycosylation profile of acetylcholinesterase (AChE) in different brain regions namely frontal and cerebral cortex and hippocampus in a rat model of dementia. The C/W ratio was calculated as percentage of AChE that did not bound to Con A (C) divided by percentage of AChE activity that did not bound to WGA (W). A retrograde dementia was induced by administering scopolamine (i.p.) 5 min prior to the first trial in passive avoidance test. No significant increase in transfer latency time on second trial after 24 h as compared to first trial indicated dementia. Tacrine, an AChE inhibitor, was studied for its effect on scopolamine dementia and AChE glycosylation pattern. C/W ratio exhibited specific alterations in the AChE glycosylation in cortex and hippocampus. In dementic rats among other brain regions there was significant increase in C/W ratio in cerebral cortex and hippocampus as compared to the control. The C/W ratio in Tacrine treated dementic rats was significantly decreased in cerebral cortex as compared to the control. Partially purified G4 and G1 molecular isoforms of AChE in rat brain revealed a differential binding pattern to lectins. The C/W ratio of G4 isoform was significantly increased in dementia as compared to control group while, G1 isoform remained unchanged. Tacrine treatment significantly increased and decreased the C/W ratio of G4 and G1 isoform, respectively as compared to the control group. Results obtained thus suggest that glycosylation of AChE was affected by alteration in learning and memory process – deficit (Scopolamine) and enhancement (Tacrine treatment).

Action of Transcription Factors in the Control of Transferrin Receptor Expression in Human Brain Endothelium

Brain endothelium has a distinctive phenotype, including high expression of transferrin receptor, p-glycoprotein, claudin-5 and occludin. Dermal endothelium expresses lower levels of the transferrin receptor and it is absent from lung endothelium. All three endothelia were screened for transcription factors that bind the transferrin receptor promoter and show different patterns of binding between the endothelia. The transcription factor YY1 has distinct DNA-binding activities in brain endothelium and non-brain endothelium. The target-sites on the transferrin receptor promotor for YY1 lie in close proximity to those of the transcription initiation complex containing TFIID, so the two transcription factors potentially compete or interfere. Notably, the DNA-binding activity of TFIID was the converse of YY1, in different endothelia. YY1 knockdown reduced transferrin receptor expression in brain endothelium, but not in dermal endothelium, implying that YY1 is involved in tissue-specific regulation of the transferrin receptor. Moreover a distinct YY1 variant is present in brain endothelium and it associates with Sp3. A model is presented, in which expression from the transferrin receptor gene in endothelium requires the activity of both TFIID and Sp3, but whether the gene is transcribed in different endothelia, is related to the balance between activating and suppressive forms of YY1.

Synthetic peptides mimicking antigenic epitope of Helicobacter pylori urease.

Short peptides resembling the Helicobacter pylori urease antigen (UreB F8 Ser-Ile-Lys-Glu-Asp-Val-Gln-Phe) with deleted aspartic acid and glutamic acid residues, anchored through a triazine linker via the N-terminal moiety to cellulose plate were prepared. The peptides were used for binding of antibodies from sera of patients with medically confirmed atherosclerosis. Recognition of the peptides was also tested with anti-Jack beans urease antibodies. The important role of a Gly-Gly spacer separating the peptides from the cellulose support was shown. Different patterns of binding of antibodies from H. pylori infected patients and anti-Jack bean urease antibodies were observed only in the case of pentapeptides. The peptide Gly-Gly-Leu-Val-Phe-Lys-Thr was recognized by most of the tested sera.

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus.

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions.

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses.

Characterization of the cts4 repressor mutation in transposable bacteriophage Mu

Mu cts4 was isolated more than 30 years ago and was the first available thermoinducible derivative of transposable phage Mu. We have characterized the cts4 mutation and the corresponding mutant protein. Contrary to previously characterized thermoinducible Mu prophages (e.g., Mu cts62), Mu cts4 lysogenizes at reduced frequency even at 30 ∘ C. The cts4 mutation (Leu129Val) was located in this central repressor region. The cts4 protein was thermosensitive for operator DNA binding in vitro. Temperature-dependent changes in protein–protein cross-linking patterns in the absence of DNA were detected for purified wild type, cts62 and cts4 repressor proteins. The cts4 protein exhibited a subtly different electrophoretic profile, which became more marked at higher temperatures, from both the wild type and cts62. In addition the cts4 repressor generated a significantly different pattern of binding to DNA fragments carrying the early operator region. Consistent with the predicted involvement of the central leucine-rich region of the Mu repressor in the formation of multimeric forms, the cts4 mutation thus appeared to affect protein–protein interactions.

Anti-myelin associated glycoprotein antibodies: variability in patterns of IgM binding to peripheral nerve

We previously found that serums with anti-sulfatide antibodies have several different patterns of binding to neural tissue. In this study, we asked whether serums with anti-myelin associated glycoprotein (MAG) antibodies also have similar variations in patterns of tissue binding. We examined binding to peripheral nerve in 49 serums with IgM anti-MAG antibodies and 13 serums with IgM anti-sulfoglucuronyl paragloboside (SGPG) antibodies but no MAG binding. We correlated patterns of binding with titers of IgM binding to MAG and SGPG measured by ELISA methods. Our results show that IgM in most anti-MAG serums stained areas of non-compact myelin, including the periaxonal and outer myelin membranes and Schmidt–Lanterman incisures. However, other patterns included IgM binding to areas of compact myelin and to non-myelin structures including axons and endoneurial macrophages. IgM in anti-SGPG serums bound to axons or macrophages, but rarely to myelin-related structures. A total of 11/62 (18%) of serums had IgM binding to axons, six with anti-MAG antibodies and five with anti-SGPG antibodies. The majority of these serums (73%) had SGPG titers greater than MAG titers when measured by ELISA. We conclude that anti-MAG serums have several different binding patterns to neural tissue, including axonal binding, especially when anti-MAG antibodies cross-react with SGPG. These different binding patterns may relate to the ability of anti-MAG serum IgM to bind both MAG and SGPG or to other molecules with a sulfated glucuronic acid epitope that are present in peripheral nerve.

1] In vivo modulation of G proteins and opioid receptor function by antisense oligodeoxynucleotides

The work in our laboratory has been designed to characterize the transducer mechanisms coupled to neurotransmitter receptors in the plasma membrane. Particular attention has been paid to the physiological/pharmacological effects mediated by the opioid system. Antisense oligodeoxynucleotides have proved useful in correlating opioid receptor clones with those defined pharmacologically. The involvement of the cloned opioid receptors mu, delta, and kappa in analgesia has been determined by means of in vivo injection of ODNs directed to the receptor mRNAs. Using this strategy the classes of G-transducer proteins regulated by each type/subtype of opioid receptor in the promotion of antinociception have also been characterized. After displaying different patterns of binding to their receptors, opioids trigger a variety of intracellular signals. The physiological implications and therapeutic potential of these findings merit consideration.

The interaction of fresh and frozen-thawed ram spermatozoa with oviducal epithelial cells in vitro.

In order to investigate the interaction of fresh and frozen-thawed spermatozoa with oviduct epithelial cells, spermatozoa were co-incubated with ovine oviduct epithelial cell monolayers (OECM) derived from either complete oviducts, at any stage of the oestrous cycle (Experiments 1 and 2), or from different regions of the oviduct at different stages of the cycle (Experiment 3). Fresh and frozen-thawed spermatozoa displayed different patterns of binding to, and release from, the OECM. Frozen-thawed spermatozoa immediately bound to the complete oviduct OECM and were released after 2 h. A small proportion of fresh spermatozoa bound immediately, increasing to a maximum after 2 h, and were gradually released thereafter. When only the cells that were released from the OECM were observed by chlortetracycline staining in Experiment 2, it was found that the presence of an OECM increased the number of capacitated fresh spermatozoa while decreasing the number of capacitated frozen-thawed spermatozoa. Overall, the OECM advanced the membrane state of both types of spermatozoa from uncapacitated to acrosome-reacted. Fresh and frozen-thawed spermatozoa bound to OECM derived from the cells of the isthmus and the ampulla in similar proportions. However, more spermatozoa were capacitated when incubated with OECM derived from isthmic rather than ampullary cells. Higher proportions of fresh spermatozoa bound to, and were acrosome-reacted following incubation with OECM derived from post- rather than pre-ovulatory tracts. Such differences were not observed for frozen-thawed spermatozoa. The findings reported in this study show that fresh and frozen-thawed spermatozoa behave differently when in contact with oviduct cells in vitro. This may be a consequence of the more advanced membrane state of the frozen spermatozoa upon thawing.

Localization of the PAK1-, WASP-, and IQGAP1-specifying Regions of Cdc42

The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.

Lectin-HPMA copolymer conjugates: potential oral drug carriers for targeting diseased tissues

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-lectin conjugates were investigated for potential use as targeted oral drug carriers for treatment of inflammatory conditions such as colitis. Wheat germ agglutinin (WGA)-HPMA copolymer and peanut agglutinin (PNA)-HPMA copolymer and fluorescein isothiocyanate (FITC)-labeled WGA- and PNA-HPMA copolymer conjugates were synthesized. Conjugate dissociation constants (Kd) for lectin-carbohydrate binding determined by frontal affinity chromatography indicated that no activity reduction of the lectins occurred during the synthesis of these conjugates. Kd values measured were in good agreement with literature findings for similar lectin-carbohydrate interactions, on the order of 10−5 M−1. Biorecognition of these conjugates by healthy rat intestinal tissue resulted in differential HPMA copolymer-lectin conjugate binding patterns in the same tissue. HPMA copolymer-WGA conjugate showed strong binding in the healthy rat intestinal tissues, while the HPMA copolymer-PNA conjugate showed minimal, but specific binding. This differential binding suggests that site-specific drug delivery via specific lectin recognition may be feasible for treatment of colon inflammation or cancer.

Lectin-HPMA copolymer conjugates: potential oral drug carriers for targeting diseased tissues

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-lectin conjugates were investigated for potential use as targeted oral drug carriers for treatment of inflammatory conditions such as colitis. Wheat germ agglutinin (WGA)-HPMA copolymer and peanut agglutinin (PNA)-HPMA copolymer and fluorescein isothiocyanate (FITC)-labeled WGA- and PNA-HPMA copolymer conjugates were synthesized. Conjugate dissociation constants (K d ) for lectin-carbohydrate binding determined by frontal affinity chromatography indicated that no activity reduction of the lectins occurred during the synthesis of these conjugates. K d values measured were in good agreement with literature findings for similar lectin-carbohydrate interactions, on the order of 10 -5 M -1 . Biorecognition of these conjugates by healthy rat intestinal tissue resulted in differential HPMA copolymer-lectin conjugate binding patterns in the same tissue. HPMA copolymer-WGA conjugate showed strong binding in the healthy rat intestinal tissues, while the HPMA copolymer-PNA conjugate showed minimal, but specific binding. This differential binding suggests that site-specific drug delivery via specific lectin recognition may be feasible for treatment of colon inflammation or cancer.

Differential regulation and transcriptional control of immediate early gene expression in forskolin-treated WEHI7.2 thymoma cells.

Agents that increase intracellular cAMP are frequently growth inhibitory for lymphocytes and induce apoptosis in cortical thymocytes by regulating gene expression. In the present study, immediate early gene expression was examined in WEHI7.2 thymoma cells undergoing cAMP-mediated apoptosis. Temporal differences in c-fos, junB, and inducible cAMP early repressor (ICER) steady-state mRNA levels were observed after forskolin exposure. Maximal induction of c-fos and junB occurred within 1 h, returning to basal levels by 3.5 h. In contrast, a 1.5-h time lag was observed before ICER transcript levels increased, reaching maximal levels after 3.5 h. This rise in expression, correlating with the decrease in c-fos and junB levels, preceded apoptotic DNA fragmentation by 1.5 h. Transient expression of ICER promoter constructs demonstrated that cAMP responsiveness occurred through cAMP-autoregulatory response element (CARE)3/4, two of the four proposed response elements in the ICER promoter. In contrast to the cAMP-responsive cell line JEG-3, CARE1/2 was not functional for cAMP-activated transcription in WEHI7.2 cells. An observed differential binding pattern of WEHI and JEG nuclear extracts to these elements may account for the cell-specific differences in expression patterns. To determine the role of endogenous ICER in regulating gene expression, cells were treated with two sequential doses of forskolin after which ICER and c-fos mRNA levels were measured. The high levels of cAMP-induced ICER expression dramatically reduced a second induction of c-fos. These data suggest that ICER expression may function as an antioncogene to attenuate the expression of certain protooncogenes, thereby preventing transformation and oncogenesis due to continuous overexpression. Moreover, inhibition of growth-stimulatory genes may be required for the activation of the cell death machinery in specific cells.

Fibrinogen Coating Density Affects the Conformation of Immobilized Fibrinogen: Implications for Platelet Adhesion and Spreading

Adhesion of platelets to immobilized fibrinogen appears to play an important role in a variety of physiologic and pathologic phenomena. We previously observed that the fibrinogen concentration used to coat polystyrene wells affected the morphology and distribution of GP IIb/IIIa receptors on the surface of platelets adherent to the fibrinogen. One possible explanation for these differences is that fibrinogen immobilized at high density adopts a different conformation than fibrinogen immobilized at low density. To address this possibility, we studied the binding of a panel of anti-fibrinogen monoclonal antibodies (mAbs) to fibrinogen immobilized at different coating densities. Three different patterns of binding were observed: 1) a linear increase in binding to wells coated with 1-10 microg/ml fibrinogen, followed by a lesser increase or plateau at higher fibrinogen concentrations (mAbs Fd4-4E1, Fd4-7B3, 1D4, 4-2); 2) minimal reactivity at all fibrinogen concentrations (mAbs GC4-1A12, 2C34); 3) a biphasic response, with a linear increase up to 10 microg/ml fibrinogen and then a significant decline in binding at higher fibrinogen concentrations (mAbs 311, 31A9, FPA 19/7, 9C3, 1C5-A5/2, 44-3). The patterns of mAb binding to fibrinogen immobilized from plasma were similar. Most mAbs that demonstrated a biphasic response bound poorly or not at all to soluble fibrinogen, while mAbs that demonstrated a linear/plateau response were able to bind soluble fibrinogen. At equal surface densities, mAbs that bound biphasically, particularly mAb 1C5-A5/2, were more reactive to urea-denatured than native fibrinogen. mAbs 1C5-A5/2 and 44-3 are specific for gamma 1-78 and 95-265, respectively, suggesting that the fibrinogen gamma-chain may be sensitive to changes in conformation induced by immobilization. In summary, these data suggest that fibrinogen immobilized at 1-10 microg/ml adopts a conformation unlike soluble fibrinogen, while fibrinogen immobilized at > 30 microg/ml adopts a more solution-like conformation. These differences in fibrinogen conformation may partially account for the ability of platelets to bind to immobilized fibrinogen without the addition of agonist, as well as the differences in spreading and GPIIb/IIIa distribution on platelets adherent to high- versus low-density immobilized fibrinogen.

Muscarinic toxins with selectivity for muscarinic receptor subtypes: Differential binding patterns in cloned and native receptors

Muscarinic toxins with selectivity for muscarinic receptor subtypes: Differential binding patterns in cloned and native receptors

Changes in glycoconjugate-lectin binding characteristics of in vitro and in vivo mouse blastocysts.

To investigate the influence of embryo culture, on blastocysts cultured from the zygote stage, on blastocyst cell surface glycoconjugates as an indicator of cellular metabolism. Eleven fluorescein isothiocyanate (FITC)-labelled lectins, which recognize different oligosaccharide structures, were applied to zona pellucida intact and zona pellucida free blastocysts which had developed either in vivo or in vitro. The differential binding patterns for the zona pellucida, trophectoderm and inner cell mass were determined using fluorescence microscopy. With the exception of wheat germ agglutinin (WGA) and Limax flavus agglutinin (LFA) the remaining nine lectins bound less intensely to the zona pellucida of the in vitro blastocysts compared with the in vivo blastocysts. The trophectoderm from both groups bound seven lectins with equal intensity, of the remaining four Lotus tetragonolbus agglutinin (LTA), Bandeiraea simplicifolia 4 (BSB4), Phaseolus vulgaris agglutinin P (PHA-P) and Limulus polyphemus agglutinin (LPA) demonstrated differential binding. The inner cell mass from the two blastocyst groups bound only three lectins identically, peanut agglutinin (PNA), Bandeiraea simplicifolia 4 (BSB4) and Dolichos biflorus agglutinin (DBA). Oviductal glycoproteins contribute to the zona pellucida glycoprotein content during early development, however embryo culture from the zygote stage may not provide adequate substrates for normal inner cell mass glycoconjugate biosynthesis.

Differences in the binding of blocking anti-CD11b monoclonal antibodies to the A-domain of CD11b.

CD11b/CD18 (Mac-1) is a leukocyte integrin that plays a critical role in neutrophil adhesion and the initiation of acute inflammatory responses. Several Mac-1 blocking mAbs bind to the A-domain of CD11b, a approximately 200 amino acid region in the N-terminal portion of the protein that is involved in ligand binding and Mac-1 functional activity. We examined several CD11b blocking mAbs for different patterns of binding to A-domain. We used human/murine chimeric CD11b expression constructs and deletions of the A-domain to examine binding. We describe the binding characteristics of mAbs 60.1, LM2/1, LPM19C, M170, 44, and 904. All of these mAbs, except for 60.1, bind to the C-terminal half of the human A-domain (CD11b181-316). mAb 60.1 was unique in that it required regions of the N- and C-terminal ends of the A-domain for binding. mAbs 60.1, LPM19C, 904, and 44 all required the A-domain to be intact for binding. This suggests that these CD11b mAbs recognize a conformational epitope. LM2/1 was capable of binding to a fragment of the A-domain, CD11b285-300. Inasmuch as this system has been used to define different mAb binding sites, it may be used to analyze specific ligand binding sites in the A-domain of CD11b.

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb.

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.