Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies.

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.     

Multiple forms and cellular localization of Drosophila DNA topoisomerase II

Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073-1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself.     

Clathrin structure characterized with monoclonal antibodies. II. Identification of in vivo forms of clathrin

Clathrin was isolated from detergent-solubilized, biosynthetically radiolabeled cells by immunoprecipitation with anti-clathrin monoclonal antibodies. Immunoprecipitates obtained after pulse-chase labeling demonstrated that after biosynthesis the LCa light chain of clathrin could be found either complexed to heavy chain or in a free pool (not associated with heavy chain) which decreased steadily over time. More than half of the free LCa disappeared within the first hour after biosynthesis, but some was still detectable after several hours. Incorporation of clathrin LCa light chain and heavy chain into coated vesicles was coordinate and increased up to 4 h after biosynthesis. Comparison of these kinetics suggested that once incorporated into coated vesicles, LCa and heavy chain did not dissociate, even during depolymerization of the vesicle. There was also little apparent degradation of clathrin found in coated vesicles for up to 22 h after biosynthesis. Immunoprecipitation with anti-clathrin monoclonal antibodies was carried out after fractionation of continuously radiolabeled cell lysates using two different sizing columns. These experiments indicated that the triskelion form of clathrin that has been isolated from coated vesicles in vitro also exists in vivo. They also confirmed the existence of a transient but detectable pool of newly synthesized free LCa light chain.     

8-methoxycaffeine inhibition of Drosophila DNA topoisomerase II.

We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase II and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug.     

Regional heterogeneity of human spermatozoa detected with monoclonal antibodies

The regional antigenic heterogeneity of human spermatozoa is confirmed with 6 monoclonal antibodies raised against ejaculated human spermatozoa. The topographical localization of the antigenic determinants suggests the existence of at least 6 domains on the human spermatozoon. Different fixatives had severe detrimental effects on the antigen-antibody binding. On live human spermatozoa, each antibody bound to a distinct region: acrosome, equatorial segment, entire tail, neck, midpiece and terminal piece. The antigens detected on the acrosome, equatorial segment and entire tail were surface components, whereas the other three were intracellular structures. The determinant present along the entire tail was a sperm-coating antigen. The molecular weights of the recognized antigens were estimated with the Western blot technique. Immunostaining of individual ejaculates established that the percentages of positive cells were 12-56% for the acrosome, 8-35% for the equatorial segment, 90-100% for the entire tail, 20-52% for the neck, 9-35% for the midpiece and 36-90% for the terminal piece. In addition, labelling of motile and immotile spermatozoa showed differences in the percentages of positive cells, with 5 out of 6 monoclonal antibodies, or in the fluorescence intensity, with the last one labelling the entire tail.     

Antigens of selected Acanthamoeba species detected with monoclonal antibodies

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.     

Antigenic variation of influenza A virus nucleoprotein detected with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in the nucleoprotein of influenza A viruses. We found that the nucleoprotein molecule of the WSN/33 strain possesses at least five different determinants. Viruses of other influenza A virus subtypes showed antigenic variation in these nucleoprotein determinants, although changes in only one determinant were detected in H0N1 and animal strains. The nucleoprotein of human strains isolated from 1933 through 1979 could be divided into six groups, based on their reactivities with monoclonal antibodies; these groups did not correlate with any particular hemagglutinin or neuraminidase subtype. Our results indicate that antigenic variation in the nucleoproteins of influenza A viruses proceeds independently of changes in the viral surface antigens and suggest that point mutations and genetic reassortment may account for nucleoprotein variability.     

Neutrophil activation detected by monoclonal antibodies

Monoclonal antibodies have been produced against three neutrophil-associated membrane proteins (p 90, p 170, and p 70) expressed at different maturation stages of the cells. The reactivity of the antibodies against p 90 (B13.9) and p 170 (CLB-gran 10), as measured by quantitative flow cytofluorometry, increased after stimulation of the neutrophils by the calcium ionophore A23187, by phorbol myristate acetate, or by the chemoattractant formylmethionyl-leucyl-phenylalanine in combination with cytochalasin B. This increase is regulated independently of the simultaneously increased expression of the C3bi receptor, because neutrophils of a patient deficient for the C3bi receptor showed a normal increase in membrane expression of p 90 and p 170. Neutrophil cytoplasts were not inducible to increased membrane expression, suggesting that the cytoplasts lack the internal pool of these proteins. The reactivity of the antibody against p 70 (CLB-gram 5) was not affected by activation. The antibodies B13.9 and CLB-gran 10 may be useful to detect neutrophil activation.     

Developmental regulation of Drosophila DNA topoisomerase II

Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Heterogeneity of growth-hormone receptors detected with monoclonal antibodies to human growth hormone.

The specificity of hormone-receptor interactions has been examined with the aid of monoclonal antibodies (MABs) (EB1, EB2, QA68 and NA71) defining four non-overlapping antigenic determinants on human growth hormone (hGH). The results indicate that growth-hormone receptors in liver obtained from different sources differ with regard to their affinities and relative numbers; they may also differ with respect to the region of the growth-hormone molecule to which they bind. Antibody NA71 effectively inhibited hormone binding to all receptor preparations tested, although with various degrees of potency. Monoclonal antibody EB1 demonstrated a graded inhibition with respect to its ability to block 125I-hGH binding to receptors from various sources, the maximum inhibition being seen in receptor preparations from mouse and ovine liver and the minimum in rat liver. MABs EB2 and QA68 also showed various abilities to inhibit hormone-receptor interaction, depending on the origin of the receptor preparation. Furthermore, the receptor-binding characteristics of hormone-antibody complexes were dependent on whether the binding-site preparation was derived from pregnant, lactating or 'normal' animals. A particularly striking difference between the ability of hormone-MAB complexes to bind to receptors from different sources was seen for microsomes (microsomal fractions) derived from livers of animals of the 'Little' mouse strain. These animals become progressively obese, and it was shown that MABs were considerably more effective in inhibiting 125I-labelled hGH binding to microsomes from phenotypically obese mice than to those derived from their non-obese littermates. The results can be explained by the presence of multiple receptor types for GH, the relative proportions of which vary according to the physiological state of the animal, and possibly between species.     

Basement membrane diversity detected by monoclonal antibodies

Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno) chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Basement membrane diversity detected by monoclonal antibodies

Abstract. Human fetal membranes or pepsin solubilized proteins thereof were used as immunogens in the production of monoclonal antibodies to basement membrane-associated components. Some of the antibodies obtained reacted with all basement membranes in indirect immunofluorescent-cent microscopy, others reacted with all epithelial but not with endothelial basement membranes, and yet other antibodies reacted only with certain epithelial basement membranes in these tests. The reactivities of the antibodies demonstrate that different basement membranes are (immuno)chemically different and contain unique components in addition to ubiquitous components such as type IV collagen and laminin     

Carbohydrate sequences detected by murine monoclonal antibodies

The carbohydrate sequences of cell surface glycolipids change during differentiation and oncogenic transformation. To detect these structural changes, murine monoclonal antibodies have been produced in many different laboratories. Some of these antibodies are used to distinguish various cell types such as normal and transformed cells, while others are used to analyze developmentally regulated antigens. Recently, the structures of many of these carbohydrate antigens have been determined. The availability of these well-defined monoclonal antibodies will be useful for the study of the regulation and function of glycoconjugates.     

DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.     

Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.     

Nuclease protection by Drosophila DNA topoisomerase II. Enzyme/DNA contacts at the strong topoisomerase II cleavage sites

A DNA consensus sequence for topoisomerase II cleavage sites was derived previously based on a statistical analysis of the nucleotide sequences around 16 sites that can be efficiently cleaved by Drosophila topoisomerase II (Sander, M., and Hsieh, T. (1985) Nucleic Acids Res. 13, 1057-1072). A synthetic 21-mer DNA sequence containing this cleavage consensus sequence was cloned into a plasmid vector, and DNA topoisomerase II can cleave this sequence at the position predicted by the cleavage consensus sequence. DNase I footprint analysis showed that topoisomerase II can protect a region of approximately 25 nucleotides in both strands of the duplex DNA, with the cleavage site located near the center of the protected region. Similar correlation between the DNase I footprints and strong topoisomerase II cleavage sites has been observed in the intergenic region of the divergent HSP70 genes. This analysis therefore suggests that the strong DNA cleavage sites of Drosophila topoisomerase II likely correspond to specific DNA-binding sites of this enzyme. Furthermore, the extent of DNA contacts made by this enzyme suggests that eucaryotic topoisomerase II, in contrast to bacterial DNA bacterial DNA gyrase, cannot form a complex with extensive DNA wrapping around the enzyme. The absence of DNA wrapping is probably the mechanistic basis for the lack of DNA supercoiling action for eucaryotic topoisomerase II.     

DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.