Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex.

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.

Che-1 modulates the decision between cell cycle arrest and apoptosis by its binding to p53.

The tumor suppressor p53 is mainly involved in the transcriptional regulation of a large number of growth-arrest- and apoptosis-related genes. However, a clear understanding of which factor/s influences the choice between these two opposing p53-dependent outcomes remains largely elusive. We have previously described that in response to DNA damage, the RNA polymerase II-binding protein Che-1/AATF transcriptionally activates p53. Here, we show that Che-1 binds directly to p53. This interaction essentially occurs in the first hours of DNA damage, whereas it is lost when cells undergo apoptosis in response to posttranscriptional modifications. Moreover, Che-1 sits in a ternary complex with p53 and the oncosuppressor Brca1. Accordingly, our analysis of genome-wide chromatin occupancy by p53 revealed that p53/Che1 interaction results in preferential transactivation of growth arrest p53 target genes over its pro-apoptotic target genes. Notably, exposure of Che-1(+/-) mice to ionizing radiations resulted in enhanced apoptosis of thymocytes, compared with WT mice. These results confirm Che-1 as an important regulator of p53 activity and suggest Che-1 to be a promising yet attractive drug target for cancer therapy.

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.

Evaluation of 50 probands with early-onset Parkinson's disease for Parkin mutations.

BACKGROUND: Early onset PD has been associated with different mutations in the Parkin gene, including exon deletions and duplications. METHODS: The authors performed an extensive mutational analysis on 50 probands with onset of PD at younger than 50 years of age. Thirteen probands were ascertained from a registry of familial PD and 37 probands by age at onset at younger than 50 years, blind to family history. Mutational analysis was undertaken on the probands and available family members and included conventional techniques (single strand conformation polymorphism analysis and sequencing) and a newly developed method of quantitative duplex PCR to detect alterations of gene dosage (exon deletions and duplications) in PARKIN: RESULTS: Using this new technique, the authors detected eight alterations of gene dosage in the probands, whereas 12 mutations were found by conventional methods among the probands and another different mutation in an affected family member. In total, the authors identified compound heterozygous mutations in 14%, heterozygous mutations in 12%, and no Parkin mutation in 74% of the 50 probands. We expanded the occurrence of Parkin mutations to another ethnic group (African-American). CONCLUSION: The authors systematically screened all 12 Parkin exons by quantitative PCR and conventional methods in 50 probands. Eight mutations were newly reported, 2 of which are localized in exon 1, and 38% of the mutations were gene dosage alterations. These results underline the need to screen all exons and to undertake gene dosage studies. Furthermore, this study reveals a frequency of heterozygous mutation carriers that may signify a unique mode of inheritance and expression of the Parkin gene.

Parkin mutations in a patient with hemiparkinsonism-hemiatrophy: a clinical-genetic and PET study.

The authors describe a 37-year-old woman with early-onset hemiparkinsonism (HP) and ipsilateral body hemiatrophy (HA). Genetic analysis revealed a missense mutation (Arg275Trp) and a duplication of exon 7 of parkin. The complementary metabolic and receptor pattern of PET ligands corresponded to that typically found in idiopathic PD, although tracer binding asymmetry was lacking. Parkin mutations should be considered in HPHA, particularly when there is a younger age at onset and dystonia is an early sign.

Species-specific localization of actin in mammalian spermatozoa: fact or artifact?

Actin has been characterized and localized in sperm cells of many mammals. Nevertheless, the reported localizations obtained by different methods and/or antibodies varied from species to species and even for the same species. To clarify the question, sperm actin distribution was reinvestigated under uniform technical conditions. Immunogold post-embedding procedures were performed using a polyclonal and two monoclonal antibodies of known specificity to localize actin in spermatids and spermatozoa of rabbit, mouse, rat, monkey, and human. In these species, actin (F-actin) was detected with the three antibodies between the nucleus and the acrosome of round and elongating spermatids. Species-specific changes occurred in maturing spermatids. In the rabbit, actin labeling decreased and disappeared from the tip to the base of the subacrosomal layer. In testicular and epididymal spermatozoa actin was detected only with a monoclonal antibody (Amersham) successively in the neck, postacrosomal area, and subacrosomal bulges. In mouse late spermatids a transitory labeling of the neck was detected only with the polyclonal antiactin. In testicular and epididymal spermatozoa an actin labeling was observed in the principal piece of the tail. In rat, monkey, and human sperm cells actin remained undetected. These results suggest that there is a redistribution of actin in late spermatids and spermatozoa which is a species-specific process but not an artifact of methodological origin. Thus, a function for actin in sperm, if any, remains to be demonstrated.

Characterization of the endogenous protein kinase activity of the hepatitis B virus.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

Differential distribution of glutamylated tubulin in the flagellum of mouse spermatozoa.

