Ultrastructural and cytochemical studies of nucleolus-like bodies in neurons of rat sympathetic ganglia

Intracytoplasmic fibrillar inclusions, generally referred to as nucleolus-like bodies (NLBs) were studied by means of ultrastructural cytochemistry. The structure of these bodies was visualized by several different staining procedures: conventional electron microscopy and preferential staining methods for localization of various proteins including ribonucleoproteins, basic proteins, glycoproteins and phosphorylated proteins. The results of the cytochemical tests indicate that NLBs have an essentially proteinaceous nature. They consist of ribonucleoproteins, basic proteins and glycoproteins but do not contain phosphorylated proteins. These findings suggest that NLBs are, at least partially, of the same nature as nucleoli and coiled bodies. The origin of NLBs and their possible functional role is briefly discussed.     

Cytochemical observations of the coiled bodies in neurons of rat sympathetic ganglia

Coiled bodies were investigated by means of ultrastructural cytochemistry. Preferential staining methods for localization of various proteins (ribonucleoproteins, basic proteins, phosphoproteins and glycoproteins) and DNA were applied. The results of cytochemical tests revealed that coiled bodies have a proteinaceous nature. They are composed of ribonucleoproteins, probably of nucleolar origin. They also contain phosphoproteins and glycoproteins but lack cytochemically detectable DNA. Coiled bodies present ultrastructural and cytochemical characteristics similar to the fibrillar part of the nucleous and to the interchromatin granules. The origin and possible functional role of coiled bodies are briefly discussed.     

Cytochemical observations of the coiled bodies in neurons of rat sympathetic ganglia

Summary Coiled bodies were investigated by means of ultrastructural cytochemistry. Preferential staining methods for localization of various proteins (ribonucleoproteins, basic proteins, phosphoproteins and glycoproteins) and DNA were applied. The results of cytochemical tests revealed that coiled bodies have a proteinaceous nature. They are composed of ribonucleoproteins, probably of nucleolar origin. They also contain phosphoproteins and glycoproteins but lack cytochemically detectable DNA. Coiled bodies present ultrastructural and cytochemical characteristics similar to the fibrillar part of the nucleous and to the interchromatin granules. The origin and possible functional role of coiled bodies are briefly discussed.     

Fine structural cytochemical and immunocytochemical analysis of nucleic acids and ribonucleoprotein distribution in nuclei of pig oocytes and early preimplantation embryos

The fine structure of pig oocytes at the germinal vesicle (GV) stage and early preimplantation embryos (one to four blastomeres) isolated at slaughter was investigated by cytochemical and immunocytochemical methods. The distribution of nucleic acids and ribonucleoproteins (RNPs) in compact nucleoli [denominated nucleolus-like bodies (NLB) in oocytes and nucleolus precursor bodies (NPB) in early embryos] and in intranuclear bodies or granules was investigated by staining methods preferential for nuclear RNPs or using the osmium ammine or ethidium bromide-phosphotungstic acid (EB-PTA) reactions for nucleic acids. The distributions of the Sm antigen of nucleoplasmic small nuclear RNPs (snRNPs), the methyl-3 guanosine (m₃G) cap of snRNAs and the splicing factor SC-35 were detected by immunoelectron microscopy using specific antibodies. The RNP nature of both NLBs and NPBs, and of nuclear granules in oocytes and embryos, and of fibrillar strands radially projecting from NLBs was revealed. Cytochemical evidence for RNA as a component of NLBs was further provided by EB-PTA staining in combination with the enzymatic removal of RNA, or by osmium-ammine staining without previous acid hydrolysis, while the absence of DNA in NLBs was established by Feulgen-like osmium-ammine staining. In addition, autoradiography demonstrated the absence of [6-³H]thymidine incorporation into NPBs. Other autoradiographic evidence attested the accumulation of RNA in NLBs of oocytes after a 60 min in vitro pulse of [5-³H]uridine. Immunoelectron microscopy using specific antibodies revealed the occurrence of nucleoplasmic snRNPs in both NLBs and NPBs. The presence of snRNA in NLB was confirmed by means of an antibody recognizing the m₃G-cap structure. Another spliceosomal component, the protein SC-35 was also detected in NLBs. Among the numerous and variable intranuclear granules occurring mostly in aggregates, the Sm antigen was clearly detected only in the interchromatin granule-type component. Some Sm labeling was occasionally seen in other categories of larger granules. No reaction was detected over any granules when using the anti-m₃G-cap antibody. The aggregates consisting of large granules and a finely fibrillar component were intensely immunolabeled by the anti-SC-35 splicing factor probe. Our observations suggest that the compact nucleoli, known to be present before and after fertilization in mammals (NLBs of oocytes and NPBs of early embryos), represent nuclear structural elements containing nonnucleolar, spliceosomal components.     

