Detection of sugar-binding proteins in membrane-depleted nuclei

Nuclear sugar-binding proteins were detected in membrane-depleted nuclei isolated from hamster BHK cells and mouse L 1210 leukemia cells by means of fluorescein-labelled neoglycoproteins. In fluorescence microscopy, the fluorescence was seen throughout the nucleus but was generally brighter over the nucleoli than over the rest of the nucleus. Flow cytofluorometry analysis demonstrated the presence of nuclear sugar-binding proteins for synthetic glycoproteins associated with different sugar residues. Among the nine neoglycoproteins used, four neoglycoproteins (namely alpha-rhamnosylated, alpha-glucosylated, N-acetyl-beta-glucosaminylated and alpha-mannosylated-6P-serum albumin) strongly labelled nuclei. Various controls strongly argue for the specificity of the nuclear labelling. The possibility that some of the sugar-binding proteins might correspond to endogenous nuclear lectins is considered.     

Structural aspects of intranuclear matrix disintegration upon RNase digestion of HeLa cell nuclei

The organization of the intranuclear elements observed in histone-depleted (2 M NaCl-extracted) HeLa cell nuclei was investigated by means of electron microscopy and two-dimensional gel electrophoresis. This work was mainly aimed at verifying whether or not an intranuclear skeleton or matrix existed, which could explain the stable attachment of RNA to the residual nuclear structure after high-salt extraction, and its three-dimensional organization. We compared the ultrastructure and the polypeptide composition of RNA-containing and RNA-depleted (RNase-treated) nuclear residues, and we visualized intermediate stages of RNase action on the intranuclear material. We showed that this material was made of two types (fibrillar and granular) of salt-resistant RNP components equally sensitive to RNase when the enzyme was used prior to high-salt extraction. At least in our material and under our experimental conditions, no intranuclear matrix could be distinguished from the residual RNP material. Our results further suggest that formation of such a matrix is a path-dependent phenomenon.     

Ultrastructure of chick erythrocyte nuclei undergoing reactivation in heterokaryons and enucleated cells

The reactivation of chick erythrocyte nuclei after Sendai virus induced fusion of chick erythrocytes with intact or anucleate rat myoblasts or rat epithelial cells was studied by electron microscopy. Both in heterokaryons and in reconstituted cells formed by the fusion of chick red cells with anucleate rat L6 myoblasts the amount of highly condensed chromatin in the chick nuclei decreased with time after fusion at the same time as the proportion of dispersed chromatin increased. Nuclear organelles, typical of active nuclei but absent in the nuclei of unfused erythrocytes, appeared during reactivation. The percentage of chick nuclei containing a nucleolus was low 24 h after fusion but increased so that almost all nuclei contained one or more nucleoli 120 h after fusion. In reconstituted cells the frequency of nucleoli was much lower than in heterokaryons. In other respects, the erythrocyte nuclei introduced into anucleate rat cells underwent a normal reactivation and appeared to be well integrated with the cytoplasm. Thus, the nuclear envelope consisted of two normal leaflets in direct contact with the cytoplasm. Nuclear pores were observed in front of interchromatin channels. A normal cytoplasmic geometry appeared to be re-established since the Golgi apparatus occupied a position close to the poles of the chick nucleus.     

Effect of the ionic environment on the transcriptional activity of rat liver nucleoli

The localization of various RNA polymerase activities in the nucleoplasm and the nucleolus was investigated in isolated nuclei and in isolated nucleoli by means of a combination of cell fractionation, biochemical analysis and ultrastructural autoradiography. This resulted in four chief conclusions.

1. 1. The two main RNA polymerase activities can be localized clearly in the interphase nucleus: at low ionic strength, the activity is restricted to the nucleolus whereas at high ionic strength the activity is found in both nucleolar and nucleoplasmic regions.

2. 2. The preservation of the ultrastructure of the nuclei at high ionic strength is rather poor so that the physiological meaning of the activity revealed is questionable.

3. 3. Contrary to other reports, the nucleolar activity is significantly enhanced by increasing ammonium sulphate concentration in the presence of Mn²⁺.

4. 4. This increase is probably related to progressive revealing of RNA polymerase B activity within the nucleolus.

