Cyclic nucleotides affect growth and differentiation of cultured rat embryonic shields

The modified organ culture of rat embryonic shields provides favorable conditions during 2 weeks for the differentiation of main tissue types. Since the terminal differentiation in explants is inferior to that obtained in the homografts of the same shields under the kidney capsule, we tried to improve the culture medium by adding some known regulatory molecules: db-cAMP, db-cGMP, ATP, AMP, and butyric acid. These agents were added to the liquid medium in the concentration of 1 mM. In the first part of the study the explants were fixed and weighed after 8 or 14 days in vitro culture, and histological sections were examined. When the explants were treated with db-cAMP during the second week of culture, the skeletal muscle appeared more frequently in the treated series than in controls, and the weight of the treated explants was sometimes increased when compared with the control series. The db-cGMP had no effect on differentiation, but stimulated the growth of the explants when applied during the first week of culture. On the contrary, the db-cAMP when added during the first week, severely impeded the growth of explants. Other agents seem to be ineffective. In the second part, the content of cAMP and cGMP was measured in normal explants. The radioimmunoassay showed the same content of cAMP and cGMP during the entire culture period. In the third part of our study the incorporation of tritiated uridine and tritiated thymidine was measured during the second week of culture after the addition of db-cAMP. During the first days of treatment with db-cAMP the uptake of tritiated uridine and thymidine was inhibited, whereas on the seventh day the uptake was similar to that of the control. We can conclude that both cyclic nucleotides have a visible effect on growth whereas only cAMP has a positive impact on the differentiation of myotubes in cultured rat embryonic shields.     

Ocular findings in patients with chronic renal failure undergoing haemodialysis

The aim of this paper was to evaluate the ocular findings in patients with chronic renal failure (CRF) undergoing haemodialysis (HD). In 64 patients undergoing haemodialysis (30 female and 34 male), aged 24-83 years (mean 58 years) on haemodialysis 1-213 months (mean 47 months) complete ocular examination were performed: visual acuity (VA), intraocular pressure (IOP), biomicroscopic examination and fundoscopy. On right eye sixty-nine percent of patents had VA 0.6 or better, and on left eye 84% of patients had VA 0.6 or better. Mean IOP before dialysis was 15 mmHg and after dialysis was 14 mmHg. In 9 patients (14%) we found corneo-conjunctival calcium deposits. No correlation of ocular calcification and parathyroid hormone (PTH) level or calcium and phosphate product were observed. 39 (60%) patients had cataract. Hypertensive vascular changes were seen in 44 (68%) patients and in 6 (7%) patients age-related macular degeneration. Seven patients had diabetes mellitus and in 5 diabetic retinopathy was observed. Patients with CRF or who are receiving HD represent unique group of patients. Pathologic change could be found in many tissue and organs, therefore we suggest ocular examination more frequently in dialysis patients.     

SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

Heparan sulfate proteoglycans regulate autophagy in Drosophila

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.     

Of mice and men: teratomas and teratocarcinomas

Teratomas and teratocarcinomas are tumors containing tissue derivatives of all three germ-layers. They can be induced by transplantation of animal embryos to ectopic microenvironment. Development of malignant teratocarcinomas depends on embryonic stage, species-specificity and immunological competence of the host. In the man, teratomas and teratocarcinomas usually represent a subtype of germ-cell tumors but sacrococcygeal teratomas arise from the remnants of the pluripotent primitive streak. Undifferentiated embryonal carcinoma (EC) cells are responsible for the malignancy of experimental mouse teratocarcinomas. Mouse EC cells injected to the adult give rise to tumors and upon injection to early embryos to differentiated tissues--thus resembling normal mouse embryonic stem cells (mESC). Epigenetic changes rather than mutations are associated with transformation of mESC to EC cells. Human EC and ES cell-lines (hESC) contain chromosomal abnormalities and can form teratocarcinoma after transplantation. ES cells are among those proposed for cell replacement therapy in the man. Suicide gene introduction should be recommended prior to their use in vivo to ablate them in case of malignant transformation.     

JNK Signalling Controls Remodelling of the Segment Boundary through Cell Reprogramming during Drosophila Morphogenesis

Segments are fundamental units in animal development which are made of distinct cell lineages separated by boundaries. Although boundaries show limited plasticity during their formation for sharpening, cell lineages make compartments that become tightly restricted as development goes on. Here, we characterize a unique case of breaking of the segment boundary in late drosophila embryos. During dorsal closure, specific cells from anterior compartments cross the segment boundary and enter the adjacent posterior compartments. This cell mixing behaviour is driven by an anterior-to-posterior reprogramming mechanism involving de novo expression of the homeodomain protein Engrailed. Mixing is accompanied by stereotyped local cell intercalation, converting the segment boundary into a relaxation compartment important for tension-release during morphogenesis. This process of lineage switching and cell remodelling is controlled by JNK signalling. Our results reveal plasticity of segment boundaries during late morphogenesis and a role for JNK-dependent developmental reprogramming in this process.     

Changes in the placenta and in the rat embryo caused by the demethylating agent 5-azacytidine

DNA methylation is an important mechanism for regulation of gene expression during vertebrate development. 5-azacytidine is used as an experimental tool for demethylation. In this work, a single dose of 5-azacytidine (5 mg/kg body weight) was administered to rats at different stages of development. After 5-azacytidine administration on the first or third day of pregnancy, no changes were detected. After administration on the fourth day of pregnancy or later, a reduction in growth was observed. After treatment on day five and on any other day till day eleven of pregnancy, no living fetuses were found. Of those treated on day twelve, 24% of fetuses survived, but forelimb and hindlimb malformations were present. Administered on day thirteen, 5-azacytidine did not interfere with survival, but malformations were still present. From day fourteen on, 5-azacytidine caused no gross external malformations. Placentas were also influenced by 5-azacytidine. They were significantly smaller and histological evaluation showed the labyrinthine part to be severely reduced. In contrast, trophoblast giant cells were more abundant than in controls.     

GW body disassembly triggered by siRNAs independently of their silencing activity

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.