Nematosomes or nucleolus-like bodies in hypothalamic neurons,the subfornical organ and adenohypophysial cells of the rat

Summary Fibrillar intracytoplasmic bodies, generally referred to as nematosomes or nucleolar like bodies (NLBs), are not only observed in various types of neurons in the hypothalamus and subfornical organ but also in the glandular cells of the pars tuberalis and the pars intermedia hypophyses. According to their cytochemical properties the NLBs are probably of ribonucleoprotein nature. Within the neurons NLBs occur within perikarya and processes. Their presence within the neurosecretory nerve fibers of the neural lobe proves their ability to migrate within the axon. Morphologic modifications of NLBs are observed in stimulated neurons and after colchicine treatment. Colchicine causes a characteristic dense texture of NLBs and a peripheral agglomeration of mitochondria very similar to the rosette arrangement observed in oocytes. Our findings suggest a structural and functional similarity of NLBs in neurons and oocytes, in which their nucleolar origin appears obvious and where they seem to represent preribosomal material. It is very likely that the axonal migration of the NLBs reflects transport of ribosomal RNA for delayed utilization (as in oocytes).This paper is dedicated to Prof. F. Stutinsky for his 65th birthday.     

Nucleolus-like bodies in embryonal carcinoma cells of the embryoid bodies isolated from mouse teratocarcinoma

Undifferentiated embryonal carcinoma cells (EC cells) in the embryoid bodies isolated from mouse teratocarcinoma contained nucleolus-like bodies (NLBs) of smaller sizes in their cytoplasm (their sectional area averaged about 0.036 μm²). At the onset of EC cell differentiation, the average sectional area of NLBs significantly increased (about 0.107 μm²). When EC cells had differentiated into mesenchymal cells and endothelial cells of primitive blood vessels, NLBs decreased dramatically both in size and number. The possible role of NLBs in the differentiation process of EC cells is discussed.     

Rearrangement of nuclear ribonucleoproteins and extrusion of nucleolus-like bodies during apoptosis induced by hypertonic stress

Short-term hypertonic (HT) stress induces apoptotic cell death in human EUE cells in culture, as observed by electron microscopy, agarose-gel electrophoresis of low-molecular-weight DNA, DNA flow cytometry and annexin-V-propidium iodide double-staining. During HT-induced apoptosis, nuclear ribonucleoprotein (RNP)-containing structures undergo rearrangement, with the formation of Heterogeneous Ectopic RNP-Derived Structures (HERDS) which pass into the cytoplasm, as already reported for other examples of spontaneous and drug-induced apoptosis. Of special interest was the observation that nucleolus-like bodies (NLBs) which resemble morphologically nuclear functional nucleoli may be extruded into the cytoplasm of apoptotic cells and are observed inside the cytoplasmic fragments blebbing-out at the cell surface; these NLBs still contain immunodetectable nucleolar proteins (such as fibrillarin). This is an additional example of RNP-containing structures of nuclear origin which are extruded from the nucleus, in an almost "native" form, during apoptosis.     

Cytochemistry of kinetochores under electron microscopy

A cytochemical analysis has been performed on kinetochores of mouse, Allium and grasshopper under the electron microscope. The study was carried out using serial sections and cytochemical methods. Alcoholic PTA was used for basic protein staining and the EDTA method for preferential staining of ribonucleoproteins. In mouse and Allium chromosomes the kinetochore appears positively stained after PTA and EDTA. In grasshopper chromosomes, kinetochores appear as a fibrillar and less dense region and are positively stained after EDTA. Blocks from mouse treated with HCl prior to PTA stain show lower contrast in the kinetochore. When Allium cepa anthers were treated with RNase and perchloric acid (PCA) there was no positive effect after EDTA stain in the kinetochore region. It is suggested that non-DNA material takes part in the constitution of the kinetochore. This material would be made up, at least in part, of basic proteins and ribonucleoproteins.     

A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle

An mAb library was produced against proteins from the germinal vesicle (GV) of the frog Xenopus laevis; mAb 104 was selected from this library on the basis of its immunofluorescent staining of lampbrush chromosome loops. Chromosomes from several species of frogs and salamanders stained equally well. The antibody also stained the surface of numerous small granules in the GV nucleoplasm. The interior of the same granules was stained by antibodies against small nuclear ribonucleoproteins (snRNPs). mAb 104 also stained somatic nuclei from many vertebrate and invertebrate species, usually in a finely punctate pattern similar to that described for anti-snRNP and other antinuclear antibodies. The staining of somatic nuclei was much stronger during the mitotic stages than during interphase. Immunoblot analysis showed that mAb 104 recognizes a phosphorylated epitope.     

Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus.

Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins.     

The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in the absorptive cells of cultured human small-intestinal tissue as shown by silver proteinate staining

The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in cultured intestinal absorptive cell was investigated by silver proteinate staining. The results of this staining method, which is specific for carbohydrate containing macromolecules such as glycoproteins and mucopolysaccharides, showed that in the presence of the drug considerable amounts of silver proteinate-positive material accumulated in one type of lysosome-like body: the dense bodies. The staining pattern of other cell organelles was not affected by chloroquine. The presence of the drug in the culture medium also resulted in the occurrence of numerous small vesicular structures in the matrix of the dense bodies. These showed a similar size and structure to those present in the other type of lysosome-like body: the multivesicular bodies. This observation, together with earlier autoradiographical data, suggests that cell-coat material is transferred from multivesicular to dense bodies by fusion between these organelles. This study thus provides further evidence for a regulatory mechanism of cell-coat glycoprotein transport by the lysosome-like bodies in human intestinal absorptive cells.     

Phosphorylation in vivo of four basic proteins of rat brain myelin

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H₃³²PO₄. Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight `polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [³²P]phosphate–protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.     

The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in the absorptive cells of cultured human small-intestinal tissue as shown by silver proteinate staining

Summary The effect of chloroquine on the intralysosomal degradation of cell-coat glycoproteins in cultured intestinal absorptive cells was investigated by silver proteinate staining. The results of this staining method, which is specific for carbohydrate containing macromolecules such as glycoproteins and mucopolysaccharides, showed that in the presence of the drug considerable amounts of silver proteinate-positive material accumulated in one type of lysosome-like body: the dense bodies. The staining pattern of other cell organelles was not affected by chloroquine. The presence of the drug in the culture medium also resulted in the occurrence of numerous small vesicular structures in the matrix of the dense bodies. These showed a similar size and structure to those present in the other type of lysosome-like body: the multivesicular bodies. This observation, together with earlier autoradiographical data, suggests that cell-coat material is transferred from multivesicular to dense bodies by fusion between these organelles. This study thus provides further evidence for a regulatory mechanism of cell-coat glycoprotein transport by the lysosome-like bodies in human intestinal absorptive cells.     

Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase.

Human platelet glycoproteins IIb and IIIa form the receptor for fibrinogen, von Willebrand factor and fibronectin. Isolated human glycoproteins IIb-IIIa are phosphorylated by purified pp60c-src protein tyrosine kinase. Analysis of the phosphorylated proteins on SDS-PAGE showed that under reducing conditions both phosphoproteins change their relative molecular masses from 135 to 120 kDa and from 97 to 105 kDa, which are characteristic properties of glycoproteins IIb-IIIa. Phosphorylated proteins could be immunoprecipitated with an antiserum against glycoproteins IIb-IIIa but not by control serum. Some kinetic properties of the glycoprotein phosphorylations are also investigated. How the glycoprotein IIb-IIIa complex acquires its receptor activity in stimulated platelets is unknown; however, phosphorylation could be an important mechanism.     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。     

Localization of highly phosphorylated proteins in cells altered by Herpes simplex virus infection