Using a monoclonal antibody (GT 335) we previously demonstrated that glutamylation is a predominant posttranslational modification of alpha and beta tubulin isoforms in the axoneme of mouse spermatozoa (Fouquet et al., Cell Motil. Cytoskel. 27, 49, 1994). However, we noted that the staining intensity and/or distribution of glutamylated tubulin were not identical using either indirect immunofluorescence (IIF) or immunoelectron microscopy. To test this discrepancy various permeabilization procedures were performed for IIF: methanol or acetone alone or in combination, including freezing pretreatment and with or without paraformaldehyde fixation. Each procedure gave a particular labeling of sperm axoneme. The diversity of axoneme labeling in mouse spermatids and spermatozoa appeared dependent both on the absence or presence of periaxonemal sheaths and permeabilization procedures. For comparison with IIF and to avoid problematic premeabilization treatments a quantitative postembedding immunogold approach was preferred. In these conditions the labeling predominated in the middle piece of the sperm flagellum and decreased progressively in the principal piece. However, the labeling of the terminal piece was similar to that of the middle piece. These results suggested a differential glutamylated tubulin distribution along the axoneme of the mouse sperm flagellum.

Perinuclear cytoskeleton of acrosome-less spermatids in the blind sterile mutant mouse.

The perinuclear cytoskeleton of mammalian spermatids is thought to play a major role in nucleus-acrosome association and in shape changes of the head during spermiogenesis. To test these hypotheses acrosome-less spermatids in blind-sterile mutant mice were investigated for the development of the subacrosomal layer. Immunogold procedures were used for the detection of actin and calmodulin. In addition to various other abnormalities many acrosome-less round and elongating spermatids developed a subacrosomal layer with an actin and calmodulin distribution similar to that observed in normal spermatids. However, in mutant elongating spermatids the apical part of the nucleus was truncated and/or folded. The expected elongation and shaping of the nucleus only occurred in its caudal part associated with an hypertrophied and somewhat ectopic manchette. These abnormalities and those previously observed in mutant and experimental models indicated that the subacrosomal layer may form independently of the acrosome. It is suggested that the subacrosomal filamentous actin is a transitory scaffolding which might be involved in the assemblage of other proteins of the perinuclear cytoskeleton. However, by itself, this layer is not sufficient to ensure a normal shaping of the nucleus. Acrosome-nucleus interactions mediated by the subacrosomal layer seem necessary to shape the cranial spermatid head. The manchette appears to be involved only in the caudal nuclear shaping.

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.

Expression of tubulin isoforms during the differentiation of mammalian spermatozoa.

Using the GT 335 mAb we have previously demonstrated a differential expression of glutamylated tubulin isoforms during spermatogenesis and in spermatooza of the mouse and human. Moreover, the proximodistal decrease of the immunolabeling and its predominance in doublets 1-5-6, corresponding to the plane of the flagellar wave, suggested that the glutamylated tubulin could be involved in a functional heterogeneity of microtubules in peripheral doublets of the sperm flagellum. In order to characterize further the importance of glutamylated tubulin in the sperm model, we analyzed tubulin isoforms by immunoblotting and quantitative immunogold, using antibodies to the C-terminal domain of both subunits including non-glutamylated and glutamylated epitopes. The unique differential immunolabeling of the glutamylated tubulin was confirmed with three mAbs 406-3, 392-2 and B3, in addition to GT 335. This differential labeling was interpreted as a differential accessibility of tubulin epitopes since it was greatly reduced in human spermatozoa lacking dynein arms and after motility inhibition of normal spermatozoa by azide pretreatment. We suggest that the glutamylated tubulin interacts with other axonemal and/or periaxonemal proteins which could be involved in flagellar beating and its regulation.

Regulatory role of suppressive motifs from commensal DNA.

The microbiota contributes to the induction of both effector and regulatory responses in the gastrointestinal (GI) tract. However, the mechanisms controlling these distinct properties remain poorly understood. We previously showed that commensal DNA promotes intestinal immunity. Here, we find that the capacity of bacterial DNA to stimulate immune responses is species specific and correlated with the frequency of motifs known to exert immunosuppressive function. In particular, we show that the DNA of Lactobacillus species, including various probiotics, is enriched in suppressive motifs able to inhibit lamina propria dendritic cell activation. In addition, immunosuppressive oligonucleotides sustain T(reg) cell conversion during inflammation and limit pathogen-induced immunopathology and colitis. Altogether, our findings identify DNA-suppressive motifs as a molecular ligand expressed by commensals and support the idea that a balance between stimulatory and regulatory DNA motifs contributes to the induction of controlled immune responses in the GI tract and gut immune homeostasis. Further, our findings suggest that the endogenous regulatory capacity of DNA motifs enriched in some commensal bacteria could be exploited for therapeutic purposes.

Association of spectrin with manchette microtubules in mammalian spermatids.

In hamster and mouse spermatozoa a spectrin immunogold labeling was found under the plasma membrane in the principal piece of the flagellum. During spermatid differentiation, the spectrin labeling was associated with the manchette, a transient microtubular network involved in nuclear shaping and organelle translocation.

The cytoskeleton of mammalian spermatozoa.

The mammalian spermatozoa are endowed with a unique cytoskeleton which consists both of ubiquitous and specific proteins, some of them arising from gene haploid transcription. In the head, a dense perinuclear layer is made of basic proteins (calicin, cylicin, etc) associated with calmodulin and actin remnants. In the flagellum, the axonemal microtubules are mainly composed of glutamylated tubulin isoforms; the periaxonemal outer dense fibers and fibrous sheath are considered as related cytoskeletal structures on the basis of some common polypeptides.

In vivo fertilization after initiation of sperm motility in the hamster epididymis.

Hamster spermatozoa, collected from the caput and the cauda of the epididymis, are known to differ in their motility. They do not normally acquire fertilizing ability until they reach the proximal portion of the cauda epididymidis. The aim of the present investigation was to test the fertilizing capacity of spermatozoa from the caput epididymis after initiation. Sperm cells were incubated by adding 10 mM caffeine and 20-30 p. 100 epididymal plasma to the culture medium. Superovulated females were inseminated in utero and the eggs recovered 14 h after ovulation. In these conditions, 22 p. 100 of the ova were fertilized. The possibility that the increased motility of the caput epididymal spermatozoa might reflect an increase in fertilizing ability is discussed.

Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus.

Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown. By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles. To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA. Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles. Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein. This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation.

Development and initiation of sperm motility in the hamster epididymis.

Hamster spermatozoa were isolated from the caput, corpus and cauda epididymidis. They were observed in culture medium at 37 degrees C with a phase-contrast microscope and their motility recorded cinematographically. About 20 p. 100 of the caput epididymidis spermatozoa were motile and moved in a confined space with no forward progression. 30 p. 100 of the corpus epididymidis spermatozoa were motile, showing increased flagellar activity and moving in wide circles. 90 p. 100 of the cauda epididymidis spermatozoa were motile and moved forward. Forward motility was induced in immotile spermatozoa from the caput epididymidis by adding cyclic 3'-5' adenosine monophosphate (cAMP) phosphodiesterase inhibitors (caffeine, theophylline, IMX) and epididymal plasma. The best stimulation was initiated by 15 mM caffeine with 10 p. 100 of cauda epididymal plasma; a mean of 60 p, 100 of forward motility was obtained which lasted for one hour and then ceased. Cinematographic studies revealed that some induced sperm movements differed from the equivalent natural ones by the amplitude of the head movements. It is shown that during epididymal transit of hamster spermatozoa, the induction of forward motility requires not only an increased cAMP level but also factors from the cauda epididymal plasma. The idea that glycoprotein of epididymal origin initiates forward motility is discussed.

Forward motility protein (FMP): localization in hamster epididymal spermatozoa.

The presence of FMP was investigated by immunocytochemistry in spermatozoa from the hamster caput and cauda epididymis. Spermatozoa from the caput showed no staining whereas spermatozoa pre-incubated with FMP were stained on the acrosome. Pre-treatment of the same sperm with epididymal plasma induced staining on the principal piece of the flagellum. Spermatozoa from the cauda were stained without previous incubation both on the acrosome and on the principal piece of the flagellum. These results suggest that the action of FMP, which prevents head-to-head agglutination of motile spermatozoa and allows acquisition of forward motility, may be due to at least two proteins. The first localizes to the acrosome during epididymal transit (anti-sticking factor), whilst the second localizes to the principal piece of the flagellum (forward motility initiation factor).

Mitotic activity in monkey and rat Leydig cells.

The testes of the monkey, Macaca fascicularis, from birth to adulthood and of rats from puberty to adulthood were examined by both light and electron microscopy to estimate the mitotic activity of the Leydig cells. In monkeys, mitotic activity was measurable only in neonates: 2.3% in Leydig cells and 0.4% in fibroblast-like cells. From measurements of 3H-thymidine incorporation, mature Leydig cells were labeled in neonates (2%) and adult animals (1%). The labeling indices were significantly increased with increasing time after isotope exposure, and some pairs of labeled cells were found. In prepubertal (28 days of age) and pubertal (45 days of age) rats, mitotic indices were 2 and 0.2%, respectively, in the Leydig cells and 0.4 and 0.1% in fibroblast-like and myoid cells, respectively. Mitoses were not detected in postpubertal rats at 63 days of age. Taken together, these results demonstrate the self-renewal ability of mature Leydig cells during normal development.