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 .

Survey of food supplementary (Pseudomonas fluorescence) and live food (Algae and brine shrimp) on Penaeus indicus larval stages

In this study ,the effects of Pseudomonas fluorescence obtained from generator pond water of Kolahi as supplementary and four algae consisting of : Chaetoceros sp, Chlorella and Skeletonema sp and Tetraselmis sp, three types of artemia as live food larval states from zoa to postlarvae (PL4 ) Penaeus indicus were investigated. The results indicate that Pseudomonas fluorescence has positive effect on Penaeus indicits larvae growth and their living food. Effective ranges at minimum and maximum were estimated. In most cases optimum dosage was approximately determined. Optimum dosage is between 50 -150 milligrams per liter for living food and Penaeus larval More than 200 milligram per liter resulted in a negative effect on the growth and survival. Also the results indicate Uromiana artemia. Requires a higher concentration of the bacteria the imported artemia. As a conclusion it is recommended to introduce Pseudonionas fluorescence as a new medium for the growth of some mentioned algae .

Measurement of liquid film thickness by optical fluorescence and its application to an oscillating piston positive displacement flowmeter

The movement of the circular piston in an oscillating piston positive displacement flowmeter is important in understanding the operation of the flowmeter, and the leakage of liquid past the piston plays a key role in the performance of the meter. The clearances between the piston and the chamber are small, typically less than 60 νm. In order to measure this film thickness a fluorescent dye was added to the water passing through the meter, which was illuminated with UV light. Visible light images were captured with a digital camera and analysed to give a measure of the film thickness with an uncertainty of less than 7%. It is known that this method lacks precision unless careful calibration is undertaken. Methods to achieve this are discussed in the paper. The grey level values for a range of film thicknesses were calibrated in situ with six dye concentrations to select the most appropriate one for the range of liquid film thickness. Data obtained for the oscillating piston flowmeter demonstrate the value of the fluorescence technique. The method is useful, inexpensive and straightforward and can be extended to other applications where measurement of liquid film thickness is required. © 2011 IOP Publishing Ltd.

Effects of gamma-aminobutyric acid, progesterone and ionophore A23187 on acrosome reaction of tree shrew sperm in vitro: examination of acrosome reaction with an improved fluorescence microscopy

A number of acrosome reaction (AR) initiators have been found to be effective in inducing AR of human, laboratory and domestic animal sperm. Using an improved simple fluorescence microscopy, effects of gamma-aminobutyric acid (GABA), progesterone and ionophore A23187 on sperm AR of tree shrew, a useful animal model in biomedical research, have been investigated. Spontaneous AR in 4.92-7.53% of viable sperm was observed. Complete AR in 10.31-18.25% of viable tree shrew sperm was obviously induced by 5 mu M and 10 mu M calcium ionophore A23187, 1 mM GABA, and 5 mu M progesterone, and there were no significant differences between their abilities to initiate complete AR. No significant differences of AR percentages between 1- and 2-h treatments with A23187, progesterone and/or GABA were observed. These results suggested that the responses of tree shrew sperm to these AR initiators are similar to that of human and other mammalian sperm. (C) 1997 Elsevier Science B.V.

Metamaterial filter for the near-visible spectrum

We demonstrate metamaterials operating in the near-visible regime based on two-dimensional arrays of gold-coated silicon nanopillars. The nanopillar arrays demonstrate a cutoff response at the metamaterial plasma frequency in accordance with theory and can be utilized for filtering applications. A plasma frequency in the near visible region of λ = 1 μm is calculated numerically for an array with a lattice constant of 300 nm and wire radius of 50 nm, with reflection measurements in agreement with numerical calculations. Such structures can be utilized for achieving negative-index based metamaterials for the visible spectrum. © 2012 American Institute of Physics.

Metamaterial filter for the near-visible spectrum

We demonstrate metamaterials operating in the near-visible regime based on two-dimensional arrays of gold-coated silicon nanopillars. The nanopillar arrays demonstrate a cutoff response at the metamaterial plasma frequency in accordance with theory and can be utilized for filtering applications. A plasma frequency in the near visible region of λ = 1 μm is calculated numerically for an array with a lattice constant of 300 nm and wire radius of 50 nm, with reflection measurements in agreement with numerical calculations. Such structures can be utilized for achieving negative-index based metamaterials for the visible spectrum.

Instability of the steady-state regime of generation in laser diodes with a broad gain spectrum

The laser-diode parameters at which the steady-state regime of generation becomes unstable are analyzed within the framework of the mode-locking model. The crucial role of the transverse inhomogeneity of the field, pumping intensity, and spectrum width in developing the instabilities of the steady-state regime of generation is demonstrated. The calculated values of the instability threshold are shown to be consistent with the experimental results. © 2008 Springer Science+Business Media, Inc.

EFFECT OF ELEVATED BICARBONATE CONCENTRATION ON GROWTH, CHLOROPHYLL A FLUORESCENCE AND ULTRA-STRUCTURE OF Microcystis aeruginosa (CYANOBACTERIUM)

The influence of bicarbonate (HCO3-) on Microcystis aeruginosa FACHB 905 was assessed in this study. Growth curves, chlorophyll a fluorescence and ultrastructure were measured at two HCO3- concentrations, 2.3 mM and 12.4 mM. A treatment of sodium chloride (NaCl) was also conducted alongside to establish the influence level of sodium. It was found that upon treatment with elevated HCO3- concentrations of 2.3 mM and 12.4 mM, cell densities were 13% and 27% (respectively) higher than controls. In photosynthetic performance, elevated HCO3- concentration initially stimulated Fv/Fm at the prophase of culture and then subsequently inhibited it. The inhibition of 2.3mM was higher than that of 12.4mM HCO3-. The maximum relative electron transport rate (ETRmax) exhibited inhibition at elevated HCO3- concentrations. DI0/CS was decreased at 2.3 mM and increased at 12.4mM. In the case of both treatments. ABS/CSI TR0/CS, ET0/CS, RC/CS0 and RC/CSm were decreased by elevated HCO3- concentrations, which indicated damage to photosynthetic apparati and an inactivation of a fraction of reaction centers. This point was also proven by ultrastructural photos. High HCO3--exposed cells lost the characteristic photosynthetic membrane arrangement compared with the control and high salinity treated samples. At the 2.3mM concentration of HCO3-. damage to photosynthetic apparati caused decreased photosynthetic activity. These findings suggested that elevated HCO3- concentration stimulated the growth and photosynthesis of M. aeruginosa FACHB 905 in a short time. Exposure to high HCO3- concentrations for a longer period of time will damage photosynthetic apparatus. In addition, the ultrastructure indicated that elevated HCO3--concentration lead to photosynthetic apparati damage. In our experiment, it was observed that the inhibition effect of 2.3mM HCO3- was higher than that of 12.4mM HCO3-. We hypothesized that M. aeruginosa FACHB 905 induced a protective mechanism under high concentrations of HCO3-.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.