Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris.

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.     

Anti-schistosome monoclonal antibodies of different isotypes--correlation with cytotoxicity

Five monoclonal antibodies specific towards Schistosoma mansoni antigens were prepared by fusion of spleen cells of infected and immunized mouse with the murine myeloma NS-1 cells. Three of the five antibodies belonged to the IgG1 class, one was an IgM and the fifth one was an IgE. The IgE monoclonal antibody designated 54.10, induced antigen-specific degranulation of rat basophilic cell line, a property which served as the basis for the screening assay. Its biological function was demonstrated by a specific macrophage activation that led to killing of schistosomula; no such killing was obtained with anti-schistosome antibodies of other classes or with IgE of different antigenic specificity. The second monoclonal antibody of biological significance was an IgG1, designated 27.21 which is reactive in the immunofluorescence staining of surface antigens on intact schistosomula. All three monoclonal antibodies that belonged to the IgG1 class were effective in mediating killing of schistosomula by complement, with the highest effect exerted by 27.21. It is thus apparent that the 27.21 monoclonal antibody is directed against a densely distributed surface antigen on the schistosomula membrane which is possibly involved in the protective immunity. Preliminary data showed that immunoprecipitation with the 27.21 antibodies results in the isolation of three major protein bands, of 60 kd, 50 kd, 19 kd, respectively.     

Expression of Neural Antigens in Normal Xenopus Embryos and Induced Explants

Monoclonal antibodies were raised against neural tissues of Xenopus larvae. Three monoclonal antibodies, named NEU-1, NEU-3, and NEU-4, were specific for neural tissue and first bound to neural cells at stage 25 after neural tube formation (NEU-1 and NEU-3) or at stage 31 (NEU-4). These antibodies bound to differentiating neural cells, but not to germinal neuroepithelial cells. NEU-1 and NEU-3 recognized antigens in cell bodies as well as neural fibers of neural cells, and these antigens were distributed throughout the central nervous system. NEU-4 bound to antigens in granular materials in neural cells, and these antigens were present in head and trunk regions but not in the tail region.
These three antibodies were used as neural markers in two types of induction experiments, in which 1) the animal pole region and the dorsal blastopore lip from stage-10 gastrulae were combined, or 2) the animal pole region and the vegetal pole region from stage-8 blastulae were combined. In both experiments, most conjugated explants expressed the NEU-1, NEU-3, and NEU-4 antigens, although the expression of NEU-4 antigen was delayed compared with those of the NEU-1 and NEU-3 antigens. These results show that these antibodies are useful as markers in neural induction experiments.     

Tissue specific nuclear antigens in the germinal vesicle ofXenopus laevis oocytes

Summary A library of hybridoma cell lines has been established which produce monoclonal antibodies against antigens from the germinal vesicle ofXenopus laevis oocytes. Many of the antigens are also found in the nuclei ofXenopus embryonic cells in culture. The fate of two of these antigens during embryogenesis was traced by immunofluorescence on embryo and tadpole sections. Early in development these antigens appear to be evenly distributed in the nuclei of all cells. In later stages they gradually disappear from most embryonic structures but are strongly accumulated in the nuclei of some specific cell types and organs.     

The expression of epidermal antigens in Xenopus laevis

Five kinds of monoclonal antibodies that are specific for the epidermis of Xenopus embryos were produced. Epidermis-specific antibodies were used to investigate the spatial and temporal expressions of epidermal antigens during embryonic and larval development. The cells that were recognized by the antibodies at the larval stage are as follows: all of the outer epidermal cells and cement gland cells were recognized by the antibody termed XEPI-1, all of the outer and inner epidermal cells, except the cement gland cells, were recognized by XEPI-2 antibody, the large mucus granules and the apical side of the outer epidermal cells, except for the ciliated epidermal cells, were recognized by XEPI-3 antibody, the large mucus granules and basement membrane were recognized by XEPI-4 antibody, and the small mucus granules contained in the outer epidermal cells as well as extracellular matrices were recognized by the antibody termed XEPI-5. All of the epidermal antigens, except XEPI-4, were first detected in the epidermal region of the late gastrula or early neurula. The XEPI-4 antigen was first detected in stage-26 tail-bud embryos. None of these antigens were expressed by the neural tissues at any time during embryonic development. Only the XEPI-2 antigen continued to be expressed after metamorphosis, while the expression of the other antigens disappeared during or before metamorphosis. The specificity of the antibodies allowed us to classify the epidermal cells into four types in early epidermal development. The four types of epidermal cells are (1) the outer epidermal cells that contain small mucus granules, (2) the ciliated epidermal cells, (3) the outer epidermal cells that contain large mucus granules and (4) the inner sensorial cells.     

