Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors

BACKGROUND: Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS: The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS: In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS: This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.     

Dynamic organization of microtubules and microtubule-organizing centers during the sexual phase of a parasitic protozoan, Lecudina tuzetae (Gregarine, Apicomplexa)

Lecudina tuzetae is a parasitic protozoan (Gregarine, Apicomplexa) living in the intestine of a marine polychaete annelid, Nereis diversicolor. Using electron and fluorescence microscopy, we have characterized the dynamic changes in microtubule organization during the sexual phase of the life cycle. The gametocyst excreted from the host worm into seawater consists of two (one male and one female) gamonts in which cortical microtubule arrays are discernible. Each gamont undergoes multiple nuclear divisions without cytokinesis, resulting in the formation of large multinucleate haploid cells. After cellularization, approximately 1000 individual gametes are produced from each gamont within 24 h. Female gametes are spherical and contain interphase cytoplasmic microtubule arrays emanating from a gamma-tubulin-containing site. In male gametes, both interphase microtubules and a flagellum with "6 + 0" axonemal microtubules extend from the same microtubule-organizing site. At the beginning of spore formation, each zygote secretes a wall to form a sporocyst. Following meiotic and mitotic divisions, each sporocyst gives rise to eight haploid cells that ultimately differentiate into sporozoites. The ovoid shaped sporocyst is asymmetric and forms at least two distinctive microtubule arrays: spindle microtubules and microtubule bundles originating from the protruding apical end corresponding to the dehiscence pole of the sporocyst. Because antibodies raised against mammalian centrosome components, such as gamma-tubulin, pericentrin, Cep135, and mitosis-specific phosphoproteins, react strongly with the microtubule-nucleating sites of Lecudina, this protozoan is likely to share common centrosomal antigens with higher eukaryotes.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Human transformations of the Wadden Sea ecosystem through time: a synthesis

Todays Wadden Sea is a heavily human-altered ecosystem. Shaped by natural forces since its origin 7,500 years ago, humans gradually gained dominance in influencing ecosystem structure and functioning. Here, we reconstruct the timeline of human impacts and the history of ecological changes in the Wadden Sea. We then discuss the ecosystem and societal consequences of observed changes, and conclude with management implications. Human influences have intensified and multiplied over time. Large-scale habitat transformation over the last 1,000 years has eliminated diverse terrestrial, freshwater, brackish and marine habitats. Intensive exploitation of everything from oysters to whales has depleted most large predators and habitat-building species since medieval times. In the twentieth century, pollution, eutrophication, species invasions and, presumably, climate change have had marked impacts on the Wadden Sea flora and fauna. Yet habitat loss and overexploitation were the two main causes for the extinction or severe depletion of 144 species (~20% of total macrobiota). The loss of biodiversity, large predators, special habitats, filter and storage capacity, and degradation in water quality have led to a simplification and homogenisation of the food web structure and ecosystem functioning that has affected the Wadden Sea ecosystem and coastal societies alike. Recent conservation efforts have reversed some negative trends by enabling some birds and mammals to recover and by creating new economic options for society. The Wadden Sea history provides a unique long-term perspective on ecological change, new objectives for conservation, restoration and management, and an ecological baseline that allows us to envision a rich, productive and diverse Wadden Sea ecosystem and coastal society.     

Drosophila Ric-8 regulates Galphai cortical localization to promote Galphai-dependent planar orientation of the mitotic spindle during asymmetric cell division

Localization and activation of heterotrimeric G proteins have a crucial role during asymmetric cell division. The asymmetric division of the Drosophila sensory precursor cell (pl) is polarized along the antero-posterior axis by Frizzled signalling and, during this division, activation of Galphai depends on Partner of Inscuteable (Pins). We establish here that Ric-8, which belongs to a family of guanine nucleotide-exchange factors for Galphai, regulates cortical localization of the subunits Galphai and Gbeta13F. Ric-8, Galphai and Pins are not necessary for the control of the anteroposterior orientation of the mitotic spindle during pl cell division downstream of Frizzled signalling, but they are required for maintainance of the spindle within the plane of the epithelium. On the contrary, Frizzled signalling orients the spindle along the antero-posterior axis but also tilts it along the apico-basal axis. Thus, Frizzled and heterotrimeric G-protein signalling act in opposition to ensure that the spindle aligns both in the plane of the epithelium and along the tissue polarity axis.     

