Mechanism of Iron Transport in Mycelia Sterilia EP-76

The cyclic trihydroxamic acid, N, N\u27, N\u27 \u27-triacetylfusarinine C, produced by Mycelia sterilia EP-76, is shown to be a ferric ionophore for this organism. The association constant for ferric-N, N\u27, N\u27 \u27-triacetylfusarinine C complex was determined to be log K=32.5. Other iron chelating agents, such as rhodotorulic acid, citric acid, or the monomeric subunit of triacetylfusarinine C, N-acetyl- fusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating agents that transport iron into the cell. Ferrichrome, but not ferrichrome A, was also capable of delivering iron and gallium to this organism, but not by an exchange mechanism. Unlike triacetylfusarinine C, the 14C-ligand of ferrichrome was retained by the cell. A mid-point potential of -690 mV versus the saturated silver chloride electrode was obtained for the ferric-N, N\u27, N\u27 \u27-triacetylfusarinine C complex, indicating that an unfavorable reduction potential was not the reason for utilizing a hydrolytic mechanism of intracellular iron release from the ferric triacetylfusarinine C chelate. The iron transport system recognizes only the -cis coordination isomer of ferric-N, N’, N\u27 \u27-triacetylfusarinine C metal ligand complex even though the -cis configuration predominates in solution. Ferrichrome and ferric-N, N\u27, N\u27 \u27-triacetylfusarinine C are both transported by the same receptor

A comparative study of chitin synthase activity in two Mortierella species

An in vitro investigation of some important factors controlling the activity of chitin synthase in cell-free extracts of two Mortierella species has been carried out. Mixed membrane fractions from mycelial homogenates of Mortierella candelabrum and Mortierella pusilla were found to catalyse the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into an insoluble product characterized as chitin by its insolubility in weak acid and alkali, and the release of glucosamine and diacetylchitobiose on hydrolysis with a strong acid and chitinase, respectively. Apparent Km values for UDP-GlcNAc were 1.8 mM and 2.0 mM for M. pusilla and ~ candelabrum, respectively. Polyoxin D was found to be a very potent competitive inhibitor with values of the constant of inhibition, Ki' for both species about three orders of magnitude lower than theKm for UDP-GlcNAc. A divalent cation, Mg+2 , Mn+2 or Co+2 , was required for activity. N-acetylglucosamine, the monomer of chitin, stimulated the activity of the enzyme. The crude enzyme preparation of ~ candelabrum, unlike that of ~ pusilla, showed an absolute requirement for both Mg+2 and N-acetylglucosamine. Large differences in response to exogenous proteases were noted in the ratio of active to inactive chitin synthase of the two species. A fifteen fold or greater increase was obtained after treatment with acid protease (from Aspergillussaitoi) as compared to a two- to four-fold activation of the M. pusilla membrane preparation treated similarly. During storage at 4°C over 48 hours, an endogenous activation of chitin synthase of ~ pus ilIa was achieved, comparable to that obtained by exogenous protease treatment. The high speed supernatant of both species inhibited the chitin synthase activity of the mixed membrane fractions. The inhibitor of ~ pus ilIa was effective against the pre-activated enzyme whereas that of M. candelabrum inhibited the activated enzyme. Several possibilities are discussed as to the role of the different factors regulating the enzyme activity. The suggestion is made from the properties of chitin synthase in the two species that in vivo a delicate balance exists between the activation and inactivation of the enzyme which is responsible for the pattern of wall growth of each fungus

Stereochemical aspects of iron transport in Mycelia sterilia EP-76.

Chromic complexes of N,N',N''-triacetylfusarinine C have been prepared and examined for biological activity in Mycelia sterilia EP-76. The iron transport system of this organism recognizes only the lambda coordination isomer of Cr(III)-triacetylfusarinine C even though the delta configuration predominates in solution. Chromium is excreted into the medium following triacetylfusarinine C-mediated uptake of the metal. Hydrogenation of the double bonds conjugated to the hydroxamic acid functions of triacetylfusarinine C yields four chromatographically distinct ferric complexes. Two of the complexes have the lambda configuration, while the other two have the opposite (delta) configuration. The complexes differ in effectiveness as siderophores in M. sterilia EP-76, the lambda isomers being most active

