Chloramphenicol inhibits hormone-dependent induction of cytoplasmic mRNA coding for the mitochondrial enzyme, carbamyl phosphate synthetase, in Rana catesbeiana tadpoles.

In tadpoles injected with thyroxine (T4), synthesis of carbamyl phosphate synthetase 1 is induced so that within 7-8 days this single polypeptide represents one of the most abundant proteins in the liver. Translational assays in vitro showed that liver RNA from control animals had very low levels of translatable mRNA coding for the enzyme whereas carbamyl phosphate synthetase mRNA activity was prominent in liver from tadpoles which had been treated with T4 for just 2 days. When the primary translation product of carbamyl phosphate synthetase mRNA was immunoprecipitated from a messenger-dependent rabbit reticulocyte cell-free system programmed with total liver mRNA, it migrated on SDS-polyacrylamide gels more slowly than the in vivo form of the enzyme and otherwise demonstrated characteristics which were very similar to the precursor for carbamyl phosphate synthetase previously described in rat liver. If tadpoles were treated for 2 days with T4 plus an inhibitor of mitochondrial protein synthesis, chloramphenicol, T4-dependent induction of both enzyme synthesis and translatable carbamyl phosphate synthetase mRNA activity were repressed by 45-65%. The two measurements, synthesis in vivo and mRNA activity in vitro, were made on the same liver and correlated closely in their response to chloramphenicol. The data suggest that a product of mitochondrial protein synthesis may be involved in mediating hormonal regulation of the nuclear gene coding for carbamyl phosphate synthetase.

Nucleolar DNA organization in the interphase nuclei of mouse fibroblasts.

The organization of nucleolar DNA in interphase nuclei of somatic cells was studied at the ultrastructural level using oxidized DAB as a nucleic acid stain. Some finely filamentous networks of DNA-containing structures were observed within the nucleolar fibrillar component. They originated from the perinucleolar shell of condensed chromatin and from a chromatinic area with a honeycomb like structure. The latter was significantly associated with nucleoli and is believed to be a part of the nucleolar organizer region.

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells.

The binding of urokinase (u-PA) to its cell surface receptor (u-PAR) is critical for tumor cell invasion. Here, we report that the distribution of this binding by a u-PAR antagonist ATF-HSA inhibits in vitro the motility of endothelial cells in a dose-dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF-HSA did not affect the cell proliferation. ATF-HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u-PAR antagonist may be a new approach to control angiogenesis.

Microcinematographic and autoradiographic kinetic studies of bone cell differentiation in vitro: matrix formation and mineralization.

Matrix formation and mineralization have been reported in vitro with cells isolated from rat calvaria bones by collagenase digestion (Nefussi et al., 1985). In the current study, kinetics of bone nodule formation and osteoblastic cell differentiation were studied in this in vitro system using an improved microcinematographic device and flash and follow-up labeling autoradiographic techniques. Microcinematographic analysis showed the formation of bone nodules within 24 h. The initial event observed was the change in the top cells layer which became alkaline phosphatase positive. Matrix synthesis occurred a few hours after this. The autoradiographic results demonstrated the formation of an integrated system where osteoblasts and osteocytes were active and synthesized a collagen matrix and mineralized it in a similar time sequence than in vivo.

Leishmania mexicana: destruction of isolated amastigotes by amino acid esters.

Amino acid esters can destroy intracellular as well as isolated amastigotes of Leishmania mexicana amazonensis. In the present study we examined, using a tetrazolium reduction assay, the toxicity of the esters for amastigotes isolated from mouse lesions. Parasite killing by the "prototype" compound L-leucine methyl ester at 1 mM concentration and at pH 7.3 took place within 15-30 min. Time-lapse cinematographic observations showed that the amastigotes rounded up and became less phase-dense before they rapidly broke down. Ammonium chloride, ethylamine or monensin, known to raise the intracellular pH, reduced the sensitivity of the amastigotes to L-Leu-OMe. This finding suggests that an acidified compartment is involved in the destruction of the parasites. The leishmanicidal activity of a series of L-amino acid esters was also investigated. The ED50 (concentration for half maximal effect) for methyl esters was (in mM): Leu (0.62), Trp (0.96), Met (1.13), Glu (2.0), Phe (2.5), and Tyr (3.8). In contrast, the methyl esters of Ile, Val, Ala, beta Ala, Gly, Ser, His, and Pro were either inactive or weakly active at 15 mM. Benzyl esters were more active than their methyl homologs: the ED50 of the benzyl esters of Leu, Val, Ile, Gly, Ala, beta Ala, and Pro were, respectively, 0.07, 0.20, 0.22, 0.88, 1.5, 2.3, and 6.7 mM. Ranks of leishmanicidal activity may reflect differences in the rates of ester uptake and trapping by the amastigotes, in the specificity of the relevant hydrolytic enzyme(s), in the accumulation and metabolic fate of the released amino acids, or in the toxicity of the amino acid or alcohol released within the amastigotes.

