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.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.     

Immunocytochemical localization of proteins in differentiating tissues of Pisum sativum

Summary Although there may be documented morphological changes during development, it is obvious that important changes in protein content occur in a vascular plant during the several stages of differentiation. In the absence of the latter information, an approach has been established for the localization of antigenic proteins in developing tissues of Pisum sativum. Monoclonal antibodies were raised to proteins extracted from pea internode tissue, and employed for the localization of three proteins in tissue sections. One of the proteins has two polypeptide subunits with molecular weights of 25,000 and 40,000 daltons, and the antibody binds to both of them. The three monoclonal antibodies produce different patterns of cellular localization in the tissue sections, as visualized by indirect immunocytochemical labeling. In another series of analyses, quantitative and qualitative differences in the protein contents of apical shoot tissue and mature internode shoot tissue have been found. These studies were based on the use of Western blots with both polyclonal (rabbit) antibodies and monoclonal (mouse) antibodies.In honour of Prof. P. van Duijn     

An alternate form of Ku80 is required for DNA end-binding activity in mammalian mitochondria

Mammalian mitochondrial DNA end-binding activity is nearly indistinguishable from that of nuclear Ku. This observation led to the hypothesis that mitochondrial DNA end-binding activity is in part dependent upon Ku80 gene expression. To test this hypothesis, we assayed for Ku activity in mitochondrial extracts prepared from the xrs-5 hamster cell line that lacks Ku80 mRNA expression. Mitochondrial protein extracts prepared from this cell line lacked the DNA end-binding activity found in similar extracts prepared from wild-type cells. Azacytidine-reverted xrs-5 cells that acquired nuclear DNA end-binding activity also acquired mitochondrial DNA end-binding activity. Western blot analysis of human mitochondrial protein extracts using a monoclonal antibody specific for an N-terminal epitope of Ku80 identified a protein with an apparent molecular weight of 68 kDa. This mitochondrial protein was not detected by a monoclonal antibody specific for an epitope at the C-terminal end of Ku80. Consistently, while both the N- and C-terminal Ku80 monoclonal antibodies supershifted the nuclear DNA end-binding complex on an electrophoretic mobility shift assay, only the N-terminal monoclonal antibody supershifted the mitochondrial DNA end-binding complex. To confirm that the 68 kDa Ku protein was not a consequence of nuclear protein contamination of mitochondrial preparations, highly purified intact nuclei and mitochondria were treated with proteinase K which traverses the pores of intact nuclei but gains limited access into intact mitochondria. Ku80 in purified intact nuclei was sensitive to treatment with this protease, while the 68 kDa Ku protein characteristic of purified intact mitochondria was resistant. Further, immunocytochemical analysis revealed the co-localization of the N-terminal specific Ku80 monoclonal antibody with a mitochondrial-targeted green fluorescence protein. Mitochondrial localization of the C-terminal Ku80 monoclonal antibody was not observed. These data are consistent with the hypothesis that a C-terminally truncated form of Ku80 is localized in mammalian mitochondria where it functions in a DNA end-binding activity.     

Immunocytochemical localization in normal and transformed human cells in tissue culture using a monoclonal antibody to the src protein of the Harvey strain of murine sarcoma virus

Using a rat monoclonal antibody directed against the p21 src protein of the Harvey strain of Murine Sarcoma Virus (MSV), we have examined the reactivity of human cells in tissue culture using immunofluorescence and electron microscopy. Qualitative results indicated that untransformed mouse and human fibroblastic cells have undetectable amounts of p21; these levels were greatly increased after transformation with Harvey MSV. A group of human tumor cell lines adapted to tissue culture were examined and almost all of the epithelial tumor lines showed significant localization with this antibody. Notable exceptions were two melanoma cell lines which were negative for p21 by immunofluorescence. When normal human epithelial cells derived from esophageal or foreskin epithelium were examined, the antibody showed significant reactivity with subconfluent growing cells. After the normal cells were allowed to become quiescent, the reactivity with this antibody decreased. All of the localization seen by fluorescence was in a distribution consistent with the previously demonstrated location of p21 scr on the inner aspect of the plasma membrane. Electron microscope localization showed labeling for this antigen on the inner surface of the plasma membrane in both transformed mouse cells and in the human tumor cell lines MCF-7 and HTB-2 (RT4). These results suggest that the amounts of p21-like proteins detectable in human epithelial tumor cells do not necessarily reflect their malignant potential, but may be related to their epithelial nature. The loss of detectable localization at quiescence suggests that p21 levels decrease when these epithelial cells stop growing, and raises the possibility that an analog of p21 may be used by these human epithelial cells to regulate cell growth.     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

Ultrastructural Localization of a Bean Glycine-Rich Protein in Unlignified Primary Walls of Protoxylem Cells

