RESUMEN
BACKGROUND: Ferroportin (Fpn), a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms. METHODS: 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. RESULTS: The expression was confirmed by appearance of fluorescence in Hek 293 T cells. 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 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids. CONCLUSION: The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies.
RESUMEN
The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.
Asunto(s)
Antígenos de Protozoos/inmunología , Leishmaniasis Cutánea/inmunología , Leucocitos Mononucleares/inmunología , Vacunas Antiprotozoos/farmacología , Células TH1/inmunología , Análisis de Varianza , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Proliferación Celular , Humanos , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-5/inmunología , Leishmaniasis Cutánea/terapia , Activación de Linfocitos , Vacunas Sintéticas/farmacologíaRESUMEN
Leishmania parasites like other kinetoplastids are unable to synthesize purines de novo and so are reliant on a salvage pathway for recycling ribonucleotides. A stage specific class one nuclease enzyme, 3'-Nucleotidase/nuclease, has been implicated in salvage of preformed purines in Leishmania insect stage promastigote via hydrolysis of 3'-nucleotides and nucleic acids. Although a similar activity is known to exist in amastigotes which reside in infected mammalian cells, the homologous gene and the corresponding protein responsible for carrying out this function have not been well characterized. Using primers specific for conserved regions of trypanosomatid class one nucleases, a gene encoding a novel class one nuclease from amastigotes of Leishmania major (LmaC1N) was cloned and sequenced. The coding sequence consists of 951 bp encoding a 316 amino acid protein with a predicted molecular mass of 35,300 Da. Analysis of the deduced amino acid sequence showed that LmaC1N is highly homologous to other class I nucleases and contains all five conserved regions reported for promastigotes 3'-Nucleotidase/nuclease. Analysis by reverse transcriptase polymerase chain reaction and Western blotting demonstrated that expression of LmaC1N gene is regulated in a stage-specific manner. Whereas the gene appeared to be silenced in promastigotes, high level expression in amastigotes implied an important function in support of parasite survival and multiplication in the mammalian cells.
Asunto(s)
Leishmania major/genética , Nucleotidasas/genética , Purinas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting/métodos , Clonación Molecular/métodos , Medios de Cultivo , Regulación de la Expresión Génica/genética , Leishmania major/enzimología , Datos de Secuencia Molecular , Nucleotidasas/metabolismo , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de SecuenciaRESUMEN
Mislabeled umbilical cord blood specimens were identified as an important and difficult problem to solve. Quality improvement principles were employed after education-based interventions failed to achieve measurable improvement. A small interdisciplinary working group of key stakeholders investigated, designed, and evaluated interventions for a solution. This article describes a system-based change where substantial qualitative and quantitative improvements were measured. The success of the change is attributed to the involvement and commitment by key stakeholders and use of systems reengineering principles.