110 research outputs found
Determinants of removal and reappearance in plasma non-transferrin bound iron
No full textTransferrin is a plasma iron transport protein responsible for binding iron released from the breakdown of red cells in macrophages and delivering this iron to developing red cells in the bone marrow. In health, about one-third of the two iron binding sites on transferrin molecules are saturated with iron. In iron overload, transferrin becomes fully saturated and iron is found in the plasma in forms which are not bound to transferrin, defined as nontransferrin bound iron (NTBI). In this thesis, the factors which determine the appearance and removal of NTBI have been examined. The effects of iron chelation therapy with desferrioxamine (DFO) have been compared both in vivo and in vitro. In order to do this, a novel modification of an existing HPLC based assay system has been developed. This assay has been designed following the discovery during the work on this thesis of an in vitro 'shuttle' effect of iron between nitrilotriacetic acid (NTA) and DFO. This 'shuttle' leads to a falsely low measurement of NTBI, or a falsely fast apparent kinetic of NTBI removal, unless the free metal binding sites on DFO are blocked prior to the assay procedure. An aluminium blocking step has been developed whereby the remaining metal binding sites of DFO are blocked with an excess of aluminium prior to the NTBI assay. Using this approach, the kinetics of NTBI removal by DFO in vitro are relatively slow as is NTBI removal in vivo. NTBI removal by DFO is both concentration and time dependent but in iron overload NTBI removal is not complete at 10 μM DFO (a clinically relevant plasma concentration) even after 8 hours. Having defined the aluminium blocking method in vitro, the kinetics of NTBI removal by a variety of clinical DFO regimens has been examined. DFO levels have also been measured using a novel immunoassay system which measures the iron bound form of DFO, namely ferrioxamine (FO). The removal of NTBI with 8 hour subcutaneous infusions of DFO (standard therapy) has been compared with NTBI removal with twice daily intramuscularly DFO boluses. Surprisingly, these studies suggest that NTBI removal as quantitated by reduction in the area under the curve is as efficient with the bolus regimen as 8 hour subcutaneous infusions. The kinetics of NTBI removal have been also examined with prolonged intravenous DFO therapy and suggest that NTBI removal may not be possible with conventional intravenous therapy. In a further study, the kinetics of removal with long acting depot DFO have been compared with NTBI removal with conventional therapy. The effects of a novel polymeric form of DFO (HES- DFO) on NTBI removal have been examined which suggest that this form of DFO may result in significant quantities of loosely bound and potentially toxic forms of iron building up in plasma
Selection of drug resistant mutants from random library of <it>Plasmodium falciparum </it>dihydrofolate reductase in <it>Plasmodium berghei </it>model
No full textAbstract Background The prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development. Methods A system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected. Results The principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine. Conclusion This study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.</p
Genetic Engineering of Talaromyces marneffei to Enhance Siderophore Production and Preliminary Testing for Medical Application Potential
No full textSiderophores are compounds with low molecular weight with a high affinity and specificity for ferric iron, which is produced by bacteria and fungi. Fungal siderophores have been characterized and their feasibility for clinical applications has been investigated. Fungi may be limited in slow growth and low siderophore production; however, they have advantages of high diversity and affinity. Hence, the purpose of this study was to generate a genetically modified strain in Talaromyces marneffei that enhanced siderophore production and to identify the characteristics of siderophore to guide its medical application. SreA is a transcription factor that negatively controls iron acquisition mechanisms. Therefore, we deleted the sreA gene to enhance the siderophore production and found that the null mutant of sreA (ΔsreA) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1H- and 13C-NMR techniques and identified as a coprogen B. The compound showed a powerful iron-binding activity and could reduce labile iron pool levels in iron-loaded hepatocellular carcinoma (Huh7) cells. In addition, the coprogen B showed no toxicity to the Huh7 cells, demonstrating its potential to serve as an ideal iron chelator. Moreover, it inhibits the growth of Candida albicans and Escherichia coli in a dose-dependent manner. Thus, we have generated the siderophore-enhancing strain of T. marneffei, and the coprogen B isolated from this strain could be useful in the development of a new iron-chelating agent or other medical applications
