Conditions for fibronectin fibril formation in the early Xenopus embryo

A longitudinal study on exposure to tobacco smoke among adolescents was carried out in Turin (North-Western Italy) in January-February 1992 and in January-February 1993. In 1992, 394 schoolchildren aged 14-16 years were enrolled in a study protocol which consisted in answering a standardized questionnaire, measurement of urinary cotinine and testing of lung function (flow-volume curve--[FVC] and forced expiratory volume in I sec.--[FEV1]). In 1993, 333 schoolchildren from the same group repeated the survey. By comparison to urinary cotinine, findings obtained showed a reduction of increase, from 1992 to 1993, of -0.57% (p = 0.082) for FVC, and -0.66% (p = 0.05) for FEV1. Assuming that the systematic selection bias did not seem to have occurred, findings, obtained from a multiple regression analysis, showed that active and passive exposure to tobacco smoke, as measured by urinary cotinine, had a significant effect on lung growth (as measured by FEV1) in adolescents; this effect, though small, was dose-related.     

The snail repressor establishes a muscle/notochord boundary in the Ciona embryo

Previous studies have identified a minimal 434 bp enhancer from the promoter region of the Ciona Brachyury gene (Ci-Bra), which is sufficient to direct a notochord-specific pattern of gene expression. Here we present evidence that a Ciona homolog of snail (Ci-sna) encodes a repressor of the Ci-Bra enhancer in the tail muscles. DNA-binding assays identified four Ci-Sna-binding sites in the Ci-Bra enhancer, and mutations in these sites cause otherwise normal Ci-Bra/lacZ transgenes to be misexpressed in ectopic tissues, particularly the tail muscles. Selective misexpression of Ci-sna using a heterologous promoter results in the repression of Ci-Bra/lacZ transgenes in the notochord. Moreover, the conversion of the Ci-Sna repressor into an activator results in the ectopic induction of Ci-Bra/lacZ transgenes in the muscles, and also causes an intermixing of notochord and muscle cells during tail morphogenesis. These results suggest that Ci-Sna functions as a boundary repressor, which subdivides the mesoderm into separate notochord and tail muscle lineages.     

Induction of blood cells in Xenopus embryo explants

A Xenopus-specific anti-leukocyte monoclonal antibody designated XL-2 was isolated and used to identify leukocytes in tailbud embryos and activin A-treated explants of blastula animal cap. XL-2 bound to a 135-kDa polypeptide in western blots of protein extracts from adult thymocytes, tailbud embryos, tadpoles, and explants. In cell suspensions, it immunostained the cell surface of all types of adult leukocytes including lymphocytes, monocyte/macrophages, thrombocytes, and granulocytes. At embryonic stage 24, immunocytochemistry revealed XL-2-positive leukocytes, the earliest time at which such cells have been recognized. Whole-mount staining of tailbud embryos and tadpoles showed a widely dispersed population of XL-2-reactive leukocytes, many of which had elongated shapes and ameboid pseudopodia. In activin A-treated animal caps, XL-2 recognized a subpopulation of cells within the lumen of the central fluid-filled cavity as well as cells in the interstitium of mesenchymal and mesothelial components of the explant. Together, activin A and human interleukin-11 induced 100% of explants to form lumenal blood cells. Compared to activin A alone, murine stem cell factor plus activin A significantly increased the numbers of XL-2-reactive leukocytes and erythrocytes. These results support the view that activin A induces leukocyte and erythrocyte progenitors during Xenopus embryogenesis.     

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos. The results indicate that Vg1 expression is essential for normal endodermal development and the induction of dorsal mesoderm in vivo.     

Ribonucleoprotein formation by the ORF1 protein of the non-LTR retrotransposon Tx1L in Xenopus oocytes

The Tx1L elements constitute a family of site-specific non-LTR retrotransposons found in the genome of the frog Xenopus laevis . The elements have two open reading frames (ORFs) with homology to proteins of retroviruses and other retroelements. This study demonstrates an expected activity of one of the element-encoded proteins. The RNA binding properties of ORF1p, the product of the first ORF of Tx1L, were examined after expression from RNA injected into Xenopus oocytes. Using sucrose gradient sedimentation and non-denaturing gel electrophoresis, we show that ORF1p associates with RNA in cytoplasmic ribonucleoprotein (RNP) particles. Discrete RNPs are formed with well-defined mobilities. The ORF1p RNPs are distinct from endogenous RNPs that contain stored oocyte mRNAs and two specific endogenous mRNAs do not become associated with ORF1p. ORF1p appears to be capable of associating with its own mRNA and with other injected RNAs, independent of specific recognition sequences. Although nuclear localization of ORF1p was anticipated, based both on the supposed mechanism of transposition and on the presence of a potential nuclear localization signal, no significant fraction of the protein was found in the oocyte nucleus. Nonetheless, the RNA binding capability of ORF1p is consistent with the proposed model for transposition of non-LTR retrotransposons.     

Direct binding of follistatin to a complex of bone-morphogenetic protein and its receptor inhibits ventral and epidermal cell fates in early Xenopus embryo

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.     

An ascidian gene encoding an SH2-domain protein is expressed in the notochord cells of the embryo

OBJECTIVE: The purpose of this study is to explore the apparent social mediation of a postpartum somatic illness, na tadoka ni vasucu, occurring among ethnic Fijian women. METHOD: During their first two postpartum days, 85 consecutive newly delivered ethnic Fijian women were recruited for a prospective study on na tadoka ni vasucu at the Sigatoka District Hospital in Nadroga, Fiji. Subjects underwent translated structured interviews and responded to the Kellner Symptom Questionnaire and to visual analog scales to assess social supports and occurrence of mood symptoms or an episode of na tadoka ni vasucu in the postpartum period. Semistructured ethnographic interviews were also conducted with subjects who reported an episode of na tadoka ni vasucu. Data were collected in the initial postpartum days and again at 2 to 5 months postpartum; 82 women completed the study. RESULTS: Na tadoka ni vasucu is a somatic syndrome occurring in 9% (N = 7) of this sample. Both quantitative and narrative data demonstrate that this syndrome is associated with perceived inferior social supports. Despite its relatively infrequent occurrence and benign clinical course, the disorder is a subject of serious social concern within the Fijian community. CONCLUSIONS: Although na tadoka ni vasucu seems to be clinically trivial, because of its cultural salience it is nonetheless able to mobilize intensive social surveillance and care for the postpartum mother. The moral concern generated by this culturally marked disorder, as well as its association with perceived inferior social supports, suggest a dialectical relationship between somatic idiom and its social context.     

Collagen II is essential for the removal of the notochord and the formation of intervertebral discs

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.     

N-acetyl-cysteine causes a late re-specification of the anteroposterior axis in the Xenopus embryo

N-acetyl cysteine is an agent which has been shown to interrupt signal transduction processes linking a wide range of stimuli to the activation of NF-kappa B in mammalian cells. We have investigated its effect on the early development of Xenopus embryos by injecting it into blastulae, using concentrations comparable to those effective on cultured cells. High concentrations at the late blastula or early gastrula stage suppress posterior and enhance anterior development, yielding embryos with enlarged cement glands and otherwise consisting of little except head in extreme cases. Reducing the amount of N-acetyl cysteine injected leads to progressively more posterior structures developing. Injection into one- or two-cell embryos gives similar phenotypes, but of reduced severity and the cement gland is not so enlarged. Explants of animal cap cells taken several hours after injection develop to give large amounts of cement gland material. We have examined the expression of a number of genes in the anteriorised embryos. Posterior markers and Xsna are reduced. Noggin and Goosecoid mRNA are up-regulated through the gastrula and persist at these levels until at least the late neurula stage, whereas in controls Noggin is much lower and Goosecoid is absent at these stages. The most anteriorised phenotype may be a consequence of this changed expression.     

Ultrastructure of the neural canal closure in the chicken embryo

We have studied the neuroectoderm of the chicken embryo, from the beginning of somitic segmentation up to the state at which it has seven somities, i.e. the period covering the passage from the 'neural canal' into the 'neural tube'. This paper was devoted mainly to the study of the ultrastructural cytodifferentiation which takes place during the stages in which the neural canal closes up, and at the level of the first area of contact between the 'neural crests'-roughly at the level of the third somite. We used eggs from a hen of the White Leghorn breed, incubated at 38 degrees C, from which we extracted chicken embryos after 24-30 h of incubation, corresponding to Hamburger-Hamilton's stages 7, 8 and 9. Thus we were able to obtain several series of embryos with three, six and seven somites. The neural canal, or tube, at the level of the third somite, was fixed in glutaraldehyde at 6.25% for 30 min and postfixed in 1% osmium tetroxide for 2 h embedded in Araldite, and the sections were then stained with lead citrate. We observed that the vacuoles in the free edge of the neural canal gradually disappear as the canal closes up, while we gradually witness the appearance of the 'closure apparatus' (or the safety or occlusion apparatus) of what is beginning to form the ependymal epithelium, and the first rudimentary outlines of the cilia. All these changes begin to be observed at the seven-somite stage, i.e. when the neural canal is beginning to close up. The 'closure apparatus' consists of a number of intercellular joint complexes, of the 'close-join't type, between which we observe a number of fine filaments, like a terminal velum', or veil, which we call 'interconnecting filaments'. In the 'raphe', whereby contact is established between the neural crests, we observe the initial stages of fusion between the vacuolated edges, with the plasmatic membrane of these cells forming very fine cytoplasmic 'tongues' which interdigitate with cells from the opposite neural crest and finally constitute the so-called close joints.     

Transcription factor GATA-1 and erythroid development

A double-blind trial was conducted in 385 patients with suspected bacterial intra-abdominal infections to compare the efficacy and safety of ampicillin-sulbactam with cefoxitin. Patients were randomized to receive either 3 g ampicillin-sulbactam (2 g ampicillin-1 g sulbactam), or 2 g cefoxitin, every 6 hours. To be evaluable, patients had to demonstrate positive culture evidence of peritoneal infection at the time of operation. A total of 197 patients were evaluable for clinical efficacy. The two treatment groups were comparable in demographic features and in the presence of risk factors for infection. Clinical success (absence of infection and of adverse drug reaction) was observed in 86% of patients in the ampicillin-sulbactam group and 78% in the cefoxitin group. Eradication of infection occurred in 88% of the ampicillin-sulbactam group and 79% of the cefoxitin group. There were no differences in the nature or frequency of side effects observed in the two groups. Ampicillin-sulbactam demonstrated no difference in safety or efficacy when compared with cefoxitin in the treatment of serious intra-abdominal infections of bacterial origin.     

Conservation of the developmental role of Brachyury in notochord formation in a urochordate, the ascidian Balocynthia roretzi

The notochord is one of the characteristic features of the phylum Chordata. The vertebrate Brachyury gene is known to be essential for the terminal differentiation of chordamesoderm into notochord. In the ascidian, which belongs to the subphylum Urochordata, differentiation of notochord cells is induced at the late phase of the 32-cell stage through cellular interaction with adjacent endoderm cells as well as neighboring notochord cells. The ascidian Brachyury gene (As-T) is expressed exclusively in the notochord-lineage blastomeres, and the timing of gene expression at the 64-cell stage precisely coincides with that of the developmental fate restriction of the blastomeres. In addition, experimental studies have demonstrated a close relationship between the inductive events and As-T expression. In the present study, we show that overexpression of As-T by microinjection of the synthesized As-T RNA results in the occurrence, without the induction, of notochord-specific features in the A-line presumptive notochord blastomeres. We also show that overexpression of As-T RNA leads to ectopic expression of notochord-specific features in non-notochord lineages, including those of spinal cord and endoderm. These results strongly suggest that the developmental role of the Brachyury is conserved throughout chordates in notochord formation.     

Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins

Metalloproteinases (MMPs) are implicated in neointima formation and hence vein graft failure. Gene transfer to elevate local levels of tissue inhibitor of metalloproteinases (TIMPs) is therefore a potential treatment. In this study, we have used lumenal application of a replication-defective recombinant adenovirus to overexpress TIMP-2 and observe the effects on neointimal thickening in a well characterised human saphenous vein organ culture model. Increased TIMP-2 expression was localised to lumenal surface cells but nevertheless increased total functional TIMP-2 secretion after 14 days culture from 4.0 +/- 2.0 to 21.8 +/- 2.9 ng/mg wet weight/day (P < 0.05, n = 3). In situ zymography revealed a marked inhibition of gelatinolytic activity by TIMP-2 gene transfer throughout the vein segments. Neointima formation and neointimal cell numbers were reduced 79% and 71%, respectively (P < 0.05; n = 8). TIMP-2 overexpression had no effect on smooth muscle cell proliferation, secretion of pro-MMP-2 or -9 and did not inhibit the processing of pro-MMP-2 to its active form. Our data indicate that TIMP-2 overexpression reduces neointimal thickening, primarily by inhibiting MMP activity and hence smooth muscle cell migration.     

The KH domain protein encoded by quaking functions as a dimer and is essential for notochord development in Xenopus embryos

Mutations in the mouse indicate that quaking gene function is essential for both embryogenesis and for development of the nervous system. Recent isolation of the mouse quaking gene identified a putative RNA-binding protein containing a single KH domain. We have previously isolated the Xenopus homolog of quaking, Xqua, and shown that the sequence is highly conserved through evolution. Here, we report experimental data on the biochemical function of the quaking protein and its role during development. We demonstrate that the quaking protein expressed during early embryogenesis, pXqua357, can bind RNA in vitro, and we have mapped the regions of the protein that are essential for RNA binding. We present evidence that pXqua can form homodimers and that dimerization may be required for RNA binding. Oocyte injection experiments show that pXqua357 is located in both the nucleus and cytoplasm. In the Xenopus embryo, Xqua is first expressed during gastrulation in the organizer region and its derivative, the notochord. In later stage embryos, Xqua is expressed in a number of mesodermal and neural tissues. We demonstrate that disruption of normal Xqua function, by overexpression of a dominant inhibitory form of the protein, blocks notochord differentiation. Xqua function appears to be required for the accumulation of important mRNAs such as Xnot, Xbra, and gsc. These results indicate an essential role for the quaking RNA-binding protein during early vertebrate embryogenesis.     

Nitric oxide inhibits tissue factor synthesis, expression and activity in human monocytes by prior formation of peroxynitrite

This article discusses 3 different strategies for dealing with the harmful consequences of drug use and other risky behaviors: We can discourage people from engaging in the behavior (prevalence reduction), we can encourage people to reduce the frequency or extent of the behavior (quantity reduction), or we can try to reduce the harmful consequences of the behavior when it occurs (harm reduction). These strategies are not mutually exclusive; this article offers a framework for integrating them. The framework is useful for examining frequent claims that harm reduction "sends the wrong message." Opposition to harm reduction is based in part on a recognition of potential trade-offs among the strategies, but it is also fueled by several more symbolic psychological factors. Strategies for successfully integrating prevalence reduction, quantity reduction, and harm reduction are explored.     

Spinach thioredoxin m inhibits DNA synthesis in fertilized Xenopus eggs

A role for thioredoxin in metazoan DNA synthesis has been assessed by injecting rapidly dividing Xenopus eggs with purified heterologous thioredoxins, which might act as inhibitors if they were to replace resident thioredoxins in some but not all reaction steps. Of 10 tested proteins, spinach chloroplast thioredoxin m is the most potent inhibitor. Eggs cleave and produce cells lacking nuclei. DNA synthesis is severely reduced. Development arrests before gastrulation. In egg extracts, thioredoxin m inhibits incorporation of radioactive dCTP into DNA of sperm nuclei and M13 phage. Inhibition exceeds 90% when thioredoxin m and M13 DNA are preincubated together. The data support the interpretation that thioredoxins normally participate in initiation of metazoan DNA synthesis.