Dissociated limb bud cells of chick embryos can express lens specificity when reaggregated and cultured in vitro

Limb bud cells of chick embryos (stages 23–24) were dissociated into single cells, reaggregated, and cultured in vitro for about a week. δ-Crystallin, generally thought to be a lens-specific protein in the chick, was detected in the aggregates by indirect immunofluorescent staining, double immunodiffusion test, and immunoelectrophoresis with specific antiserum against δ-crystallin. Cells containing δ-crystallin were distributed in epidermal cell clusters and also in mesenchymal tissues surrounding cartilage nodules in the aggregates. Those cells in mesenchymal tissues were shown to have originated from the mesoderm of the limb bud, and those in epidermal cell clusters probably originated from the ectoderm. The possible cellular origin of this appearance of δ-crystallin was discussed.     

几种经济动物着丝粒间接免疫荧光的研究

检定间期细胞核中的着丝粒,除少数植物间期细胞核外,一直是一个问题。本工作用抗着丝粒抗血清,借间接免疫荧光染色法,对经济动物牛、羊、猪、兔和鸡的间期核进行了研究,发现在这些动物的间期核中都可检测出着丝粒,这为今后的着丝粒实验研究提供了基础。     

Pi Zpratt : A new alpha1-antitrypsin allele in an American negro family

Summary A low-power laser-UV microbeam of wave-length 257 nm was used for microirradiation of a small part of the nucleus of Chinese hamster cells. Following fixation in interphase or in the subsequent metaphase indirect immunofluorescent staining was performed with antiserum to photoproducts of DNA treated with far UV light.The results show that antibodies specific for UV-irradiated DNA can be used for a direct detection of laser-UV microirradiation-induced DNA photolesions. The potential usefulness of this method for investigation of the spatial arrangement of chromosomes in the interphase nucleus is discussed.     

Characterization of the intermediate (10 nm) filaments of cultured cells using an autoimmune rabbit antiserum.

An antiserum has been found in a nonimmunized rabbit which reacts strongly with a system of filaments in various fibroblasts, epithelial cells, macrophages and neuroblastoma. These filaments are distinct from the actin microfilament bundles visualized by an antibody against actin, and they are not affected by brief treatment with cytochalasin B. The pattern of these filaments somewhat resembles that described for microtubules, but the filaments could be clearly distinguished from microtubules by a comparison of their respective immunofluorescent patterns during cell division. In response to the drugs colcemid and vinblastine, the filaments reacting with this preimmune serum condense to form a compact perinuclear coil of fibers, a distribution and behavior in agreement with that previously described for the 10 nm or intermediate filaments studied by electron microscopy. Further evidence supporting our conclusion that this antiserum reacts with intermediate filaments is provided by a comparison of electron micrographs and the immunofluorescent patterns from parallel cell cultures. To identify the antigens reacting with this antiserum we have used the new technique of immuno-autoradiography on SDS gels of whole cell extracts. Two reactive polypeptide chains have been identified with apparent molecular weights of 56,000 and 30,000 daltons.     

Identification and characterization of a melanocyte-specific novel 65-kDa peripheral membrane protein.

In order to study proteins of the melanosome, we developed a panel of antisera against various protein fractions of melanosomes from B16 melanoma cells. An antiserum raised against a Triton X-100 insoluble fraction of melanosomes recognized a 65-kDa protein in melanocytes from mice homozygous for the buff mutation, but not in their wild type counterparts. Further studies were conducted using a specific, second generation antiserum raised against the purified protein. The protein was also detected in melanocytes cultured from albino mice, but absent in cultured mouse cell lines not of melanocyte origin. Density gradient centrifugation of subcellular organelles and indirect immunofluorescent cell staining, indicated that the protein was associated with melanosomes and vesicles. The protein on intact organelles could be made soluble using sodium carbonate, and digested with proteases in the absence of detergent suggesting that it was a peripheral membrane protein localized on the cytosolic face of organelle membranes. Metabolic labelling of cells and N-glycosidase F digestion of cell extracts indicated that the protein was not N-glycosylated. Based on its intracellular localization and biochemical defects in the buff mouse, a potential role has been suggested for the 65-kDa protein in intracellular membrane trafficking.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Identification of a chymotrypsin-like proteinase in human mast cells

An antiserum was produced against a chymotryptic proteinase purified from human skin. The antiserum did not cross-react with human leukocyte cathepsin G and elastase, rat mast cell proteinase I, and human skin tryptase. Indirect immunofluorescent staining of frozen skin sections to localize the proteinase showed cytoplasmic staining of cells scattered about the papillary dermis and around blood vessels and appendages. Restaining these sections with toluidine blue revealed that the fluorescently stained cells contained metachromatically staining granules, the major distinguishing feature of mast cells. A similar correlation was found in lung tissue. Ultrastructural studies employing the ferritin bridge technique to immunologically identify the proteinase additionally localized the proteinase to mast cell granules. Biochemical and immunochemical characterization of chymotryptic activity solubilized from isolated human lung mast cells identified a chymotryptic proteinase that may be identical to the skin chymotryptic proteinase. These studies establish that human skin mast cells contain a chymotrypsin-like proteinase that is a granule constituent and provide evidence that indicates a comparable proteinase is also present in lung mast cells.     

Demonstration of a tumor-associated surface antigen in Marek's disease.

Surface antigenic markers were detected on three classes of Marek's disease (MD) tumor cells, i.e., MD lymphoma cells, cultured cells of the MSB-1 lymphoblastoid cell line, and JMV lymphoblastic leukemia cells, by indirect membrane immunofluorescent staining with serum from chickens immunized with JMV cells or from rabbits immunized with MSB-1 cells. This surface antigen was not detected on normal chicken lymphocytes, RPL-16 tumor cells (tranedormed by an avian RNA virus, or MD virus-infected fibroblasts that were positive for viral membrane antigen (MA). Furthermore, the surface antigen appeared unrelated to embryonic or histocompatibility antigens. This antigen is provisionally designated as a Marek's disease tumor-associated surface antigen (MATSA). The MATSA's on JMV, MSB-1 and MD lymphoma cells were related but not identical as demonstrated by antiserum titration, absorption and blocking tests with homologous and heterologous systems.     

Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis

UV irradiation of quiescent human fibroblasts immediately triggers the appearance of the nuclear protein cyclin/proliferating cell nuclear antigen (PCNA) as detected by indirect immunofluorescent staining after methanol fixation. This was found to be independent of new synthesis of cyclin/PCNA by two-dimensional gel analysis and cycloheximide treatment. The intensity of the immunofluorescent staining of cyclin/PCNA observed in UV-irradiated cells corresponded with the UV dose used and with the DNA repair synthesis detected by autoradiography. The nuclear staining remains as long as DNA repair activity is detected in the cells. By extracting the UV-irradiated quiescent cells with Triton X-100 and fixing with formaldehyde, it was possible to demonstrate by indirect immunofluorescence rapid changes in the cyclin/PCNA population after irradiation, a small proportion (5-10%) of which is tightly associated to the nucleus as determined by high salt extraction. By incubating at low temperature and depleting the ATP pools of the cells before UV irradiation, we have demonstrated that the changes in cyclin/PCNA distribution observed involve at least two different nuclear associations.     

Immunohistochemical localization of a synaptic-vesicle antigen in a cholinergic neuron under conditions of stimulation and rest

Summary An antiserum against a specific component (a glycosamino glycan) of the cholinergic synaptic-vesicle of Torpedo marmorata has been used to investigate the localization of the component in the cell body, its movement within the electromotor axon and its fate within the nerve terminal upon electrical stimulation. After immunofluorescent staining, spots are observed throughout the cytoplasm of the lobe perikarya, although they are concentrated in the region of the axon hillock. Ligation of the electromotor nerves leading from the lobe to electric organ produces a proximal build-up of material which stains readily with the antivesicle antiserum, indicating that the vesicle antigen is transported from the cell body to the nerve terminal. A marked increase in indirect immunofluorescent staining of the electric organ is observed in the nerve ending upon electrical stimulation. We interpret this result as fusion of the vesicles with the presynaptic plasma membrane and exteriorization of the vesicle antigen to the extracellular space, thereby facilitating its staining. After recovery of the system the fluorescence declines, a result that is consistent with the reinternalization of the vesicle antigen into the core of reformed vesicles. The results support a mechanism whereby vesicles recycle within the nerve terminal and transmitter is released by exocytosis.     

Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity

Bovine brain-derived growth factor (BDGF) is very similar to endothelial cell growth factor and brain-derived acidic fibroblast growth factor in terms of pI (5.7) and molecular weight (approximately 17,000). BDGF has a wide spectrum of cell specificity, including bovine aorta endothelial cells and Swiss mouse 3T3 cells. BDGF stimulates the phosphorylation of a 135-kDa protein in plasma membranes of 3T3 cells. The optimal concentration for stimulation of phosphorylation is close to the Kd of 125I-BDGF binding to receptor, suggesting that the BDGF-stimulated 32P-labeled 135-kDa protein may be the BDGF receptor. The alkaline stability of this 32P-labeled 135-kDa phosphoprotein and phosphoamino acid analysis of the acid hydrolysates indicate that the phosphorylation occurs at tyrosine residues. The molecular size of BDGF receptor is estimated as approximately 135 kDa by cross-linking 125I-BDGF to its receptor in 3T3 cells, using a bifunctional reagent, ethylene glycolbis(succinimidylsuccinate). Both BDGF-stimulated phosphorylation and 125I-BDGF binding to receptor can be inhibited by protamine. These results suggest that the BDGF receptor is a 135-kDa protein which is associated with a protein tyrosine kinase activity.     

Perinuclear location and recycling of epidermal growth factor receptor kinase: immunofluorescent visualization using antibodies directed to kinase and extracellular domains

This paper describes studies on the migratory behavior of epidermal growth factor (EGF) receptor kinase using antibodies that are specific for either the kinase domain or the extracellular domain of the receptor. Antiserum was raised to a 42,000-D subfragment of EGF receptor, which was shown earlier to carry the kinase catalytic site but not the EGF-binding site. Another antiserum was raised to the pure intact 170,000-D EGF receptor. The specificities of these antibodies were established by immunoprecipitation and immunoblotting experiments. The domain specificity was examined by indirect immunofluorescent staining of fixed cells. The anti-42-kD peptide antibody could bind specifically to EGF receptors of both human and murine origin and was found to be directed to the cytoplasmic part of the molecule. It did not bind to EGF receptor-negative cells, which contained other types of tyrosine kinases. The antibodies raised against the intact receptor recognized only EGF receptor-specific epitopes and were directed to the extracellular part of the molecule. The anti-receptor antibodies described above were used to visualize the cyclic locomotory behavior of EGF receptor kinase under various conditions of EGF stimulation and withdrawal. The receptor was examined in fixed and permeabilized cells by indirect immunofluorescent staining. The results demonstrate the following: (a) the receptor kinase domain migrates to the perinuclear region upon challenge with EGF; (b) both extracellular and cytoplasmic domains of the receptor are involved in migration as a unit; (c) withdrawal of EGF results in rapid recycling of the perinuclear receptors to the plasma membrane; (d) this return to the cell surface is inhibited by methylamine, chloroquine, and monensin; and (e) neither the internal migration nor the recycling process is blocked by inhibitors of protein biosynthesis.     

Immunofluorescent study of DNA polymerase alpha in rat cells and tissues

An indirect immunofluorescent test based on globulin preparation from a highly specific antiserum against rat liver DNA polymerase alpha was used to direct the enzyme in sections of various tissues of the rat. The immunofluorescent staining was found in cells of the thymus and the wall of intestine crypt, in sparse cells of the intestinal muscular layer, and in cells of the embryo skin epithelium. In sections of liver the intensity of staining and the number of stained cells increased significantly during regeneration. The immunoglobulins did not interact with the cytoplasm and nuclei of skeletal muscle myotubes, with the epithelial cells at the top of intestinal villi, and with erythrocytes. The intracellular localization of the fluorescence observed was of two general types: 1) staining in the region of the nuclear envelope and/or in the cytoplasm; 2) an additional intranuclear staining. The staining of the first type is characteristic of the cells of intact liver and of leyomyocytes. It was also observed in the proliferating cells of thymus and crypt wall, and in cultured myogenic L6 cells. Cells of the embryo skin epithelium, the satellite cells in the skeletal muscle, and about one half of the regenerating liver cells appeared to have the second type of staining. These data serve an indication of possible histotypical differences in in the intracellular localization of DNA polymerase alpha in proliferating cells. It is proposed that the presence of DNA polymerase in resting cells is in association with their ability to respond to the mitogenic stimulus.     

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.     

Brain-derived growth factor is a chemoattractant for fibroblasts and astroglial cells

Bovine brain-derived growth factor (BDGF), a 16-17 kDa protein with biochemical properties resembling brain-derived acidic fibroblast growth factor (acidic FGF) and endothelial cell growth factor, was found to have potent chemotactic activity for bovine ligament fibroblasts, human skin fibroblasts and rat astroglial cells, maximal at 100-200 pg/ml. The chemotactic activity was completely blocked by protamine sulfate (5 ug/ml), an inhibitor of receptor-binding and mitogenic activity of BDGF. BDGF did not stimulate migration of human monocytes. These results indicate that the effects of BDGF 'in vivo' might extend to mesenchymal cell recruitment.     

Deposition of Basement Membrane Proteins in Attachment and Neurite Formation of Cultured Murine C-1300 Neuroblastoma Cells

The deposition of the basement membrane glycoproteins, laminin, fibronectin, and type IV procollagen was studied by indirect immunofluorescence microscopy during the attachment and differentiation of murine C-1300 neuroblastoma cells. A typical cytoplasmic perinuclear staining for the basement membrane antigens was seen both in undifferentiated and differentiated cells. Freshly seeded suspended cells lacked surface fluorescence but in two hours after plating, distinct punctate laminin deposits became discernible on the ventral surface of the cells. Notably, in sparsely seeded undifferentiated cultures, the cell-associated extracellular laminin deposits could only be detected under the primary attaching cells, whereas daughter cells in clonal cell colonies lacked such fluorescence. In cultures induced to neurite formation with dibutyryl cyclic AMP, laminin deposition was also detected in association with the growing cytoplasmic extensions. No distinct differences were found between the secreted proteins of cultures of differentiated and nondifferentiated neuroblastoma cells, but the patterns of fucosylation of high-molecular weight proteins in the two cultures were markedly different. We conclude that cultured neuroblastoma cells both synthesize, secrete and deposit laminin. The distribution of laminin during neuroblastoma cell attachment and neurite extension suggests that this glycoprotein may be involved in cell–to–substratum interactions in C-1300 cell cultures.     

Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.     

Transforming growth factor activity of bovine brain-derived growth factor

Bovine brain-derived growth factor (BDGF), whose biochemical properties resemble those of endothelial cell growth factor (ECGF) and brain-derived acidic fibroblast growth factor (acidic FGF), is able to promote colony formation of normal rat kidney fibroblasts (NRK cells) in soft agar. As in the case of transforming growth factor beta (TGF beta), EGF potentiates the anchorage-independent growth promoting activity of BDGF. In the presence of EGF (5 ng/ml), the optimal concentration of BDGF for stimulation of anchorage-independent of NRK cells is approximately 0.5 ng/ml. At higher concentrations, BDGF becomes inhibitory. The anchorage-independent cell growth promoting activity of BDGF differs from that of TGF beta in acid and reducing agent stability.     

Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells.

Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.