Improving case ascertainment of a population-based birth defects registry in New York State using hospital discharge data

BACKGROUND: The assessment of the data quality of population-based registration systems is essential to understanding the reliability and usefulness of disease surveillance and research findings resulting from the use of registry data. Since the New York State Congenital Malformations Registry (CMR) uses passive case ascertainment, the completeness of the registry data is an important aspect of the quality of information. This paper presents the results of hospital audits, which were conducted to capture the unreported cases using hospital discharge files, and evaluates the effectiveness of the audits. METHODS: Children age 2 years or younger and diagnosed with reportable birth defects for the birth years 1998-2000 were selected from hospital discharge files of all reporting hospitals in the New York Statewide Planning and Research Cooperative System (SPARCS) and matched to the CMR database for the same birth year period.The unmatched reports from the SPARCS hospital discharge files that the CMR possibly missed were sent to hospitals, requesting submission of the missed reports. Two audits on all reporting hospitals in New York State were conducted: 1) 1998 and 1999 birth cohorts audited from June 2000 to March 2002, and 2) 2000 birth cohort audited from November 2001 to November 2002. RESULTS: Hospital audits using SPARCS hospital discharge data identified 5,460 reports that the CMR missed for the selected 66 hospitals analyzed. About 86% of these reports had reportable conditions and were added to the CMR, which comprised 21.4% of all reports from the 66 hospitals for the birth years 1998-2000. The number of reports that would have been missed without audits decreased from the 1998 and 1999 birth cohort (25.1%) to the 2000 birth cohort (13.9%). Low reporting rates and, thus, a high percent of added reports, were found for hospitals with a relatively small number of annual reports and for some specific birth defects such as chromosomal anomalies, anencephalus and congenital anomalies of the urinary system. CONCLUSION: The current study demonstrates that using hospital discharge data to improve case ascertainment is a valuable and effective method of enhancing birth defect surveillance, particularly for those hospitals with low reporting rates.     

Gastroschisis, low maternal age, and fetal morbidity outcomes

OBJECTIVE: To determine if the risk for fetal growth inhibition among gastroschisis-afflicted fetuses is heightened among younger gravidas (teen mothers). METHOD: This was a retrospective cohort study on live-born infants with isolated gastroschisis delivered in New York State from 1983 through 1999. We compared infants of mature (>20 years) mothers with those of younger (<20 years) mothers with respect to the following indices of fetal morbidity outcomes: low birth weight and very low birth weight, preterm and very pre-term, and small for gestational age. We used adjusted odds ratios to approximate relative risks. RESULTS: A total of 368 infants with isolated gastroschisis were analyzed. The two groups differed in terms of mean gestational age at delivery [Mean + standard deviation(SD) for infants with gastroschisis born to mature mothers = 37.2 weeks +/- 2.8 versus 36.3 weeks + 3.6 for those of teenage mothers(p = 0.01)], as well as mean birth weight [mean birth weight +/- SD for infants with gastroschisis born to mature mothers = 2562.4 grams +548.8 versus 2367.9 grams +/- 645.2 for those of younger mothers (p = 0.004)]. Infants of teen mothers were about twice as likely to be of low birth weight (OR = 1.70; 95% CI = 1.05-2.77) and about three times as likely to be born very preterm when compared to those of mature mothers (OR = 2.80; 95% Cl = 1.02-8.00). No significant differences were observed with respect to very low birth weight, pre-term and small for gestational age. CONCLUSION: Low maternal age appears to be a risk factor for low birth weight and very preterm birth among gastroschisis-affected fetuses. This information is potentially useful for planning by care providers and in counseling affected parents.