On the track of a human circulating mesenchymal stem cell of neural crest origin.

The neural markers present in the normal circulating monocytoid cells able, in pathological situations, to trans-differentiate into different mesenchymal-type cells, confirm the hypothesis previously raised that these cells derive from the neural crest. In culture, the normal cells display a great plasticity very reminiscent of microglial cells in culture. Almost a quiescent cell in normal individuals, this monocytoid cell shows its division potentialities in pathological situations of fibrosis and cancer (chondrosarcoma) where it is found to spontaneously proliferate. While the normal neofibroblasts are rapidly recognized and destroyed by fibrophagic T-lymphocytes, the pathological cells escape this control and, as a result, they accumulate in vitro giving rise to a tissue sometimes organized as nodules. Although basically the transdifferentiation process is similar in all the pathological situations of fibrosis and cancer studied so far, the end-result phenotype evokes the pathology the patient is suffering from. It evokes osteoblasts in a case of osteomyelosclerosis, chondroïdocytes in a case of chondrosarcoma, myelofibroblasts in a case of fibrosis of lung and kidney in a patient under ciclosporine treatment. Hence, this circulating monocytoid cell is a multipotent cell with great division potentiality. These are characteristics of stem/preprogenitor cells. Since this circulating monocytoid cell also bears the neural markers we called it a monocytoid ectomesenchymal stem/preprogenitor cell. Therefore, the existence of an ectomesenchymal system is discussed here. The circulating monocytoid ectomesenchymal stem/preprogenitor cell might be involved in the normal cicatrisation process while the fibrophagic T lymphocytes might be involved in its termination. Impairment of this controlled mechanism might result in the development of fibrosis and/or cancer such as chondrosarcoma in vivo. Interestingly, at least in vitro, proliferation is restricted to the monocytoid cell before transdifferentiation takes place. In this model, fibrosis and cancer might share some common steps going from the proliferation of the monocytoid cells to their transdifferentiation into mesenchymal-type cells and the accumulation of these transdifferentiated cells in the tissues. Then, cancer might be distinguished from fibrosis by the additional acquisition of the ability to proliferate by the transdifferentiated cells. The monocytoid ectomesenchymal stem/preprogenitor cell might also be involved in brain neurodegenerative diseases characterized by an accumulation of microglia. The circulating monocytoid ectomesenchymal stem/preprogenitor cell appears as a target for gene therapy in pathological situations of fibrosis and/or cancer where it proliferates out of control. If the normal cell can be expanded and if its transdifferentiation can be directed, the circulating monocytoid ectomesenchymal stem/preprogenitor cell may become a useful tool for cellular therapy, in case of failure in wound healing and tissue regeneration.

Regulation by phagic T-lymphocytes of a (pluripotent?) organ stem cell present in adult human blood. A beneficial exception to self-tolerance.

