Transgenic major histocompatibility complex class I antigen expressed in mouse trophoblast affects maternal immature B cells.

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.

T-antigen interactions with chromatin and p53 during the cell cycle in extracts from xenopus eggs.

The role of SV40 large tumor T-antigen in replication of viral DNA is well established, but it is still unclear how T-antigen triggers cellular replication and cell transformation in non-permissive cells. Here, we used Xenopus egg extracts which reproduce most nuclear events linked to the cell cycle in vitro to analyze its interaction with genomic chromatin during the cell cycle. We show that T-antigen associates with chromatin before the nuclear membrane formation, and further demonstrate that the nuclear membrane is not necessary for its import into the nucleus. We show that the interaction of T-antigen with the endogenous chromatin does not occur at replication foci nor at RPA pre-replication centers. Immunoprecipitations as well as sucrose gradient experiments, indicate that the endogenous pool of p53 interacts with T-antigen. In addition, a transient association of both proteins with the nuclear matrix is observed during the ongoing DNA synthesis. These data are discussed in view of the T-antigen and p53 activity during the cell cycle.

Nuclear import of p53 during Xenopus laevis early development in relation to DNA replication and DNA repair.

The role of p53 in transcriptional activation of genes involved in cell cycle progression is well established. However, the wide range of functions attributed to this gene suggests that some of them might be unrelated to transcription. Here we investigated p53 localization and recruitment to chromatin during Xenopus early development when 12 rapid cell cycles occur without transcription of the genome. We show that after fertilization, part of the large store of p53 previously stored in the cytoplasm of the oocyte is imported into the nucleus. This import was further analyzed in relation with DNA replication and DNA repair using cell-free systems from Xenopus eggs. Formation of a nuclear lamina envelope is necessary for the import of p53 into the nucleus. p53 associates both with decondensed DNA and the nuclear lamina envelope, but no colocalization with prereplication or replication complexes is observed. We show that UV- or gamma-damaged nuclei recruit p53 as well as replication protein A (RPA) in large common foci. Together, these data suggest that p53 plays a role in the regulation of the accelerated S phases that occur during Xenopus early development, in a manner that does not rely on its transcription-mediated activity.

A functional analysis of p53 during early development of Xenopus laevis.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

Stabilization and expression of high levels of p53 during early development in Xenopus laevis.

We previously isolated a p53 cDNA from a Xenopus oocyte library. To determine if p53 has a function in the developmental period, we have studied its expression at the RNA and protein levels during the early development of Xenopus laevis. Two p53 transcripts (3 and 2.2 kb) are expressed from the beginning of Xenopus oogenesis, and the major one (2.2 kb) reaches a level of 7 x 10(5) to 7 x 10(6) transcripts per mature oocyte. After fertilization only the 2.2-kb RNA is detected, but its level decreases and at the neurula stage p53 RNA becomes undetectable. The p53 protein is highly expressed during Xenopus development, in contrast to an undetectable level in Xenopus cells in culture. Most of the p53 protein is synthesized during late oogenesis and a stage VI oocyte contains 7 x 10(11) molecules of p53 protein. This maternal p53 store is maintained at a constant level during Xenopus development, at least until the tadpole stage. This high level of expression is mainly due to stabilization of the p53 protein. Unusually for p53, the protein is strictly located in the cytoplasm of oocytes and this localization might indicate that it is stored in an inactive form at this stage. These data are discussed relative to previous observations made in transgenic mice.

Posttranscriptional regulation of c-myc RNA during early development of Xenopus laevis.

The remarkable stability of c-myc during oogenesis contrasts with its degradation during the early developmental period in Xenopus laevis. Three evolutionary conserved motifs found in the 3'-untranslated region of Xenopus c-myc RNAs have been analyzed for a possible role in c-myc RNA degradation. No specific degradation was observed when these sequences were cloned downstream of a reporter gene and the corresponding RNAs were injected into fertilized eggs. The relation between polyadenylation and degradation of c-myc mRNA has been examined during early development. c-myc is adenylated during early oogenesis, and a dramatic de-adenylation occurs in full grown oocytes. Consequently, the de-adenylation of c-myc mRNA that occurs in eggs might be a requirement for its degradation after fertilization, but is not sufficient to trigger its degradation.