Antioxidant and free radical scavenging activity of iron chelators

Inside the human body, reactive derivatives of oxygen, known as reactive oxygen species (ROS) such as the superoxide radical (O2·−), hydroxyl radical (·−OH) and hydrogen peroxide (H2O2), are constantly generated. The ROS easily cause oxidative damage to various biomolecules such as proteins, lipids and DNA leading to various disease conditions. Iron chelators function as antioxidants by scavenging ROS and also reduce the amount of available iron thereby decreasing the quantity of ·−OH generated by Fenton reactions. In this study, the antioxidant activity of the iron chelators: caffeic acid (CA), 2,3-dihydroxybenzoic acid (DHBA), desferroxamine B (FOB) and benzohydroxamic acid (BHA) were determined using five different in vitro antioxidant assays. The antioxidant assays used were: iron binding ability, reducing ability using the potassium ferricyanide reduction method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, H2O2 scavenging activity and ·−OH scavenging activity. The standard used for the iron binding ability was Na2EDTA whereas vitamin C was used as a standard for the remaining assays. The iron chelators showed a concentration dependent increase in their radical scavenging activities as well as their reducing ability. At the concentration of 1 mM, FOB had the highest iron binding ability of 93.7% whereas DHBA had the lowest iron binding ability of 5.0% compared to the standard Na2EDTA which had 94.8%. The iron chelators, with the exception of BHA, showed good reducing ability than vitamin C. Caffeic acid showed significant DPPH, hydrogen peroxide and hydroxyl radical scavenging activities of 84.7%, 99.8% and 14.5%, respectively. All the iron chelators were observed to show significant activities in all five antioxidant assays

Is there any relationship between haptoglobin phenotypes and retinopathy among Ghanaian diabetics?

This study was conducted to examine the possible association between Haptoglobin (Hp) phenotypes and diabetes retinopathy in some Ghanaians to determine whether a specific Hp phenotype predisposes diabetics to retinopathy. A total of 110 diabetics were enrolled into the study. Blood samples were taken from each participant and analyzed for Hp phenotypes and selected biochemical indices – fasting blood glucose, blood pressure, AST and ALT. The results indicated that most diabetic retinopathics were of the Hp 2-2 phenotype, whereas most of diabetics without retinopathy were of the Hp 1-1, Hp 2-1 or Hp 2-1M phenotypes. No diabetic with retinopathy had Hp 2-1M phenotype. The odds ratio of having retinopathy in a diabetic with an Hp 2-2 phenotype was 3.42 times greater than Hp 0-0. Contrary to what has been reported elsewhere, this study suggests that, among Ghanaians, the Hp 2-2 phenotype may be a major risk factor for the development of diabetic retinopathy, whereas Hp 1-1 and Hp 2-1M appear protective

Impact of Successful Weight Loss Maintenance on Serum Lipids and Glucose Concentrations of Previous Participants of a Weight Loss Programme in Accra, Ghana

Background and Aim. There is a need to investigate the long-term impact of successful weight loss maintenance on blood lipids and glucose concentrations in populations within Africa, where obesity and cardiovascular disease (CVD) rates are increasingly becoming a public health threat. The aim of this study was to compare the serum lipid and glucose concentrations of successful and unsuccessful weight loss maintainers who previously participated in the Nutriline Weight Loss Programme (NWLP) in Accra, Ghana. Methods. 112 participants were randomly selected to participate in this cross-sectional study. Baseline and end of weight loss programme anthropometric and programmatic data were accessed via the NWLP archival database. On follow-up, anthropometric data, physical activity, dietary behaviour, serum lipid, and glucose indices were taken. Successful weight loss maintainers (SWLM) were defined as those achieving at least 5% weight loss below the baseline weight at follow-up, otherwise unsuccessful (UWLM). Results. The adjusted serum total cholesterol (TC) concentration was significantly lower for SWLM (5.17 ± 0.99 mmol/L) compared to UWLM (5.59 ± 1.06 mmol/L). Serum low-density lipoprotein (LDL), high-density lipoprotein (HDL), fasting blood glucose (FBG), and glycosylated haemoglobin (HbA1c) concentrations for SWLM versus UWLM did not differ significantly and were as follows: 3.58 ± 0.92 mmol/L versus 3.87 ± 0.99 mmol/L, 1.22 ± 0.38 mmol/L versus 1.17 ± 0.32 mmol/L, 4.48 ± 0.72 mmol/L versus 4.73 ± 1.00 mmol/L, and 5.52 ± 0.39% versus 5.59 ± 0.59%, respectively. Triglyceride (TG) concentration was significantly (P<0.001) lower for SWLM (0.79 ± 0.28 mmol/L) compared to UWLM (1.17 ± 0.51 mmol/L). After adjusting for covariates, it was no longer significant. Additionally, there was no significant association between weight loss maintenance success and having a normal status for selected lipids and glucose parameters. Conclusion. SWLM had a significantly lower serum TC compared to UWLM. In addition, a greater proportion of SWLM had normal values for TC, TG, HbA1c, and LDL out of the six parameters measured although not statistically significant