Stem cells isolated from adult human blood are able to give rise to several different kinds of cell types such as mesenchymal cells, including striated muscle cells, hepatocytes, and endothelial-cells. Because independently studied by authors whose interests focused on particular tissue types, these stem cells have been described as different. However, they might well represent one unique population of pluripotent stem cells in homeostatic equilibrium with the 'reserve' stem cells buried in organs. In the blood, these stem cells have a monocytic phenotype. In in vitro culture, once they have adhered, they spontaneously differentiate into diverse types of cells reminiscent of embryonic stem cells in culture. Normally, they are almost quiescent cells. But under precise circumstances such as wound-healing, they may proliferate and migrate to the right organ to give rise there to the right type of cells, in order to participate in the repair process. Indeed, such a powerful stem cell needs to be tightly controlled. We illustrate here, by time-lapse videocinematography, how a special subpopulation of T-lymphocytes, for which we coined the name 'phagic T-lymphocytes' (PTLs), destroys these stem cells as soon as they differentiate in vitro, i.e., without the purpose of a repair. These stem cells express constitutively HLA-DR molecules and therefore can act as antigen-presenting cells able to activate phagic T-lymphocytes. The targets of these activated phagic T-lymphocytes are the differentiated stem cell themselves. Phagic T-lymphocytes are attracted by the stem cells, circulate around them, then penetrate and circulate inside them until the latter 'explode'. This mechanism of destruction by phagic T-lymphocytes is unique and seems to be normally restricted to stem cells. It represents a beneficial exception in self-tolerance since it avoids the accumulation of these stem cells out of healing purposes. Interestingly, in disorders such as fibrosis and/or some malignant proliferations, these stem cells proliferate, escape destruction by phagic T-lymphocytes and, as a consequence, accumulate, giving rise to a 'tissue' when cultured in vitro.

Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations.

Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

Leishmania (L.) amazonensis: fusion between parasitophorous vacuoles in infected bone-marrow derived mouse macrophages.

[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.

Nucleolus and large nucleolar aggregates of condensed chromatin in interphase nuclei of L 929 cells.

Three-dimensional reconstructions show that the nucleoli from L 929 cells are associated with one or several large aggregates of chromatin displaying a honeycomb-like structure. The form and the number of both nucleoli and honeycomb structures vary as the cells emerge from the resting state and enter exponential growth. Quantitative data show that the number of honeycomb structures decreases as the number of nucleoli diminishes; both numerical regressions are significant. In addition, the nucleoli and the honeycomb structures enlarge when the cells enter the exponential growth phase. In resting cells the number of honeycomb structures is correlated to the number of nucleoli. Therefore we conclude that the large nucleolar mass of condensed chromatin, which in L 929 cells displays a honeycomb structure, contains a portion of the nucleolar organizing region.

Correspondence of two nuclear networks observed in situ with the nuclear matrix.

In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter-chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten-fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.

The spatial organization of nucleolar DNA in phytohemagglutinin-stimulated lymphocytes of the guinea pig.

The ultrastructural changes in the spatial organization of nucleolar DNA in lymphocytes during phytohemagglutinin (PHA) stimulation was studied in guinea pigs by means of oxidized diaminobenzidine (DAB) at low pH as a differentially contrasting stain for nucleic acids and by the use of reconstruction of serial sections. The extended DNA filaments situated inside the fibrillar area originate from a large aggregation of heterochromatin, which is closely associated with the nucleolus, and from the perinucleolar shell of condensed chromatin. It is suggested that these two distinct regions of chromatin might be associated with different functions.

Cell shape change reveals the cyclic AMP-mediated action of follicle stimulating hormone, human chorionic gonadotrophin and vasoactive intestinal peptide in primary cultured human granulosa-lutein cells.

Granulosa cells are known to be the site of action of various hormones and agents that regulate ovarian function. This study was conducted to evaluate the effects of gonadotrophins, vasoactive intestinal peptide (VIP), prostaglandin (PG) F2 alpha and angiotensin II on the cyclic AMP (c-AMP) signalling transduction pathway in human granulosa-lutein cells. Exposure to agents that elevate c-AMP or mimic c-AMP action caused the cells to become rounded in a process that was rapid and reversible. We were able to demonstrate this cell rounding process in the presence of gonadotrophins and VIP, but not in the presence of PGF 2 alpha or angiotensin II. In addition, incubation of the cells with various selective phosphodiesterase (PDE) inhibitors revealed that the PDE type IV isoform, but not type III, catalyses c-AMP degradation in human granulosa-lutein cells. Alteration in c-AMP-dependent cytomorphology appears to be a convenient method to analyse the regulation of c-AMP-mediated events in the human granulosa-lutein cells.

