Involvement of L-DNase II in nuclear degeneration during chick retina development.

During the development of the neural retina, 50% of the neurons die physiologically by apoptosis. In the chick embryo, the apoptotic wave starts at E8 and ends at E18, with a peak at E11. The onset of apoptosis is accompanied by the activation of several degradative enzymes. Among these, the activation of the endonucleases leads to the degradation of the genomic DNA of the cell which is thought to be the final event in apoptosis. Here, we have investigated the endonucleases activated during apoptosis associated with retinal development. We have found that Ca2+-Mg2+-dependent endonucleases, as well as acid endonucleases are activated. The results obtained in vitro using purified nuclei from chicken retina indicate that the endonuclease activity resulting from the activation of L-DNase II, an acid DNase is responsible for most of the DNA degradation observed in these cells.

Sarcolectin: complete purification for molecular cloning.

A great variety of vertebrate cells contain detectable amounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.

Sarcolectin (SCL): structure and expression of the recombinant molecule.

Interferons (IFNs) are major cytokines, responsible for down-regulating cell growth and for promoting cell differentiation. The sarcolectin (SCL) protein presented here blocks in the cells the established IFN-dependent interphase and stimulates DNA synthesis, probably in co-ordination with more specific growth factors or hormones. The SCL-DNA structure is closely related to that of cytokeratine K2C7 intermediate filaments, but the SCL is a monomer, or sometimes a dimer, which is excreted into the serum, where it is frequently bound to albumin. Its specific biological functions are carried by the beta sheets, and can be found on the two terminal domains of the molecule, the lectinic properties being located mainly on the N-terminus. The recombinant SCL molecule possesses the same biological functions as the native one, since it inhibits the IFN-dependent antiviral state both in human and in mouse cell cultures. On the contrary, antibodies raised against amino acids 41-55 located on the N-terminal domain of SCL inhibit this antagonistic effect. We postulate that the IFN and SCL proteins, because of their opposite biological functions, are in balance and are part of a feedback system operating the regulation of normal growth. In pathological cases, SCL could play a role in the development of tumors, as we have found in juvenile osteosarcomas or in AIDS cases.

Developmental expression of nitric oxide synthase isoform I and III in chick retina.

During our studies on the multiple possible functions of nitric oxide (NO) in chick retinal development and physiology, we have demonstrated the presence and the activity of NO synthase (NOS-I and III) in certain neuronal populations (photoreceptors, amacrine cells in the inner nuclear and ganglion cells) and also in synaptic-rich regions in the developing chick retina. Both enzymes, detected by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, immunohistochemistry and Western blotting, appeared between embryonic days 6 and 12, and followed a spatial and temporal pattern of expression which correlated with the differentiation of the neuronal layers. Evaluation of the conversion of [3H]-labeled arginine to [3H]-citrulline, confirmed the presence of a calcium-dependent NOS activity in the cytosolic and particulate retinal extracts during the development. This pattern of NOS expression suggests that the regulated release of NO during key phases of development might be one mechanism involved in the regulation of retinal differentiation.

Localization and structure of v-mos in transformed mouse fibroblasts reverted by long-term interferon treatment to nonmalignancy.

We have reported previously that Moloney virus-transformed cells, when treated for over 200 passages in the presence of low concentrations of mouse interferon-alpha/beta, can be reverted to a stable nonmalignant status. The cells recover full contact inhibition and are unable to raise tumors when grafted in nude mice. In the present report, we show that whether reverted or malignant, these cells contain deleted v-mos oncogenes, which have lost 392 nucleotides. The truncated oncogenes contain a reduced and modified open reading frame but are able, however, to induce tumors when transfected in mouse 3T3 cells. As there is no difference either in the location or in the structure of this modified v-mos, whether yielded by reverted or malignant cells, we postulate that both cell lines derive from the same population and this modification does not play any role in the reversion process obtained through prolonged IFN-dependent selection. We suggest that reversion could be an epigenetic phenomenon, involving the constitutive synthesis of IFN-beta only in the reverted and not in the malignant cells. The continued persistence of such noncancerous cells could result at least partly from a balance involving the expression of v-mos, IFN-beta, and an IFN antagonist, sarcolectin. These reverted cells can undergo an unlimited number of passages, but they must be trypsinized before day 5 in confluent cultures. Thereafter, the cells stop dividing, cannot proliferate anymore, progressively show signs of apoptosis, and die.

Involvement of DNase II in nuclear degeneration during lens cell differentiation.

The characterization of DNase II and DNase I activity was undertaken to discriminate their different roles in physiological nuclear degradation during lens fiber cell differentiation. The activity of both nucleases determined in a new assay allows to discriminate DNase II from DNase I in the same extract. In fibers, both types of nuclease activities are found and appear higher than in epithelial cells. Specific polyclonal antibodies directed against these two nucleases reveal by Western blot analysis the presence of various DNase isoforms. DNase II like-nuclease, present in fibers, is represented by three major bands (60,23, and 18 kDa), which are not detected, at least for two of them (60 and 23 kDa), in epithelial cells. DNase I like-nuclease pattern in fiber cells shows a single 32-kDa band, while several bands can be detected in epithelial cells. Immunocytochemistry studies show both nucleases present in lens cell sections. DNase II is, as usual, in cytoplasm of epithelial cells, but it appears strikingly concentrated in the nuclei of fibers. DNase I is always concentrated in nuclei of epithelial and fiber cells. DNA degradation observed in agarose gels shows that DNase II-activating medium cleaves the DNA from fiber cells more efficiently than DNase I-activating buffer. In addition, DNase II antibody is able to prevent this degradation. These results suggest a specific involvement of DNase II in nuclear degradation during lens cell differentiation.

Retinoic acid-induced heparin binding protein (RIHB) binds to embryonal chondrocytes and cartilage primarily via proteoglycans.

Retinoic acid-induced heparin binding protein (RIHB) is a highly basic, secreted polypeptide expressed during early chick embryogenesis. We have characterized the binding of 125I-labeled RIHB to embryonal chondrocytes in culture. No saturable, high-affinity binding can be observed on these cells. Furthermore, no 125I-labeled RIHB was internalized into the chondrocytes at 37 degrees C. The low-affinity binding of 125I-labeled RIHB observed can be competed with another heparin binding factor, fibroblast growth factor 2, as efficiently as with unlabeled RIHB. The binding can also be almost completely inhibited by preincubation of the 125I-labeled RIHB with heparin or with a monoclonal antibody which recognizes the heparin binding site of both RIHB and HBNF. When cross-linking experiments are performed with 125I-labeled RIHB, specific RIHB-containing high-molecular-weight complexes are observed; however, these represent only a very small fraction of the bound material. Immunohistochemical analyses of embryonic wing cartilage demonstrate that a significant fraction of bound RIHB can be removed from unfixed tissue simply by rinsing with phosphate-buffered saline. The remaining RIHB can be removed partially by incubation with heparitinase I or III and completely when the incubation is performed with chondoitinase A, B, C. These results demonstrate that RIHB binds to embryonal chondrocytes and cartilage primarily through proteoglycans of both heparan sulfate and chondroitin sulfate types.

[Mechanism of proliferation of cells transformed by the Moloney mouse sarcoma virus after stable reversion to a non-malignant state]. / Mécanisme de la multiplication des cellules transformées par le virus du sarcome de Moloney après réversion stable à l'état non malin.

Prolonged interferon (IFN) treatment can convert Moloney sarcoma-transformed mouse Balb C fibroblasts to a stable non-malignant status. The cells recover a number of differentiated features, which are maintained even when IFN is permanently omitted from the tissue culture medium. We show here that reversion could be at least in part attributed to constitutive IFN beta synthesized only in the reverted cells. The continued replication of these cells, although unable to induce tumours in athymic mice, could be the result of the maintained expression of an IFN antagonist termed sarcolectin (SCL), a balance being struck between the effects of v-mos oncogene, interferon beta and SCLs. In agreement with Lampl et al. [11], we suggest that normal cell growth is discontinuous, consisting of sudden bursts followed by prolonged arrests which could be necessary to promote differentiation during the ensuing interphase.

Retinoic acid-induced heparin-binding factor (RIHB) mRNA and protein are strongly induced in chick embryo chondrocytes treated with retinoic acid.

Retinoic acid-induced heparin-binding factor (RIHB) is a highly basic polypeptide expressed during early chick embryogenesis. We have examined the induction of RIHB by retinoic acid in chondrocytes isolated from the sterna of Day 15 chick embryos and the effects of exogenous RIHB on these cells. There is an induction of RIHB mRNA in chondrocytes which is dose dependent, with maximal levels of expression observed with concentrations of retinoic acid in the 10(-6) M range. RIHB mRNA is first observed 16 h after commencement of treatment, is maximal after 24-48 h, and is completely attenuated after 5 days. This transient pattern of expression is very similar to that of type X collagen; however, RIHB induction precedes that of type X collagen by about 24 h. The expression of both RIHB and type X collagen precedes the drop in keratan sulfate:chondroitin sulfate proteoglycan and type II collagen expression and the surge of fibronectin expression. The induction of RIHB mRNA is accompanied by an increased synthesis of the protein. No RIHB can be detected in untreated chondrocytes; however, large amounts are produced by cells treated with 5 x 10(-7) M retinoic acid. The protein is recovered mainly in the culture medium and bound to the extracellular matrix. Only a small amount can be detected in cell extracts. RIHB can be detected in the culture medium after 16-24 h and, unlike the mRNA, persists over the 5-day period examined. The effect of exogenous RIHB (purified from chick embryos) on chondrocyte proliferation and morphology was examined. When added to the culture medium in concentrations of up to 500 ng/ml RIHB had no effect on [3H]thymidine incorporation or cell morphology. Thus, RIHB is not the direct mediator of retinoic acid for these cells, but is strongly induced during the treatment.

Sarcolectin and interferon in the regulation of cell growth.

Sarcolectin is an endolectin present in a great variety of conjunctival tissues (muscles, cartilage, sarcomas), but also in brain or placental extracts of vertebrates, including primates. When purified to electrophoretical homogeneity as a 65-kd protein, it agglutinates cells and has an affinity for simple sugars. In addition, it is able to inhibit the synthesis of interferon (IFN)-dependent secondary proteins and to restore cells to their status ad primum. The biological effect of Poly(I).Poly(C)-induced feedback interferon is inhibited by the addition of sarcolectins, which also abolishes cellular refractoriness to repeated IFN induction. Similarly, sequential association of, first, Poly(I).Poly(C); 4-5 h later, sarcolectin restores the full capacity of both to promote cell growth, unrestrained by IFN. Indeed, the secondary proteins which are in the process of being synthesized are inhibited. In a great variety of animal cells, sarcolectin can also initiate growth after it has been blocked by IFN. This is not an all-or-none effect, but a balance may be struck by IFN and sarcolectin, depending on their respective concentrations and specific activity. We propose that the coordination of these cellular functions of Poly(I).Poly(C), IFN, and sarcolectin takes place in the form of a triangular growth-regulatory cycle and postulate that they thus maintain a balance during differentiated normal tissue development.

Interferon- and sarcolectin-dependent cellular regulatory interactions.

Interferons, via specific membrane-bound receptors, induce various cellular functions of which antiviral protection is the most extensively studied. We have previously reported the existence of interferon antagonists (referred to as sarcolectins) in various tissue extracts from placental blood, cartilage, brain, muscle, or from sarcomas. These sarcolectins have been fully characterized and purified to homogeneity. In interferon-treated cells, they restore virus sensitivity 4-6 h after the establishment of antiviral protection. In the present study we investigate the effect of sarcolectins on the steady state levels of two double-stranded RNA dependent enzymes, 2-5A (p chi (A2'p)nA) synthetase and protein kinase. Several authors have previously emphasized the role of these enzymes in the mechanism of interferon's antiviral action. Interferon promotes a 4-8 fold increase in protein kinase and 2-5A synthetase in cells. Addition of sarcolectin 5 h after interferon results in a dramatic reduction in the steady state levels of both these enzymes, as shown by their decreased activity and yield observed in Western blot assays. The degradation of the antiviral response in sarcolectin-treated cells might therefore be at least partially attributed to a reduced synthesis of protein kinase and 2-5A synthetase. Since there are no direct interactions between sarcolectins and interferon or its receptors, it can be postulated that sarcolectins exert their effect through these interferon-dependent proteins. We postulate that the opposing biological effects of interferon and sarcolectins strike a balance which may, however, be modified in one direction or the other, depending on their respective concentrations.

Detection of vesicular stomatitis virus (VSV) RNA in the central nervous system of infected mice by in situ hybridization.

Using in situ hybridization with a cloned DNA probe specific for the VSV G protein, viral RNA was detected and localized in CNS tissue of mice infected i.c. with either wild or ts G 31 VSV mutant. In both cases, brain and spinal cord neurons were the only cells seen to contain viral RNA. Virus-positive neurons were observed enclosed in spongious areas induced by the ts VSV mutant. These results suggest that the VSV shows a strong tropism for the neuronal cell and indicate that the vacuole formation might be associated with the expression of the VSV G protein gene in infected neurons.

Cell distribution and antigenic properties of mammalian sarcolectins.

Sarcolectins are present in a great variety of tissues from mammalian origin. Such substances were observed to be secreted from cultures of human embryonic fibroblasts, human osteosarcoma and rat Rous sarcoma transformed cells and could be extracted from TG 180 Crocker Sarcoma or normal human placenta. All sarcolectins tested here, were comparable by their physicochemical properties to those previously reported in hamster or human sarcomas. Indeed, they are proteins or glycoproteins, resistant to pepsin and migrate in SDS-PAGE in the 65 kDa area. They agglutinate cells with an affinity for simple sugars and degrade previously established interferon-induced antiviral resistance. Considering the hamster sarcolectin as reference in this comparative study, both differences and similarities in the antigenic properties of mouse, rat and human sarcolectin variants were demonstrated. An indirect immunofluorescence assay showed that sarcolectins were specifically labelled on the cell surface but not detected in the cytoplasm after methanol or acetone permeabilization of the membrane. By electron microscopy, using immunoperoxidase labelling, sarcolectins can be localized on the surface of normal, transformed, human or rat cells. Only limited segments of normal cell membranes were labelled, while transformed cells were frequently stained on their whole surface. Other known extracellular proteins, such as fibronectin and collagen, did not share common antigenic determinants with sarcolectins.

Role of VSV G antigen in the development of experimental spongiform encephalopathy in mice.

The ts G31 VSV mutant induced spongiform encephalopathy without any inflammatory response when injected i.c. into the mouse. In electron microscopy, no virions could be detected in spongiform lesions. In contrast, with the wild VSV strain inflammatory lesions were seen, which contained viral particles in great abundance. As previously shown in vitro, when using the ts G 31 mutant at the nonpermissive temperature, the G antigen can spread from membrane to membrane to distant sites, fusing a great number of cells even in the absence of virus multiplication. Therefore, we postulate that a comparable mechanism is responsible for extensive brain lesions originating probably from a relatively small number of G antigen-producing cells. Indeed, the spongious regions seen mainly in the grey matter contained vacuoles, whose walls were clearly stained by peroxidase-labelled immune serum to G antigen, without detectable virions or inflammatory lesions. The vacuoles probably represent altered and swollen dendritic cell membranes. The relationship between spongiosis development and antigen diffusion in the absence of significant virus replication is discussed.

Detection of an interferon antagonist, sarcolectin, in human sarcomas and muscles.

In a variety of human sarcomas we detected the presence of a sarcolectin which reversed an established antiviral protection induced by interferon (IFN). For the same protein concentration, this biological activity was significantly increased when compared to that of normal muscles. All the biological characteristics were comparable to those of a sarcolectin found in hamster tissues; namely the capacity to agglutinate cells and its inhibition by specific sugars, migration in sodium dodecyl sulfate gel, and pepsin, heat and sodium dodecyl sulfate stability. Except for its anti-IFN function and cell agglutinating activity, the biological significances of this sarcolectin is presently poorly understood.

Early nuclear structural changes of transformed human amniotic cells (WISH) in presence of type IV collagen detected by the nuclear refringency assay.

An early nuclear activation of transformed human amniotic epithelial (WISH) cells triggered by type IV collagen is visualized by a modification of the nuclear refringency obtained by mercury binding on condensed chromatin. This phenomenon is quantified by the nuclear refringency test. The nuclear activation of WISH cells by basement membrane collagen is also shown by the DNase I sensitivity of chromatin and by the measurement of mRNA synthesis. These nuclear phenomena are concomitant with WISH cell attachment and laminin synthesis. Reversible effects on nuclear refringency, cell attachment, and laminin synthesis are tested by the addition and removal of different metabolic inhibitors.

Sarcolectin: an interferon antagonist extracted from hamster sarcomas and normal muscles. Isolation, characterization, and purification.

In the present work we show that sarcoma and normal hamster tissues contain a protein which agglutinates normal and transformed cells. The inhibition of agglutination by galacturonic acid and occasionally by fucose suggests a resemblance of this protein with vegetal lectins. When added 5 h after interferon, the crude semipurified and electrophoretically homogeneous preparations reduce within 20 h the antiviral state pre-established by interferon. These two biological tests have enabled us to monitor the subsequent purification steps. The isolation of the biologically active protein is greatly facilitated by its resistance to pepsin and nucleases, whereas its sensitivity to trypsin and Pronase suggests its proteinaceous character. Furthermore, the molecule is stable when heated 1-2 min at 100 degrees C in the presence or absence of sodium dodecyl sulfate. After pepsin treatment, Sephacryl G-200 gel filtration, and ion exchange chromatography on DEAE-cellulose, 25-40-fold purification can be obtained. When controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a double band (DEAE-cellulose sample) or single band (octyl-agarose sample) is detected in the 65,000-dalton region and no other contaminator is present. The eluted protein retains full biological activity when assayed by the degradation of the interferon-induced antiviral protection in the cell or titrated by cell agglutination.