Immunocytochemical evidence for centrosomal phosphoproteins in mitotic sea urchin eggs

The change in distribution of centrosomal phosphoproteins was examined in sea urchin eggs from fertilization to the first cleavage by immunofluorescence staining with the anti-phosphoprotein antibodies, MPM-1 and MPM-2. The antibodies reacted with female pronuclei in unfertilized eggs as well as centriolar complexes located at the base of sperm flagella. After insemination, male and female pronuclei fused together to form a zygotic nucleus which was visualized by staining of fertilized eggs with the antiphosphoprotein antibodies. No major change in staining pattern was detected in extracted whole eggs until mitosis. As the fertilized eggs approached mitosis, however, the antigens started to redistribute from nuclei to the perinuclear position where the mitotic centrosomes were located. Detailed immunofluorescence observation of isolated spindles revealed that the phosphoantigens were retained in isolated structures. A major 225 kd polypeptide was recognized by the antibodies, suggesting that the 225 kd protein is a phosphocomponent of centrosomes. The area recognized by the antibody in mitotic poles enlarged with the progress of mitosis, suggesting that the antigens were apparently localized in the centrosphere. Centrospheres prepared from isolated spindles by salt extraction strongly reacted with the antibodies. One or two bright dots, which may represent centrioles, were visible in the isolated centrosphere. At the end of mitosis, the antigens again appeared in the newly formed daughter nuclei. Centriole-containing cytasters and centriole-free monasters were parthenogenetically induced in unfertilized eggs (Kuriyama and Borisy, (1983) J. Cell Sci. 61: 175-189). The antibodies stained centers of both the asters whether they contained centrioles or not, indicating that the antibodies recognizes the components of the pericentriolar material.     

Germline-specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized egg's cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.     

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

Monoclonal antibodies demonstrate multiple epitopes on the O antigens of Salmonella typhimurium LPS

To investigate the complexity of the antigenic determinants presented on the surface of Salmonella typhimurium, a panel of murine monoclonal antibodies was generated and characterized. Hybridomas specific for S. typhimurium (strain TML, O antigens 1, 4, 12) were produced by immunization with acetone-killed and dried bacteria and standard fusion procedures. In this report, 15 such monoclonal antibodies, all of which bind lipopolysaccharide (LPS) extracted from S. typhimurium, are described. The fine specificity of these antibodies was assessed by examining the differential binding of each antibody to a panel of Salmonella strains, which selectively express different O antigenic determinants. This analysis defined several distinct categories of monoclonal antibodies of varying isotypes. Four anti-O:4-specific antibodies were identified. Two were specific for O:1. One antibody appears to react with the core polysaccharide of S. typhimurium LPS. Several of the monoclonal antibodies recognized LPS determinants that are presumably created by a combination of O antigens. For instance, one bound only to Salmonella strains that expressed both O:1 and O:12, whereas another bound only to those strains which expressed both O:4 and O:12. A group of three antibodies bound to any strain that simultaneously expressed O:1, O:4, and O:12. A distinct group of three monoclonal antibodies also bound strains that expressed O:1, O:4, and O:12, but only when the O:5 antigenic determinant was not present. The latter are, in that respect, S. typhimurium strain TML LPS-specific. The results of this analysis suggest that the epitopes of the S. typhimurium LPS molecule that are recognized by the host are considerably more complex than has been previously indicated by classical serology.     

Interaction of antibodies against nuclear envelope-associated proteins from rat liver nuclei with rodent and human cells

Antibodies have been prepared against the three major polypeptides of the nuclear pore complex-lamina fraction from rat liver nuclei. The three antisera prepared in chickens give similar results in indirect immunofluorescence microscopy. In rat embryo fibroblasts we observe bright fluorescence at the level of the nuclear envelope, with no fluorescence of the nuclear interior and little or no fluorescence of the cytoplasm. The nuclear envelope regions of rat hepatoma cells, mouse A9 cells, HeLa cells and rat liver nuclei also fluoresce brightly. HeLa nucleoids, which are depleted of nuclear envelope components, still exhibit specific fluorescence when reacted with these antibodies. Distribution of the antigens changes during mitosis. Fluorescence in the cytoplasm is observed following the breakdown of the nuclear envelope at prometaphase. The antigens appear to progressively accumulate at the periphery of the chromosomes until telophase. In late telophase fluorescence occurs predominantly at the periphery of the chromosomes where the new nuclear envelope is formed.     

