Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

Tidal oscillations at the head of Monterey Submarine Canyon and their relation to oceanographic sampling and the circulation of water in Monterey Bay. Annual report, Part 6, September 1972

During a 25-hour hydrographic times series at two stations near the head of Monterey Submarine Canyon, an internal tide was observed with an amplitude of 80 to 115 m in water depths of 120 and 220 m respectively. These large oscillations produced daily variations in hydrographic and chemical parameters that were of the same magnitude as seasonal variations in Monterey Bay. Computed velocities associated with the internal tide were on the order of 10 em/sec, and this tidally induced circulation may have a significant role in the exchange of deep water between Monterey Submarine Canyon and the open ocean. (PDF contains 49 pages)

Proposal to permit experimental artificial reefs inside and outside the Big Sycamore Canyon Ecological Reserve

(Document pdf contains 19 pages)

Modelling of the Subarctic North Pacific Circulation (Report of Working Group 7)

This review focuses on modelling ocean circulation and its variability in the subarctic North Pacific; it addresses issues specific to that region, and not the subject of ocean modelling in general. The performance of existing models is assessed in relation to observations in the upper ocean, intermediate waters and deep/abyssal waters. (PDF contains 87 pages)

Proposal for improving fishing opportunities off Pt. Loma by construction of an artificial reef

(Document pdf contains 42 pages)

Report of Pendleton artificial reef studies with recommendations for constructing a kelp reef

(Document pdf contains 64 pages)

Beach surveys of South Carlsbad State Beach onshore of Carlsbad artificial reef

(Document pdf contains 9 pages)

A report of biological observations at Topanga artificial reef and Santa Monica Bay artificial reef

(Document pdf contains 25 pages)

Biological surveys of five southern artificial reefs: Oceanside #1, Oceanside #2, Carlsbad, Pacific Beach, and Mission Bay

(Document pdf contains 19 pages)

Observations of the biological communities at Bolsa Chica artificial reef

Bolsa Chica Artificial Reef (BCAR) was constructed in November 1986 with 10,400 tons of concrete rubble and eight concrete and steel barges. Prior to any additional augmentation of BCAR, the u.s. Army Corps of Engineers and the California Coastal Commission required the California Department of Fish and Game (CDFG) to survey the bioloqical communities on and around BCAR. In April 1992, qualitative surveys of the biological communities were conducted on one of the eight modules at BCAR and at a nearby sand-only site. One of the modules, Module D, located in 90 feet of water (MLLW), was surveyed for fish, macroinvertebrates, and turf community organisms (small plants and sessile animals). Twelve species of fish were observed, including kelp bass (Paralabrax clathratus) and barred sand bass (P. nebulifer). Eight macroinvertebrate species were observed, rock scallops (Crassedoma giganteum) being the most abundant. The turf community was comprised of thirteen invertebrate taxa, among which erect ectoprocts (Bugula spp.) were the most numerous. Two species of foliose red algae (Rhodymenia pacifica and Anisocladella pacifica) were also observed. The reef has reached an advanced stage of successional development with fish and invertebrate communities diverse and well established. However, due,.to its depth and the turbidity of surrounding waters, this reef is not likely to ever support a diverse algal community. The diversity and abundance of fish and macroinvertebrates were, as to be expected, much lower in the nearby sand-only site. Only two species of fish and seven macroinvertebrate species were observed. Of these, only the sea pen, Stylatula elongata, was common. Overall, when compared to nearby sand-only habitats, Bolsa Chica Artificial Reef appears to contribute substantially to the local biological productivity. In addition, the concrete rubble used in BCAR' s construction appears to be performing as well as the quarry rock used in all of CDFG's experimental reefs. (Document pdf contains 22 pages)

A report of biological observations at Pacific Beach artificial reef, Oceanside artificial reef, and Santa Monica Bay artificial reef

(Document pdf contains 16 pages)

A report of biological observations at Oceanside #1 and #2 artificial reefs, Carlsbad artificial reef, Pacific Beach artificial reef, and Mission Bay Park artificial reef

(Document pdf contains 22 pages)

The ontogeny of haematopoiesis in the marine teleost Leiostomus xanthurus and a comparison of the site of initial haematopoiesis with Opsanus tau

The ontogeny of haematopoiesis in the perciform fish, spot Leiostomus xanthurus, differed from that reported as the norm for fishes, as exemplified by the cypriniform zebrafish Danio rerio, and observed in the batrachoidiform oyster toadfish Opsanus tau. Erythropoiesis in spot was first evident in the head kidney of yolk-sac larvae 3 days after hatching (DAH). No embryonic intermediate cell mass (ICM) of primitive stem cells or blood islands on the yolk were apparent within embryos. Erythrocytes were first evident in circulation near the completion of yolk absorption, c. 5 DAH, when larvae were c. 20 mm notochord length (LN). Erythrocyte abundance increased rapidly with larval development for c. 14 to 16 DAH, then became highly variable following changes in cardiac chamber morphology and volume. Erythrocytic haemoglobin (Hb) was not detected within whole larvae until they were 12 DAH or c. 31 mm LN, well after yolk and oil-globule absorption. The Hb was not quantified until larvae were >47 DAH or >7 mm standard length. The delayed appearance of erythrocytes and Hb in spot was similar to that reported for other marine fishes with small embryos and larvae. In oyster toadfish, a marine teleost that exhibits large embryos and larvae, the ICM and Hb were first evident in two bilateral slips of erythropoietic tissue in the embryos, c. 5 days after fertilization. Soon thereafter, erythrocytes were evident in the heart, and peripheral and vitelline circulation. Initial haematopoiesis in oyster toadfish conformed with that described for zebrafish. While the genes that code for the development of haematopoiesis are conserved among vertebrates, gene expression lacks phylogenetic pattern among fishes and appears to conform more closely with phenotypic expression related to physiological and ecological influences of overall body size and environmental oxygen availability.

Pendleton Artificial Reef: annual progress report presented to Southern California Edison Company

(PDF contains 82 pages.)

Blood lactate in yellowfin tuna, Neothunnus macropterus, and skipjack, Katsuwonus pelamis, following capture and tagging

ENGLISH: The staff of the Inter-American Tropical Tuna Commission for several years has been investigating the life history, population structure, behavior and ecology of the yellowfin tuna, Neothunnus macropterus, and the skipjack, Katsuwonus pelamis, in the Eastern Tropical Pacific Ocean. The tagging and subsequent recovery of these tropical tunas, to provide information on population structure, migrations, mortality rates and growth rates, are important aspects of these investigations. Broadhead (1959) and Schaefer, Chatwin and Broadhead (1961) emphasize the many difficulties involved in tagging these extremely active yet delicate fish and give considerable evidence to suggest that tagging mortality is high, perhaps as great as 60 to 80 per cent. The latter authors suggest that the rather high mortality at tagging is related to the effects of hyperactivity brought about by the tagging operation. SPANISH: El personal de la Comisión Interamericana del Atún Tropical ha estado investigando durante varios años la historia natural, la estructura de la población, los hábitos y la ecología del atún aleta amarilla, Neothunnus macropterus, y del barrilete, Katsuwonus pelamis, en el Océano Pacífico Oriental Tropical. La marcación y el subsiguiente recobro de estos atunes tropicales, lo que da información sobre la estructura de la población, los movimientos migratorios y las tasas de crecimiento y de mortalidad, son importantes aspectos de estas investigaciones. Broadhead (1959) y Schaefer, Chatwin y Broadhead (1961) destacan las muchas dificultades que hay para marcar estos peces activos en extremo pero delicados, y proporcionan considerable evidencia que sugiere que la mortalidad por la marcación es bastante alta, siendo quizás de 60 a 80 por ciento. Los autores citados sugieren que esta elevada mortalidad por la marcación está relacionada con los efectos de la hiperactividad producida por la operación de marcación.