A polyclonal antibody was used to localize a glycine-rich cell wall protein (GRP 1.8) in French bean hypocotyls with the indirect immunogold method. GRP 1.8 could be localized mainly in the unlignified primary cell walls of the oldest protoxylem elements and also in cell corners of both proto- and metaxylem elements. In addition, GRP 1.8 was detected in phloem using tissue printing. The labeled primary walls of dead protoxylem cells showed a characteristically dispersed ultrastructure, resulting from the action of hydrolases during the final steps of cell maturation and from mechanical stress due to hypocotyl growth. Primary walls of living protoxylem and adjacent parenchyma cells were only weakly labeled. This was true also for the secondary walls of proto- and metaxylem cells, which in addition showed high background labeling. Inhibition of lignification with a specific and potent inhibitor of phenylalanine ammonia-lyase did not lead to enhanced labeling of secondary walls, showing that lignin does not mask the presence of GRP 1.8 in these walls. Dictyosomes of living proto- and metaxylem cells were not labeled, but dictyosomes of xylem parenchyma cells without secondary walls, adjacent to strongly labeled protoxylem elements, were clearly labeled. These observations suggest that GRP 1.8 is not produced by xylem vessels but by xylem parenchyma cells that export the protein to the wall of protoxylem vessels.     

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.     

Characterization of DIP1, a novel nuclear protein in Drosophila melanogaster

We have recently identified in Drosophila melanogaster a new gene encoding a nuclear protein, DIP1. Here we report the developmental expression and the finding that DIP1 subcellular localization is in the nucleus and at the nuclear periphery during interphase in embryos. Interestingly, in humans, DIP1 antibody identified signals in nuclei from cultured cells and reacted with a rough 30kDa protein in Western blotting experiments, demonstrating evolutionary conservation.     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

Cross-reactivity of birch anthers and leaves with birch pollen antigens and allergens. A fine-structural immunocytochemical study using the post-embedding protein-A-gold technique

Ultra-thin sections of vegetative tissues from birch (anthers and leaves) were labeled for pollen antigens and allergens using a commercial rabbit IgG antibody preparation directed against birch pollen antigens and allergens. Antibody binding sites were visualized using the protein A-gold technique. Specific labeling occurred in anther tissue (tape-tum cells, anther wall cells) as well as in the birch leaf (assimilation parenchyma). In both types of tissue, antigens and allergens were detected throughout the living protoplast (including cell organelles such as nuclei, mitochondria, and plastids). The cellulose cell walls were always free from anti-birch-pollen IgG-binding sites. The immunological controls (normal rabbit IgG) showed a low degree of nonspecific labeling. In plant tissues belonging to genera quite different from birch (tulip anther, rhododendron leaves), after incubation with the specific IgG weak labeling was observed. The immunological basis for these results is discussed.     

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.     

Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands

Mammary glands contain a group of calcium-sensitive proteins that bind to membranes in a calcium-dependent manner. Using the calcium-dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70 kDa mammary calcium-binding protein (70 kDa M-CBP) to homogeneity. Antisera prepared to the 70 kDa M-CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M-CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M-CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pl for the 70 kDa M-CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M-CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M-CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin I/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.     

Nuclear changes accompanying cell differentiation in stems of Pisum sativum L.

Summary Nuclear DNA, nuclear protein and nuclear size have been measured in cells of the cortex, pith and vascular tissue from three successive internodes in the stem of Pisum sativum. New techniques of computer-linked cytophotometry were used to measure these parameters simultaneously in both section and squash preparations. In cortical cells no endoreduplicated nuclei were seen in the internodes measured. In cortical cells from the oldest internode measured, a population of large nuclei with the 2C DNA amount was observed which was not present in the younger internodes. In the oldest pith nuclei measured a few 8C nuclei were present, but maturing pith was most characterized by increasing nuclear size and the population of nuclei accumulating with the 4C DNA amount. Polyploid nuclei were present in all of the vascular tissue measured, including the youngest internode. Maturing vascular tissue was also characterized by increasing nuclear size. Nuclear protein measurements demonstrated a close link between nuclear protein and nuclear size and suggest that increased nuclear size, with constant DNA content, may be due to increased nuclear protein. This raises the question of the nature and function of this nuclear protein, perhaps more characteristic of differentiating cells than dividing cells.To whom offprint requests should be sent     

The production and characterization of monoclonal antibodies to a human colonic antigen associated with ulcerative colitis: cellular localization of the antigen by using the monoclonal antibody

We detected in human colon extracts a 40 kDa protein(s) that specifically reacts with tissue-bound IgG obtained from the colon of patients with ulcerative colitis or CCA-IgG. Using the hybridoma technology, we developed monoclonal antibodies to this 40 kDa protein. The specific immunoreactivity of one of the monoclonal antibodies (7E12H12, IgM isotype) against the 40 kDa protein was demonstrated both by ELISA and by immunotransblot. Competitive binding experiments showed that CCA-IgG inhibits the binding of 7E12H12 to the 40 kDa protein, suggesting the recognition of common epitope(s) on the 40 kDa protein by the monoclonal antibody and CCA-IgG. 7E12H12 was used to determine cellular localization of the 40 kDa protein. Biopsy tissue specimens from colon, esophagus, stomach, duodenum, jejunum, ileum, liver, pancreas, lungs, kidneys, salivary, and mammary glands were obtained. Tissue specimens were fixed in 4% paraformaldehyde or in 10% formalin. Sections were sequentially incubated with the hybridoma supernatant, biotinylated anti-mouse IgM, avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine. An unrelated hybridoma supernatant was used as control. The monoclonal antibody exclusively recognized colonic epithelial cells both in the crypt and on the luminal surface. Immunoreactivity was present on the plasma membrane chiefly along the basolateral areas of the cells. Plasma membrane localization of the 40 kDa protein was confirmed by immunoelectron microscopy. All colonic mucosal biopsy specimens from both adult and fetal colon reacted with the monoclonal antibody. None of the biopsy specimens from stomach, duodenum, jejunum, ileum, liver, pancreas, or non-gastrointestinal tissue reacted with the antibody, confirming the organ specificity of the 40 kDa protein. The interaction between this colonic epithelial membrane protein and the CCA-IgG may play an important role in the pathogenesis of ulcerative colitis.     

Localization of neonatal secretory proteins in different cell types of the rat submandibular gland from embryogenesis to adulthood

In the neonatal rat submandibular gland, Type III cells contain a group of related proteins that we call the B1-immunoreactive proteins (B1-IP; 23.5, 26, and 27.5 kDa). Type I cells lack these, but synthesize a different protein, Protein C (89 kDa). With maturation of the gland, these neonatal cell types are no longer seen in the seromucous acini, which are no longer reactive for the B1-IP. Here, we report the ultrastructural immunocytochemical localization of the B1-IP and Protein C over the course of development. From their first appearance in the embryo, the B1-IP and Protein C are present in different cells which become morphologically typical Type I and III cells prior to birth. At all stages, Type I cells have strong Protein C labeling and no B1 labeling. By 3 days postpartum, ultrastructurally atypical Type III cells are seen (Type IIIP); these label for the B1-IP, but also show labeling with antibody to Protein C. In the next week, as mucous cells appear in the acini, these show both B1-IP and C labeling; the B1 marker is lost by 30 days postpartum, but adult mucous acinar cells continue to show Protein C reactivity. In view of the appearance of Protein C reactivity in neonatal Type IIIP and then in mucous cells, and the presence of B1 reactivity in early but not mature mucous cells, we suggest that Type III cells differentiate into mucous cells and that Type IIIP cells are intermediates in this transformation. We see no evidence for the differentiation of either Type III or mucous cells from Type I cells, although our data cannot rule out this possibility. In adult glands, cells with B1 labeling are seen in intercalated ducts. Cells that appear to be Type I cells are also present in these ducts and label for Protein C. Double labeling for B1-IP and Protein C demonstrated that the two markers were exclusively present in different cells within intercalated ducts. This is of considerable interest, as intercalated ducts have been reported to be the stem cell population for normal and trauma-induced cellular replacement.     

Specific accumulation of polysaccharide-linked hydroxycinnamoyl esters in the cell walls of irregularly shaped and collapsed internode parenchyma cells of the dwarf rice mutant Fukei 71

We examined a novel rice mutant, Fukei 71 (Oryza sativa L.), for alterations in the levels of hydroxycinnamoyl esters that are linked to cell wall polysaccharides and lignin units. In this mutant, a recessive mutation at a single locus caused the collapse of parenchyma cells in the internodes. Light microscopy revealed that the abnormal walls of internode parenchyma cells of Fukei 71 were stained by the M?ule reaction, which is specific for syringyl units in phenolic compounds. These walls were not stained by Wiesner's reagent (phloroglucinol-HCl), which reacts cinnamaldehyde in lignin. Levels of p-coumaric acid (PCA) and ferulic acid (FA) were apparently elevated in the abnormal tissue of the mutant. Western blotting analysis with antibodies specific for phenylalanine ammonia-lyase (PAL) revealed higher levels of PAL in the abnormal parenchyma tissue of Fukei 71 than in the parenchyma tissue of the parent cultivar Fujiminori. These results and the observation that PAL was produced at a greatly elevated level indicated that the phenylpropanoid pathway that leads to the biosynthesis of polysaccharide-linked FA and PCA was abnormally activated in the irregularly shaped and collapsed internode parenchyma cells, in which the biosynthesis of lignin is normally repressed.     

Identification of possible phosducins in the ciliate Blepharisma japonicum

Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the betagamma-subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.     

Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus

Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions.