Densovirus crosses the insect midgut by transcytosis and disturbs the epithelial barrier function.

Densoviruses are parvoviruses that can be lethal for insects of different orders at larval stages. Although the horizontal transmission mechanisms are poorly known, densoviral pathogenesis usually starts with the ingestion of contaminated food by the host. Depending on the virus, this leads to replication restricted to the midgut or excluding it. In both cases the success of infection depends on the virus capacity to enter the intestinal epithelium. Using the Junonia coenia densovirus (JcDNV) as the prototype virus and the lepidopteran host Spodoptera frugiperda as an interaction model, we focused on the early mechanisms of infection during which JcDNV crosses the intestinal epithelium to reach and replicate in underlying target tissues. We studied the kinetics of interaction of JcDNV with the midgut epithelium and the transport mechanisms involved. Using several approaches, in vivo, ex vivo, and in vitro, at molecular and cellular levels, we show that JcDNV is specifically internalized by endocytosis in absorptive cells and then crosses the epithelium by transcytosis. As a consequence, viral entry disturbs the midgut function. Finally, we showed that four mutations on the capsid of JcDNV affect specific recognition by the epithelial cells but not their binding.

The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation.

We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro.

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.

Expression and activity of serum response factor is required for expression of the muscle-determining factor MyoD in both dividing and differentiating mouse C2C12 myoblasts.

To understand the mechanism by which the serum response factor (SRF) is involved in the process of skeletal muscle differentiation, we have assessed the effect of inhibiting SRF activity or synthesis on the expression of the muscle-determining factor MyoD. Inhibition of SRF activity in mouse myogenic C2C12 cells through microinjection of either the SRE oligonucleotide (which acts by displacing SRF proteins from the endogenous SRE sequences), purified SRF-DB (a 30-kDa portion of SRF containing the DNA-binding domain of SRF, which acts as a dominant negative mutant in vivo), or purified anti-SRF antibodies rapidly prevents the expression of MyoD. Moreover, the rapid shutdown of MyoD expression after in vivo inhibition of SRF activity is observed not only in proliferating myoblasts but also in myoblasts cultured under differentiating conditions. Additionally, by using a cellular system expressing a glucocorticoid-inducible antisense-SRF (from aa 74 to 244) we have shown that blocking SRF expression by dexamethasone induction of antisense SRF results in the lack of MyoD expression as probed by both immunofluorescence and Northern blot analysis. Taken together these data demonstrate that SRF expression and activity are required for the expression of the muscle-determining factor MyoD.

Chromosomes of amphibian oocytes as a model for gene expression: significance of lampbrush loops.

Amphibian lampbrush chromosome loops exhibit morphological variability in their RNP matrix. The biological significance of such variability remains unknown. In order to approach this problem, the structural organization of each RNP matrix type was analyzed in relation to transcriptional and post-transcriptional processes. First, autoradiographic and transcription inhibition studies in conjunction with macromolecular spread analysis revealed a particular transcription pattern in the most typical loops, i.e. the globular loops. This pattern was characterized by asynchronous variations in RNA synthesis in the different transcription units present in a given loop. Second, morphological and experimental studies provided evidence that the typical morphologies of different RNP matrices were interconvertible and that the differences between the different RNP matrices resulted from various degrees of tightness in packaging of transcription products. In particular, analysis of thermic-shock-induced changes in the structure of lampbrush chromosomes enabled us to visualize the progressive disorganization of dense RNP matrices into globular, granular and normal matrices. Furthermore, these studies suggested that changes in post-transcriptional processes might play a determining role in the specific morphology of the loops. In particular, the kinetics of each of these different processes, related to one another and/or proteins specific to one or another of these processes, might determine the morphological appearance of the loops. The immunological approach revealed that specific nuclear proteins might therefore interfere with each of these processes. Third, the problem of a possible relationship between the specific morphologies of lateral loops and the expression of particular DNA sequences was approached at the molecular level.

An analog of Xenopus N1N2 protein in Pleurodeles waltl.

The oocyte nucleus of Pleurodeles waltl contains a major 185-kDa protein analog of Xenopus N1N2. Rabbit antibodies were raised against the 185-kDa protein. Affinity-purified antibody directed against the acid part (pI 4.7) of the protein was prepared using antigens separated by two-dimensional electrophoresis. Specificity of the antibody was controlled on two-dimensional gels of nuclear proteins. It was shown that the 185-kDa protein was separated into 2 forms: an acid form of pI 4.7-5.3, and a base form of pI 7. Peptide maps of the 2 spots revealed that they were closely related. The antibody was tested on: a) spread nuclear content, b) on sections of embryonic stages from stage 2 to stage 34, and c) the sections of adult tissues. The 185-kDa protein appeared to be associated with the RNP matrix of a particular type of lampbrush chromosome loop, the granular loop. This protein was present in the nuclei of all embryonic cells. In adult tissues, it was present only in the nuclei of cells which presented high mitotic activity. These results confirm that, like N1N2, the 185-kDa protein interacts with the constitution of chromatin; furthermore, they provide evidence for the role of this protein in transcriptional activity.

Cold-induced changes in amphibian oocytes.

Female Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the "cold-stress proteins"; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes.

Solid-phase synthesis and cellular localization of a C- and/or N-terminal labelled peptide.

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subjected to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.

Design of carrier peptide-oligonucleotide conjugates with rapid membrane translocation and nuclear localization properties.

Peptides containing a hydrophobic motif associated with a nuclear localization signal separated by various linkers were synthesized in solid phase. The hydrophobic sequence corresponds either to a signal peptide sequence or to a fragment of the fusion peptide of GP41 while the hydrophilic sequence is that of a nuclear localization signal. The C-termini of these peptides bear a cysteamide group that was linked to a fluorescent probe. This allowed the cellular localization of the probe to be determined as a function of the peptide sequences. The labeled peptides were then incubated with fibroblasts. Using N-biotinylated derivatives we confirmed by indirect immunofluorescence that the observed localizations corresponds to those of the peptides. The presence of a linker appears to play a role in the cellular localization. One of these peptides was successfully used to target fluorescent oligodeoxynucleotides into living cells demonstrating improved cell delivery of peptide-oligodeoxynucleotide conjugates.

Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo.

We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.

Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.

Human cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis.