The selective detection of cell surface determinants by means of antibodies and acetylated avidin attached to highly fluorescent polymer microspheres

Procedures are described for the synthesis of 500 A-diameter polymer microspheres containing a novel fluorescent cross-linking agent. These microspheres have very high fluorophore concentration without quenching of the fluorescence and show very low nonspecific interaction with cells. When monoclonal anti-Thy-1.2 is attached to the fluorescent microspheres, specific binding results in 10(4) spheres being attached per thymocyte while non-specific binding is less than 1%. Similar values are obtained for an indirect staining procedure. The high non-specific binding of cationic avidin to negative cell surfaces is shown to be decreased to negligible levels by acetylation of the amine groups of the protein without decreasing its high-affinity binding to biotin. The use of acetyl-avidin (pI = 6.7) directly, or when attached to fluorescent microspheres, resulted in a highly selective detection of biotinyl groups on the erythrocyte or lymphocyte cell surface. Attachment of biotinyl groups to the hinge carbohydrates of antibodies did not affect their specificity. It allowed their detection by means of microspheres-acetyl-avidin conjugates.     

Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides.

Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.     

An improved protocol of biotinylated tyramine-based immunohistochemistry minimizing nonspecific background staining.

An immunohistochemical method using biotinyl tyramine was recently introduced to amplify weak staining signals. Despite its high sensitivity, however, tyramine-based immunostaining has been limited by its increased background staining. In this study, to develop an improved protocol of biotinyl tyramine-based immunohistochemistry minimizing the background staining, we determined which staining steps lead to the nonspecific reaction and the most appropriate blocking agents for background-provoking steps. Trypton casein peptone and distilled water with Tween-20 were shown to be most effective as a blocking agent and a rinsing solution, respectively. In conclusion, we developed an optimized protocol for biotinyl tyramine-based immunohistochemistry with minimal background staining.     

Use of streptavidin to detect biotin-containing proteins in plants

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.     

Electrogenerated chemiluminescence biosensing method for the detection of DNA methylation and assay of the methyltransferase activity

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the CpG methyltransferase (M. SssI) activity was developed on basis of enzyme-linkage reactions and ruthenium complex served as an ECL tag. The ECL biosensing electrode was fabricated by self-assembling 5'-thiol modified 32-mer single-strand DNA (ss-DNA)-tagged with ruthenium bis (2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid)-ethylenediamine on the surface of a gold electrode, and then hybridized with complementary ss-DNA to form duplex DNA (ds-DNA). When M. SssI and S-adenosylmethionine were introduced, all cytosine residues within 5'-CG-3' of ds-DNA on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by HpaII endonuclease, the un-methylated cytosines were cleaved, thus led to decrease ECL signal. The ECL intensity of ECL biosensing electrode is related to the methylation level and M. SssI activity in a fixed concentration HpaII endonuclease. The increased ECL intensity was direct proportion to M. SssI activity in the range from 0.05 to 100 U/mL with a detection limit of 0.02 U/mL. This work demonstrates that the combination of the enzyme-linkage reactions with a highly sensitive ECL technique is a great promising approach for the detection of DNA methylation level, assay of the activity of MTase, and evaluation of the capability of inhibitors for the methyltransferase.     

Solution structures of apo and holo biotinyl domains from acetyl coenzyme A carboxylase of Escherichia coli determined by triple-resonance nuclear magnetic resonance spectroscopy

A subgene encoding the 87 C-terminal amino acids of the biotinyl carboxy carrier protein (BCCP) from the acetyl CoA carboxylase of Escherichia coli was overexpressed and the apoprotein biotinylated in vitro. The structures of both the apo and holo forms of the biotinyl domain were determined by means of multidimensional NMR spectroscopy. That of the holo domain was well-defined, except for the 10 N-terminal residues, which form part of the flexible linker between the biotinyl and subunit-binding domains of BCCP. In agreement with X-ray crystallographic studies [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419], the structure comprises a flattened beta-barrel composed of two four-stranded beta-sheets with a 2-fold axis of quasi-symmetry and the biotinyl-lysine residue displayed in an exposed beta-turn on the side of the protein opposite from the N- and C-terminal residues. The biotin group is immobilized on the protein surface, with the ureido ring held down by interactions with a protruding polypeptide "thumb" formed by residues 94-101. However, at the site of carboxylation, no evidence could be found in solution for the predicted hydrogen bond between the main chain O of Thr94 and the ureido HN1'. The structure of the apo domain is essentially identical, although the packing of side chains is more favorable in the holo domain, and this may be reflected in differences in the dynamics of the two forms. The thumb region appears to be lacking in almost all other biotinyl domain sequences, and it may be that the immobilization of the biotinyl-lysine residue in the biotinyl domain of BCCP is an unusual requirement, needed for the catalytic reaction of acetyl CoA carboxylase.     

Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs.     

Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V(1a) vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.     

Structure Prediction of the Second Extracellular Loop in G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) play key roles in living organisms. Therefore, it is important to determine their functional structures. The second extracellular loop (ECL2) is a functionally important region of GPCRs, which poses significant challenge for computational structure prediction methods. In this work, we evaluated CABS, a well-established protein modeling tool for predicting ECL2 structure in 13 GPCRs. The ECL2s (with between 13 and 34 residues) are predicted in an environment of other extracellular loops being fully flexible and the transmembrane domain fixed in its x-ray conformation. The modeling procedure used theoretical predictions of ECL2 secondary structure and experimental constraints on disulfide bridges. Our approach yielded ensembles of low-energy conformers and the most populated conformers that contained models close to the available x-ray structures. The level of similarity between the predicted models and x-ray structures is comparable to that of other state-of-the-art computational methods. Our results extend other studies by including newly crystallized GPCRs.     

A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)₃²⁺)-end-labelled primers. In this way, biotin for capture and Ru(bpy)₃²⁺ for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)₃²⁺-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.     

Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes.

The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands. BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn. The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant.     

A comprehensive approach to mapping the interacting surfaces of murine amphotropic and feline subgroup B leukemia viruses with their cell surface receptors

Because mutations in envelope glycoproteins of retroviruses or in their cell surface receptors can eliminate function by multiple mechanisms, it has been difficult to unambiguously identify sites for their interactions by site-directed mutagenesis. Recently, we developed a gain-of-function approach to overcome this problem. Our strategy relies on the fact that feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) have closely related gp70 surface envelope glycoproteins and use related Na(+)-dependent phosphate symporters, Pit1 and Pit2, respectively, as their receptors. We previously observed that FeLV-B/A-MLV envelope glycoprotein chimeras spliced between the variable regions VRA and VRB were unable to use Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras. The latter study suggested that the VRA of A-MLV and FeLV-B functionally interact with the presumptive extracellular loops 4 and 5 (ECL4 and -5) of their respective receptors, whereas VRB interacts with ECL2. We also found that FeLV-B gp70 residues F60 and P61 and A-MLV residues Y60 and V61 in the first disulfide-bonded loop of VRA were important for functional interaction with the receptor's ECL4 or -5. We have now extended this approach to identify additional VRA and VRB residues that are involved in receptor recognition. Our studies imply that FeLV-B VRA residues F60 and P61 interact with the Pit1 ECL5 region, whereas VRA residues 66 to 78 interact with Pit1 ECL4. Correspondingly, A-MLV VRA residues Y60 and V61 interact with the Pit2 ECL5 region, whereas residues 66 to 78 interact with Pit2 ECL4. Similar studies that focused on the gp70 VRB implicated residues 129 to 139 as contributing to specific interactions with the receptor ECL2. These results identify three regions of gp70 that interact in a specific manner with distinct portions of their receptors, thereby providing a map of the functionally interacting surfaces.     

A new large form of transcarboxylase with six outer subunits and twelve biotinyl carboxyl carrier subunits.

A new form of transcarboxylase has been isolated which has a molecular weight of 1,200,000, an s20,w of 26 S, and contains 12 biotinyl groups. Transcarboxylase as isolated previously has a molecular weight of 790,000, an s20,w of 18 S, and contains six biotinyl groups. The larger species of enzyme consists of a central hexameric subunit with six dimeric outer subunits attached to it by biotinyl carboxyl carrier proteins, three each at the opposite faces of the central subunits. This larger species is stable at pH 5.5, but dissociates to the 18 S species at pH values near neutrality with loss of a set of three of the outer subunits with two of the biotinyl carboxyl carrier proteins still attached to each of these subunits. The dissociation to the 18 S form occurs by several rapidly reversible steps and under certain conditions of centrifugation multiple peaks are observed as a consequence of the occurrence of different forms of enzyme with variable numbers of the outer subunits attached to the 18 S enzyme. The s20,w value of the so-called 26 S enzyme varies with conditions. Isolated 18 S enzyme has been combined with isolated outer subunits to form active 26 S enzyme. The newly enzyme is a normal form but has not been isolated previously because of its dissociation to the 18 S form at neutral pH. A procedure is described for the isolation of the 26 S form in a highly purified state. The molecular weight of the enzyme has been determined by high speed meniscus depletion. In addition, a procedure is described for dissociation of the 26 S form of the enzyme and isolation of the resulting outer subunits with the biotinyl subunits still attached to it. Evidence is presented that all six outer subunits participate in the enzymatic reaction which includes the demonstration that; (a) all 12 biotins of the 26 S form of the enzyme can be carboxylated with [3-14C]methylmalonyl coenzyme A; (b) there is an increase in enzymatic activity when the outer subunits are combined with the normal 18 S enzyme with formation of the 26 S enzyme; and (c) a 26 S form of the enzyme is active which is prepared by combination of inactive 18 S trypsin-treated transcarboxylase with the outer subunits. The trypsin-treated 18 S enzyme is inactive because trypsin removes the biotin as biotinyl peptides and the 26 S enzyme is active because of the second set of active outer subunits.     

A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands.

Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.     

Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses

CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1NDK or by FIVPET (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-189.6 (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-189.6. In contrast, its inactivity toward HIV-1NDK seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIVPET. Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1NDK infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue.     

The anatomy of transcarboxylase and the role of its subunits.

Biotin enzymes in general catalyze the fixation of CO2 and in a few instances decarboxylations yielding CO2. Transcarboxylase is an exception; it catalyzes the transfer of a carboxyl group from one compound to another and CO2 is not involved. This enzyme plays an essential role in the formation of propionic acid by propionibacteria and its structure and catalytic mechanism have been extensively investigated including studies of the quaternary structure by electron microscopy. The structure is complex, consisting of three types of subunits: (1) a central hexameric subunit, (2) six dimeric outside subunits, and (3) twelve biotinyl subunits which bind the outside subunits to the central subunit. There are 12 substrate sites on the central subunit (2 per polypeptide) and 2 substrate sites on each of the dimeric outside subunits. The carboxyl is transferred between these sites via the biotin of the biotinyl subunit. The biotinyl subunit (approximately 123 residues) has been completely sequenced and it has been shown that the first 42 residues serve in binding the outside subunits to the central subunit and the remainder of the sequence is involved in placing the biotin between the subunits so that it may serve as the carboxyl carrier between the substrate sites on the central and outside subunits. It is proposed that the dual sites on the polypeptides of the central subunit have arisen as a consequence of gene duplication and fusion. An intriguing question is why such a complicated structure is required for catalysis of a rather simple reaction.     

Charged extracellular residues, conserved throughout a G-protein-coupled receptor family, are required for ligand binding, receptor activation, and cell-surface expression

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V(1a) vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg(116) (ECL1), Arg(125) (top of TMIII), and Asp(204) (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg(125) (and Lys(125)) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp(125)) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp(204) (and Glu(204)) establishes TMIII contacts required for maintaining the beta-hairpin fold of ECL2, which if broken (Ala(204) or Arg(204)) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.     

Comprehensive mapping of receptor-functioning domains in feline leukemia virus subgroup C receptor FLVCR1

Infection of cells by the highly anemogenic feline leukemia virus subgroup C (FeLV-C) is mediated by the heme exporter FLVCR1, a cell surface protein containing 12 potential transmembrane segments with six presumptive extracellular loops (ECLs). To identify FLVCR1 residues critical for mediating FeLV-C infection, we first independently isolated a human cDNA encoding the FLVCR2 protein that shares 52% identity to human FLVCR1, and we show that FLVCR2 does not function as a receptor for FeLV-C. Then, by generating specific hybrids between FLVCR1 and FLVCR2 and testing susceptibility of mouse cells expressing these hybrids to beta-galactosidase encoding FeLV-C, we identify FLVCR1 ECLs 1 and 6 as critical for mediating FeLV-C infection. Mouse cells expressing a hybrid protein containing FLVCR2 backbone with the ECL6 sequence from FLVCR1 were highly susceptible to FeLV-C infection. Using site-directed mutagenesis, we show that a single mutation of Asn463 in FLVCR2 ECL6 to an acidic Asp residue (a residue present in the corresponding position 487 in FLVCR1 ECL6) is sufficient to render FLVCR2 functional as an FeLV-C receptor. However, an Asp487Asn mutation in FLVCR1 ECL6 or substitution of the entire FLVCR1 ECL6 sequence for FLVCR2 ECL6 sequence does not disrupt receptor function. Subsequent substitutions show that residues within FLVCR1 ECL1 also contribute to mediating FeLV-C infection. Furthermore, our results suggest that FLVCR1 regions that mediate FeLV-C surface unit binding are distinct from ECL1 and ECL6. Our results are consistent with previous conclusions that infection of cells by gammaretroviruses involves interaction of virus with multiple receptor regions.     

Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase

Lectin binding patterns in normal human skin were studied using five different biotinyl lectins and avidin-horseradish peroxidase. The staining pattern was specific for each lectin. In the epidermis, peanut agglutinin (PNA) and soybean agglutinin (SBA) preferentially stained the cell membranes of keratinocytes in the spinous and granular cell layers, indicating changes in the saccharide residues during keratinocyte differentiation. In the secretory segment of an eccrine sweat gland, the superficial cells gave a strong granular staining with Ricinus communis agglutinin (RCA). Dolichos biflorus agglutinin (DBA) and SBA, on the other hand, strongly stained the basal cells. With these lectins, two types of cells in the secretory segment were clearly distinguished. These results show that (1) PNA and SBA binding sites increase during the course of keratinocyte differentiation, and (2) RCA, DBA, and SBA are good markers to distinguish two types of cells in the secretory segment of an eccrine sweat gland.     

An improved synthesis of arsenic-biotin conjugates

An amide linked conjugate of p-aminophenylarsine oxide and biotin is conveniently prepared in a one-pot procedure by the reaction of biotinyl chloride, formed in situ, with p-aminophenyldichloroarsine. The reaction of the arsine oxide-biotin conjugate with 1,2-ethanedithiol produces the stabilized dithiarsolane. These reagents are now readily available for a variety of applications.