Highly phosphorylated proteins detectable by their ability to bind bismuth ions were localized in rabbit fibroblasts before and during infection with Herpes simplex viruses type 1 and type 2. The bismuth tartrate procedure of Locke and Huie applied to glutaraldehyde-fixed cells revealed a low level of bismuth binding in a restricted portion of the normal nucleolus in non-infected cells. From 2.5-17 hr post-infection during virus development and maturation, the phosphorylated proteins were more widespread and the intensity of reaction was augmented. Bismuth deposits were then associated with virus-modified pre-existing structures including all of the nucleolar fibrils, the more abundant interchromatin granules, reduplications of some areas of the inner nuclear membrane and the Golgi apparatus. Virus-induced structures which were stained included nuclear dense bodies, the teguments of enveloped virions and the contents of extranuclear enveloped structures devoid of capsids. Following detergent-induced destruction of membranes, staining was lost from the nuclear envelope and cytoplasmic virions, which demonstrated that the highly phosphorylated proteins were tightly bound to nuclear and viral membranes. Bismuth staining of nitrocellulose sheets containing proteins extracted from whole cells revealed no reaction in normal cells but three positive bands were found in infected cells.     

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.     

Synthesis of Proteins and Glycoproteins in Cells Infected with Human Cytomegalovirus

In cytomegalovirus-infected cells, the rate of protein synthesis was detected as two peaks. One occurred during the early phase of infection, 0 to 36 h postinfection, and the other occurred during the late phase, after the initiation of viral DNA synthesis. Double-isotopic-label difference analysis demonstrated that host and viral proteins were synthesized simultaneously during both phases. In the early phase, approximately 70 to 90% of the total proteins synthesized were host proteins, whereas approximately 10 to 30% were viral, even at a multiplicity of infection of 20 PFU/cell. Virus-related proteins or glycoproteins were referred to as infected-cell specific (ICS). Two ICS glycoproteins (gp145 and 100) were clearly detectable and were synthesized preferentially in the early phase of infection. Their synthesis was concomitant with stimulation of the protein synthesis rate. In the late phase of infection, approximately 50 to 60% of the total protein synthesis was viral and approximately 40 to 50% was host. The ICS proteins and glycoproteins detected during the late phase of infection were viral structural proteins. Infectious virus was not detectable until 48 to 72 h postinfection. An inhibitor of viral DNA synthesis, phosphonoacetic acid, prevented the appearance of the late-phase ICS proteins and glycoproteins, but there was little or no effect on early ICS glycoprotein synthesis. Radiolabeled ICS proteins and glycoproteins were identified by their relative rates of synthesis, by their different electrophoretic mobilities compared with those of host proteins and host glycoproteins, and by their similar electrophoretic mobilities compared to those of proteins and glycoproteins associated with virions and dense bodies of cytomegalovirus. Structural viral antigens in the infected-cell extracts were removed by immunoprecipitation, using F(ab')(2) fragments of cytomegalovirus-specific antibodies, and identified as described above. The last two criteria were used to identify viral structural ICS proteins and glycoproteins. Although approximately 35 structural proteins were found to be associated with purified virions and dense bodies, the continued synthesis of host cell proteins complicated their identification in infected cells. Nevertheless, seven of the nine structural glycoproteins were identified as ICS glycoproteins.     

Parainfluenza virus 5 m protein interaction with host protein 14-3-3 negatively affects virus particle formation

Paramyxovirus matrix (M) proteins organize virus assembly, linking viral glycoproteins and viral ribonucleoproteins together at virus assembly sites on cellular membranes. Using a yeast two-hybrid screening approach, we identified 14-3-3 as a binding partner for the M protein of parainfluenza virus 5 (PIV5). Binding in both transfected and PIV5-infected cells was confirmed by coimmunoprecipitation and was mapped to a C-terminal region within the M protein, namely, 366-KTKSLP-371. This sequence resembles known 14-3-3 binding sites, in which the key residue for binding is a phosphorylated serine residue. Mutation of S369 within the PIV5 M protein disrupted 14-3-3 binding and improved the budding of both virus-like particles (VLPs) and recombinant viruses, suggesting that 14-3-3 binding impairs virus budding. 14-3-3 protein overexpression reduced the budding of VLPs. Using (33)P labeling, phosphorylated M protein was detected in PIV5-infected cells, and this phosphorylation was nearly absent in cells infected with a recombinant virus harboring an S369A mutation within the M protein. Assembly of the M protein into clusters and filaments at infected cell surfaces was enhanced in cells infected with a recombinant virus defective in 14-3-3 binding. These findings support a model in which a portion of M protein within PIV5-infected cells is phosphorylated at residue S369, binds the 14-3-3 protein, and is held away from sites of virus budding.