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

We have examined preimmune serum samples from a patient who progressively developed the symptoms of scleroderma CREST over a period of several years. During this period, anti-centromere antibodies (recognized by indirect immunofluorescence) appeared in the serum. Concomitant with the appearance of the anti-centromere antibodies, antibody species recognizing three chromosomal antigens in immunoblots of SDS polyacrylamide gels appeared in the patient's serum. These antigens migrate with electrophoretic mobilities corresponding to M_r=17, 80, and 140 kilodaltons (kd). Affinity-eluted antibody fractions recognizing the antigens have been prepared from sera of three other patients. Indirect immunofluorescence labeling of mitotic cells using these antibody fractions demonstrates that the antigens are centromere components. We designate them CENP (CENtromere Protein) — A (17kd), CENP-B (80kd), and CENP-C (140kd). The three CENP antigens share antigenic determinants. Immunoblotting experiments show that these patients make antibody species recognizing at least three distinct epitopes on CENP-B and two on CENP-C. Sera from different patients contain different mixtures of the antibody species.     

Immunobiochemical characterization with monoclonal antibodies of Epstein-Barr virus-associated early antigens in chemically induced cells.

Five monoclonal antibodies which are reactive to early antigens of Epstein-Barr virus have been produced by using somatic cell hybridization techniques. The specificity of the monoclonal antibodies to early antigens was demonstrated by indirect immunofluorescence, which showed that the antigens were localized to the nucleus of early antigen-induced Raji cells. Additional indirect immunofluorescence studies showed that like patient antisera to diffuse-staining early antigen, the monoclonal antibodies gave positive staining reactions after methanol fixation. One of the antibodies, 1150-4, was positive by the anti-complement immunofluorescence technique but differed with Epstein-Barr virus-associated nuclear antigen-positive patient sera in that it only stained induced cells. Different fixation methods were found to alter dramatically the appearance of the nuclear staining reactions produced by the monoclonal antibodies. Immunoprecipitation and immunoblot experiments revealed that monoclonal antibodies 1108-1 and 1129-1 recognized two polypeptides of 55,000 and 50,000 daltons (p55;50), 1173-6 and 1180-2 recognized just p50, and 1150-4 identified a 65,000-dalton nuclear protein. Immunobiochemical characterization of these viral antigens showed that p55 is a phosphoprotein, and p55;50 has strong DNA-binding activity preferentially to single-stranded DNA. Elucidation of the role of these nuclear proteins in Epstein-Barr virus infection and the events associated with Epstein-Barr virus-directed lymphocyte transformation may provide significant information on the pathogenicity of this important human virus.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.     

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.     

Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Developmental changes in unique cell surface antigens of chick embryo spinal motoneurons and ganglion cells

The monoclonal antibody technique was used to investigate neuronal heterogeneity and its developmental changes in the chick embryo trunk especially at the thoracic level. We report here four monoclonal antibodies (called SC 1, SC 2, SC 3, and SC 4) that bound to cell surface antigens. These antigens appeared to be proteins or glycoproteins because of their susceptibility to trypsin. In the spinal cord, antibody SC 3 stained all cells, but antibody SC 1 specifically stained motoneurons and ventral epithelial cells. The staining of motoneurons by antibody SC 1 was transient. It appeared at early stages (stage 16-17; Hamburger and Hamilton), but decreased markedly in intensity at older stages (stage 30-31). Antibody SC 2 did not stain cells in the spinal cord. It stained only neurons in the dorsal root and sympathetic ganglia. Antibody SC 4 stained only cells derived from the neural crest at the early stages (stage 16-20). At later stages, it stained a wider population of cells, including sensory neurons, Schwann cells, and cells in the central nervous system. In the dorsal root ganglion, antibodies SC 1 and SC 2 stained only neuronal cells whereas antibodies SC 3 and SC 4 stained both neuronal and glial cells. The dorsal root ganglionic antigens recognized by these antibodies were not expressed concurrently but appeared in a developmental sequence. Staining with antibodies SC 3 and SC 4 appeared first, then SC 1, and finally SC 2. Among these four antigens, the antigens common to both neuronal and glial cells appeared earlier than the neuron specific antigens. Thus, our monoclonal antibodies revealed heterogeneities in cell surface neuronal molecules and their transient and sequential appearance during embryonic development.     

Expression and segregation of nucleoplasmin during development in Xenopus

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.