Monoclonal antibodies to type D retrovirus (SRV-2)

Monoclonal antibodies (MoAbs) to the major gag core protein p27 and a viral protein p44 of type D retrovirus (SRV-2) were produced and used in the detection of SRV-2 antigens in infected Raji cells and in tissues from macaques with simian acquired immunodeficiency syndrome (SAIDS) and retroperitoneal fibromatosis (RF). Anti-p44 MoAb showed inhibition of syncytium formation by both SRV-1- and SRV-2-infected Raji cells.     

Isolation and partial characterization of a type II Fe receptor from a group A streptococcus

A group A streptococcal strain rich in Fc receptors was selected by an immunoblotting technique and used as the source for isolation of a functionally active Fc receptor. A variety of extraction techniques were compared including (1) heat extraction at neutral, acid or alkaline pH, (2) treatment with the enzymes mutanolysin, hyaluronidase, trypsin, papain or phage lysin, or (3) autoclaving or heating in the presence of sodium dodecyl sulfate. The most homogeneous receptor was recovered following heat extraction and contained two molecular weight forms. The major form had a molecular weight of 56 000 daltons and the minor form had a molecular weight of 38 000 daltons. These two proteins could be isolated without loss of activity by binding to and elution from a column of immobilized human IgG. An antibody prepared against a single form of the affinity purified receptor demonstrated reactivity with both molecular weight forms of the heat extracted receptor. The group A receptor was found to be both antigenically and physicochemically distinct from either the type I receptor found on the majority of Staphylococcus aureus strains or the type III Fc receptors found on the majority of group C streptococcal strains.     

An immunoblotting technique for the detection of bound and secreted bacterial Fc receptors

A rapid semiquantitative procedure that enables bacteria to be screened for surface or secreted receptors for the Fc region of human IgG is described. Surface Fc receptors were detected by direct transfer of bacterial colonies to nitrocellulose by electroblotting and then probing with ¹²⁵I-labeled human IgG in the presence of a two fold molar excess of unlabeled F(ab′)₂fragments. The blots were exposed to X-ray film and the intensity of the resulting autoradiograph was a measure of surface Fc receptors expression. This procedure reliably distinguished Staphylococcus aureus strains which expressed different levels of surface Fc receptors. When applied to the study of group A streptococci, a number of Fc receptor-positive strains were identified. Unlike the homogeneous Fc receptor expression on individual colonies of the staphylococcal strains, a wide variation in the level of Fc receptor expression was observed within a given streptococcal strain. Group A streptococcal substrains which expressed high and low levels of surface Fc receptors could be isolated from replica plates.Secreted Fc receptors were measured by a simple modification of the blotting procedure in which the nitrocellulose was placed on the opposite side of the agar from the bacterial colonies. Secreted Fc receptors was electroblotted through the agar onto nitrocellulose and probed as described above. This approach readily detected nanogram quantities of secreted type I Fc receptor (protein A) from the Staphylococcus aureus Cowan strain. None of the group A streptococcal strains tested were found to secrete detectable quantities of Fc receptors.     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Identification of Rad's effector-binding domain,intracellular localization,and analysis of expression in Pima Indians

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109–121, 178–195, 254–271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTPγS, and GDPβS but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109–121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109–121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis. J. Cell. Biochem. 65:527–541. © 1997 Wiley-Liss Inc.     

Influence of dipeptides on the interaction of immunoglobulins with bacterial Fc receptors

Binding of radiolabeled human IgG to bacteria expressing type I, type II, or type III Fc receptors in the presence of glycyl-glycine, glycyl-tyrosine, glycyl-histidine, glycyl-leucine, or glycyl-phenylalanine was studied. No inhibition of labeled human IgG binding to type I or type III Fc receptor positive bacteria was observed by any of the dipeptides. Inhibition of binding of labeled human IgG1, IgG2, and IgG4, but not IgG3, indicated the presence of two distinct Fc receptors associated with the type II Fc receptor-positive group A streptococcal strain.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.