     

Relationship between the Ag-NOR proteins and ribosomal chromatin in situ during drug-induced RNA synthesis inhibition

In human TG tumor cells, the role of silver-NOR proteins was investigated by examining their relationship with the chromatin structure during inhibition of RNA synthesis by actionomycin-D treatment. This induced segregation of the nucleoli into four distinct zones and weakened the silver reaction. The fibrillar components were found to constitute the site of silver-stained proteins segregation. Feulgen-like osmium-ammine staining revealed that the DNA disappeared from the segregated nucleoli except for a network of nonnucleosomal filaments. When Ag-NOR protein detection was combined with chromatin visualization, we found constant overlapping of the silver reaction sites with the nonnucleosomal DNA filaments. Our results indicate that certain Ag-NOR proteins are not directly linked to active rRNA synthesis, but might rather affect the structure of ribosomal genes.     

Clinical follow-up in the rat experimental model of African trypanosomiasis

Animal models of Human African Trypanosomiasis (HAT) have been developed to understand the pathogenic mechanisms leading to the passage into the neurological phase, most of them referring to histological aspects but not clinical or behavioral data. Our study aimed at defining simple clinical and/or behavioral markers of the passage between the hemolymphatic phase and the meningo-encephalitic stage of the disease. Sprague-Dawley rats (n=24) were infected with Trypanosoma brucei brucei AnTat 1.1E. Food intake and body weight were measured daily from the day of infection until death. Hematocrit was measured twice a week. Behavioral disturbances were evaluated through an Open-field test. A sudden weight loss occurred on the twelfth day after infection, due to a significant drop of food intake starting two days before. The rats developed an anemic state shown by the hematocrit measurements. The Open-field test showed them to be less active and reactive as soon as the second week after infestation. A complementary histological study observed trypanosomes and inflammatory cells in the choroid plexus at the same period. These results are in favor of central nervous system functional disturbances. The observed weight loss is discussed as being a parameter of the entry in the meningo-encephalitic phase. The rat model reproduces neurological symptoms observed in the human disease and may prove to be useful for further neurohistological and therapeutic studies.     

A link between chemokine levels and disease severity in human African trypanosomiasis

Trypanosoma brucei gambiense infection is an important public health challenge in sub-Saharan Africa. This parasitic disease is difficult to diagnose due to insidious clinical signs and transient parasitaemias. The clinical course is marked by two stages of increasing disease severity. An early systemic parasitic invasion is followed by the development of a progressive meningo-encephalitis. During this latter stage, a broad spectrum of neurological signs appears, which finally lead to a demyelinating and fatal stage if untreated. Treatment is toxic and difficult to administer when the CNS is invaded. Therefore, accurate diagnostic methods for stage determination are needed. The classically used criteria are not sufficiently specific and mechanisms of parasite invasion through the blood-brain barrier remain poorly understood. As cytokines/chemokines are involved in the early recruitment of leukocytes into the CNS, this study has focused on their potential value to define the onset of CNS involvement. Levels of monocyte chemoattractant protein-1/CCL-2, macrophage inflammatory protein-1α/CCL-3, IL-8/CXCL-8, regulated upon activation T cell expressed and secreted (RANTES)/CCL-5 and IL-1β were measured in paired sera and CSF from 57 patients and four controls. Patients were classified into three groups (stage 1, intermediate and stage 2) according to current field criteria for stage determination (CSF cell count, presence of trypanosomes in CSF and neurological signs). In sera, cytokine/chemokine levels were poorly related to disease stage. Only CXCL-8 was higher in stage 1 patients when compared with stage 2 and CCL-5 was higher in controls when compared with patients. In contrast, in CSF the expression of the selected cytokines, except CCL-5, was associated with the presence of neurological signs, demonstrating their diagnostic value. We observed a relationship between the presence of trypanosomes or trypanosome-related compounds in CSF and levels of IL-1β, CXCL-8, CCL-2 and CCL-3. These cytokines and chemokines may be triggered by the parasite and hence are potential markers of CNS invasion.     

Rotation of the cell nucleus in living cells: a quantitative analysis

Nuclear rotation is observed in a variety of cell types. However, few quantitative analyses are reported and the significance of this phenomenon is still unclear. To investigate this type of nuclear movement, we performed a quantitative analysis in mouse L-929 fibroblasts, a cell line chosen since it displays a high nuclear rotational activity. Analyses were performed using time-lapse microcinematography. The relationship between nuclear rotation and other cellular phenomena such as the cell cycle and locomotion were studied. Then, we investigated the rotation in a population of sister cells to study whether it is genetically determined. Finally, we performed a qualitative analysis of nuclear rotation in different cultured cell lines. Results show that nuclear rotations preferentially occur during the phases of the cell cycle which surround mitosis.