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 .

Effect of type of otolith and preparation technique on age estimation of larval and juvenile spot (Leiostomus xanthurus)

Otoliths of larval and juvenile fish provide a record of age, size, growth, and development (Campana and Neilson, 1985; Thorrold and Hare, 2002). However, determining the time of first increment formation in otoliths (Campana, 2001) and assessing the accuracy (deviation from real age) and precision (repeatability of increment counts from the same otolith) of increment counts are prerequisites for using otoliths to study the life history of fish (Campana and Moksness, 1991). For most fish species, first increment deposition occurs either at hatching, a day after hatching, or after first feeding and yolksac absorption (Jones, 1986; Thorrold and Hare, 2002). Increment deposition before hatching also occurs (Barkmann and Beck, 1976; Radtke and Dean, 1982). If first increment deposition does not occur at hatching, the standard procedure is to add a predetermined number to increment counts to estimate fish age (Campana and Neilson, 1985).

Studies on the preparation of fish silage. 1. Effect of quality of raw material and type of acid

An experiment was undertaken in which silver bellies (Leiognathus splendens) of different quality were used to produce silages using different concentrations of hydrochloric acid and formic acid. The quality and storage life of the various preparations are reported. Silages which keep for at least 30 days can be produced from silver belly held for 3 or 12 hours at 28°C by: 1) reducing the pH by addition of hydrochloric acid; 2) adding 0.5% formic acid and reducing the pH to 3.5 with hydrochloric acid; or 3) adding 2.5% formic acid.

Incidence of ulcer type of disease in wild fishes of Bangladesh

Disease occurred in wild fish species investigated in different water bodies like canals, ditches, beel, haor, flood plain in 17 districts of Dhaka division. Haemorrhagic lesions were observed on the body surface of affected fishes. Incidence of the disease in the investigated water bodies ranged from 0 to 100%. In total 19 fish species were found to be affected and prevalence of infection ranged from 0.0 to 100.0%. Channa punctatus and Puntius ticto were severely affected in all locations. Percentage of infection in these fishes ranged from 0.0 to 100.0. The highest infection was observed in Netrokona, Kishoreganj, and Mymensingh districts. Bacterial genera isolated from the lesions of these affected fishes were Aeromonas, Pseudomonas, Flavobacterium, Micrococcus, and Staphylococcus. Among these isolates Aeromonas was the dominant. Abundance of Aeromonas in the lesions among the investigated bacteria ranged from 75 to 90%. Five identified Aero monas lrydrophila were examined for their pathogenicity and were able to infect the experimental fish, silver barb (Puntius gonionotus). The pathogen Aeromonas hydrophila was thus considered to have an association with the outbreak of ulcer type of disease in the investigated fish species.

Modification to the bilge and sea water pumps fitted in 17.5 metre Jingha type trawlers

This paper brings out the difficulties encountered with the Bilge and sea water circulating pumps which are fitted and drawn in series along with a fresh water pump in the 17.5 m fishing trawlers.

Evolution of suitable type of bottom trawls for the medium size steel trawlers of Orissa Fisheries Department

This paper deals with the results of fishing operations conducted with conventional trawls of size 22.3 - 25.6 m and gear of 32 m long wing and bulged belly designed and developed at the Central Institute of Fishery Technology, from four medium size trawlers of Orissa Fisheries Department during 1970-71 and 1971-72 fishing seasons. By employing suitable and standard size gear there was proper utilisation of the engine power with resultant increase in the total landings of shrimps and bottom and off bottom fishes.

Design of a detachable type fish drier

The paper describes the design of a detachable type fish drier which has been designed to operate on commercial scale. The drier with a raw material capacity of one tons has been designed after effecting all improvements on the design of the existing tunnel driers available in India. The cost of one drier works out to Rs. 2,16,700/- approximately.