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

[Electron microscopic demonstration of links between DNA filaments and fibrillar areas of nucleolar RNA in interphase nuclei of L 929 cells]. / Mise en évidence en microscopie électronique, dans les noyaux interphasiques des cellules L 929, de filaments de DNA associés aux zones fibrillaires RNA des nucléoles

Electronic opacification by DAB, following ribonuclease digestion, allowed us to observe a network of DNA filaments which appeared in close relation with both intranucleolar clumps of DNA on one hand, and with the nucleolar fibrillar RNA structure on the other hand. It is inferred that the ribosomal genes may be located on these filaments of DNA.

[Ultrastructural demonstration of deoxyribonuclease-sensitive elements in fibrillar zones of the nucleoli of L 929 cell interphasic nuclei]. / Mise en évidence ultrastructurale d'éléments sensibles à l'action de la désoxyribonucléase dans les zones fibrillaires des nucléoles des noyaux interphasiques des cellules L 929

We have shown in our earlier paper (4) that a ribonuclease resistant filamentous network was associated with the fibrillar structures of the nucleolus of L 929 interphase nucleus. After desoxyribonuclease digestion and oxydised DAB opacification, the nucleolar fibrillar areas became heterogenous from an electron microscopical point of view. We have concluded that an important part of these fibrillar areas are composed of DNA and that the high electron dense fibrillar structures seen after desoxyribonuclease digestion, are the primary ribosomal gene product, just transcribed on the DNA matrix.

Modifications of the nuclear envelope of BHK cells after infection with herpes simplex virus type 1.

Numerous discrete lesions, which we have termed blebs, appeared in the nucleus of BHK cells 10 to 15 h after infection with herpes simplex virus type 1 (HSV-1). They were formed in the inner portion of the nuclear envelope by the apposition of two thickened lamellae overlying a vacuole. As demonstrated by electron microscopic studies, blebs were regular and associated with the peripheral lamina in the nucleus, averaging 3.5 blebs per micron2. They appeared to be associated with an enrichment of the 155K major capsid protein in the nuclear membrane subfractions as compared with the protein composition of nuclei and plasma membrane fractions. We propose that blebs represent the site of assembly of capsid proteins before DNA insertion and eventual envelopment.

Urokinase receptor mediates mechanical force transfer across the cell surface.

The tripartite complex formed by the urokinase receptor, urokinase, and its inhibitor is an enzymatic system that controls plasmin formation involved in degradation of extracellular matrix proteins. With the use of magnetic twisting cytometry with urokinase-coated ferromagnetic beads, we applied mechanical stress directly to the urokinase receptor on the surface of human myogenic cells in culture. The stiffness and the stiffening response measured through the urokinase receptor resembled those of integrins, which are linked mechanically to the cytoskeleton. Furthermore, stiffness decreased with disruption of actin microfilaments. These results demonstrate that the urokinase receptor is coupled mechanically to the cytoskeleton. Inhibition of the tripartite complex formation with antibodies led to a twofold increase in cytoskeletal stiffness. A stiffened cytoskeleton might impede cytoskeletal remodeling and reorganization and thus impede cell motility. Our results demonstrate that the urokinase receptor mediates mechanical force transfer across the cell surface. As such, it is a novel pathway to regulate cytoskeletal stiffness and, thereby, possibly to modulate motility of normal and abnormal adherent cells.

[Nucleolar lesions induced by ribonuclease in living cultured cells]. / Lésions nucléolaires induites par la ribonucléase dans les cellules vivantes en culture.

The nucleolar lesions provoked by the action of ribonuclease (RNase) on living chick embryo fibroblasts were studied by means of microcinematographic analysis and at the ultrastructural level using oxidized diaminobenzidine as a differentially contrasting stain for nucleic acids. This study has shown that the induction of nucleolar dispersion by RNase was only the beginning of a series of discrete steps. The following sequences are described: dispersion of the nucleolus into fragments, their reassembly, and the emission of spherules which appear of chromatin origin. At that step nucleoli are typically segregated. The alteration of the nucleolar associated chromatin seemed to be primordial in these processes. Moreover, the large mass of heterochromatin intimately associated with the nucleolus and which has been considered to be a part of the nucleolar organizer region apparently plays a chief part in the reassembly of the nucleolar fragments into a segregated nucleolus. Ribonuclease is compared to other drugs known to act on nucleolar DNA.