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.

Bifunctional lipid-transfer: fatty acid-binding proteins in plants.

A cytosolic protein, able to facilitate intermembrane movements of phospholipids in vitro, has been purified to homogeneity from sunflower seedlings. This protein, which has the properties of a lipid-transfer protein (LTP), is also able to bind oleoyl-CoA, as shown by FPLC chromatography. This finding, in addition to previous observations suggesting that a lipid-transfer protein from spinach leaves can bind oleic acid and that oat seedlings contain a fatty acid-binding protein with similar features than lipid transfer proteins, provides a clear demonstration that plant cells contain bifunctional fatty acid/lipid transfer proteins. These proteins can play an active role in fatty acid metabolism which involves movements of oleyl-CoA between intracellular membranes.

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins.

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.

Synthesis of phospholipid transfer proteins from maize seedlings.

The synthesis of phospholipid transfer proteins has been studied in vitro after isolation of poly(A)+RNAs from maize seedlings and by in vivo labelling of coleoptiles. After immunoprecipitation of translation products in wheat germ or in reticulocyte lysate systems, the analysis by electrophoresis revealed two bands of molecular mass 9 kDa and 12 kDa. The in vitro synthesized 12 kDa protein is a precursor of the 9 kDa purified protein from maize seedlings as suggested by competition experiments with the pure protein. After immunoprecipitation of in vivo labelled proteins, two bands were detected. One of them, having a molecular mass of 7 kDa, could be related to the in vitro synthesized 9 kDa protein, the other corresponding to the purified protein. Furthermore, biosynthesis of both precursors occurs on membrane-bound polysomes. Presumably a post translational process occurs, yielding to the mature forms.

Bone imaging as an aid for the diagnosis of osteopoikilosis.

An unusual case of osteopoikilosis was found in a 52-year-old black woman. The rather large diffuse focal increased densities involving the ribs, spine, and pelvis as seen in the roentgenograms were believed to represent metastatic bone lesions. The normal nuclide bone images helped revise the diagnosis to osteopoikilosis. Early recognition of osteopoikilosis would have prevented the patient's apprehension and the extensive work-up for the primary tumor.

In vitro synthesis of a plant phospholipid transfer protein: a study by high performance liquid chromatography.

In order to study the biosynthesis of a plant phospholipid transfer protein (PLTP), poly (A)+RNAs have been prepared from maize seedlings and translated in vitro with a rabbit reticulocyte lysate. The newly synthesized proteins were then separated by fast protein liquid chromatography (FPLC) followed by SDS-PAGE or by high performance liquid chromatography (HPLC) coupled to a radioactivity detector monitor. It has been showed that a radioactive band comigrating with a 14C methylated pure PLTP was detected by SDS-PAGE. This result, confirmed by direct radioactive monitoring, indicates that PLTP is actively synthesized in vitro.

Liposarcoma complicating neurofibromatosis. Report of two cases.

Liposarcoma complicating neurofibromatosis is a rare entity. Until now, only two cases have been reported in the literature. The authors present two new cases of liposarcoma arising in generalized neurofibromatosis with detailed microscopic findings. Other neoplasms associated with neurofibromatosis are also discussed.

"Tooth" sign in patellar degenerative disease.

In the total examination of the knee, degenerative changes and ossification in the quadriceps tendon easily recognized on the lateral roentgenogram may cause confusion in diagnosis when seen on the axial view. Vertical ridging of the osteophytes at the patellar insertion of the quadriceps tendon can resemble dentate structure ("tooth" sign). Two hundred and fifty examinations of the knee in different age groups were reviewed and several anatomical sections of the quadriceps-patella insertion were studied. The "tooth" sign represents the relief of severe osteophyte formation in the bundle of the quadriceps tendon at its insertion into the patella.