Anti-malarial effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and green tea extract on erythrocyte-stage Plasmodium berghei in mice
No full textAbstractObjectiveTo examine the efficacy of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) iron chelator and green tea extract (GTE) as anti-malarial activity in Plasmodium berghei (P. berghei) infected mice.MethodsThe CM1 (0–100 mg/kg/day) and GTE (0–100 mg (−)-epigallocatechin 3-gallate equivalent/kg/day) were orally administered to P. berghei infected mice for consecutive 4 days. Parasitized red blood cells (PRBC) were enumerated by using Giemsa staining microscopic method.ResultsCM1 lowered percentage of PRBC in dose-dependent manner with an ED50 value of 56.91 mg/kg, when compared with pyrimethamine (PYR) (ED50 = 0.76 mg/kg). GTE treatment did not show any inhibition of the malaria parasite growth. In combined treatment, CM1 along with 0.6 mg/kg PYR significantly inhibited the growth of P. berghei in mice while GTE did not enhance the PYR anti-malarial activity.ConclusionsCM1 would be effective per se and synergize with PYR in inhibiting growth of murine malaria parasites, possibly by limiting iron supply from plasma transferrin and host PRBC cytoplasm, and chelating catalytic iron cstitutive in parasites’ mitochondrial cytochromes and cytoplasmic ribonucleotide reductase. CM1 would be a promising adjuvant to enhance PYR anti-malarial activity and minimize the drug resistance
Protection of renal function by green tea extract during Plasmodium berghei infection
No full textAbstractImpairment of renal function from oxidative stress during malaria infection is one of the leading causes of death in endemic areas. Since blood urea nitrogen and creatinine levels in plasma can be used as markers for monitoring renal damage, this study investigated the effect of green tea extract on reduction of blood urea nitrogen and creatinine levels during malaria infection using Plasmodium berghei ANKA infected mice as in vivo model. For in vivo testing, ICR mice were infected with 1×107 parasitized erythrocytes and green tea extract was subsequently administered orally twice a day for 10 consecutive days. Parasitemia was estimated by standard microscopy, and blood urea nitrogen and creatinine levels in plasma were also measured. It was found that parasitemia kept increasing until animal death, and is strongly correlated with high blood urea nitrogen and creatinine. The highest levels of blood urea nitrogen and creatinine in plasma were found on day 10 after infection. However, blood urea nitrogen and creatinine levels in plasma were reduced and decreased significantly (p<0.01) in green tea extract treated mice, compared with untreated group. It can be concluded that green tea extract can protect and maintain renal function during malaria infection, and this extract can be developed for use as a supplement and combination therapy
Quercetin-Rich Ethanolic Extract of Polygonum odoratum var Pakphai Leaves Decreased Gene Expression and Secretion of Pro-Inflammatory Mediators in Lipopolysaccharide-Induced Murine RAW264.7 Macrophages
No full textPolygonum odoratum var. Pakphai has been used in traditional Thai medicine for the treatment of flatulence and constipation and to relieve the inflammation caused by insect bites. Quercetin (Q), which is abundant in plant-based foods, has been found to exert anti-inflammatory properties. This study evaluated the anti-inflammatory activity of P. odoratum ethanolic extract in RAW264.7 macrophage cells. Leaves were extracted with 50% ethanol, phenolics and flavonoids were then analyzed using UHPLC-QTOF-MS and HPLC-DAD. RAW264.7 cells were induced with lipopolysaccharides (LPSs). They were then treated with the extract and prostaglandin E(2) (PGE(2)), and interleukin-6 (IL-6) and tumor necrotic factor-alpha (TNF-α) concentrations were determined. Levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6 and TNF-α mRNAs were analyzed using qRT-PCR. Chemical analysis demonstrated that the extract was abundant with Q while also containing catechin, gallic acid, epicatechin gallate and coumarin. The extract increased the viability of RAW264.7 cells and dose-dependently decreased nitric oxide production, PGE(2), IL-6 and TNF-α levels in the medium from the LPS-induced RAW264.7 cell culture. Consistently, COX-2, iNOS, IL-6 and TNF-α mRNA levels were decreased in a concentration-dependent manner (p < 0.05). Thus, the quercetin-rich ethanolic extract derived from P. odoratum var Pakphai leaves can exert anti-inflammatory activity in LPS-induced RAW264.7 cells through a reduction of the pro-inflammatory mediator response
Inhibitory effect of novel iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) and green tea extract on growth of Plasmodium falciparum
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