In vitro activity and mode of action of phenolic compounds on Leishmania donovani.

BACKGROUND:Leishmaniasis is a disease caused by the protozoan parasite, Leishmania. The disease remains a global threat to public health requiring effective chemotherapy for control and treatment. In this study, the effect of some selected phenolic compounds on Leishmania donovani was investigated. The compounds were screened for their anti-leishmanial activities against promastigote and intracellular amastigote forms of Leishmania donovani. METHODOLOGY/PRINCIPAL FINDINGS:The dose dependent effect and cytotoxicity of the compounds were determined by the MTT assay. Flow cytometry was used to determine the effect of the compounds on the cell cycle. Parasite morphological analysis was done by microscopy and growth kinetic studies were conducted by culturing cells and counting at 24 hours intervals over 120 hours. The cellular levels of iron in promastigotes treated with compounds was determined by atomic absorption spectroscopy and the effect of compounds on the expression of iron dependent enzymes was investigated using RT-qPCR. The IC50 of the compounds ranged from 16.34 μM to 198 μM compared to amphotericin B and deferoxamine controls. Rosmarinic acid and apigenin were the most effective against the promastigote and the intracellular amastigote forms. Selectivity indexes (SI) of rosmarinic acid and apigenin were 15.03 and 10.45 respectively for promastigotes while the SI of 12.70 and 5.21 respectively was obtained for intracellular amastigotes. Morphologically, 70% of rosmarinic acid treated promastigotes showed rounded morphology similar to the deferoxamine control. About 30% of cells treated with apigenin showed distorted cell membrane. Rosmarinic acid and apigenin induced cell arrest in the G0/G1 phase in promastigotes. Elevated intracellular iron levels were observed in promastigotes when parasites were treated with rosmarinic acid and this correlated with the level of expression of iron dependent genes. CONCLUSIONS/SIGNIFICANCE:The data suggests that rosmarinic acid exerts its anti-leishmanial effect via iron chelation resulting in variable morphological changes and cell cycle arrest

In vitro anti-trypanosomal effects of selected phenolic acids on Trypanosoma brucei.

African trypanosomiasis remains a lethal disease to both humans and livestock. The disease persists due to limited drug availability, toxicity and drug resistance, hence the need for a better understanding of the parasite's biology and provision of alternative forms of therapy. In this study, the in vitro effects of phenolic acids were assessed for their trypanocidal activities against Trypanosoma brucei brucei. The effect of the phenolic acids on Trypanosoma brucei brucei was determined by the alamarBlue assay. The cell cycle effects were determined by flow cytometry and parasite morphological analysis was done by microscopy. Effect on cell proliferation was determined by growth kinetic analysis. Reverse Transcriptase quantitative Polymerase Chain Reaction was used to determine expression of iron dependent enzymes and iron distribution determined by atomic absorption spectroscopy. Gallic acid gave an IC50 of 14.2±1.5 μM. Deferoxamine, gallic acid and diminazene aceturate showed a dose dependent effect on the cell viability and the mitochondrion membrane integrity. Gallic acid, deferoxamine and diminazene aceturate caused loss of kinetoplast in 22%, 26% and 82% of trypanosomes respectively and less than 10% increase in the number of trypanosomes in S phase was observed. Gallic acid caused a 0.6 fold decrease, 50 fold increase and 7 fold increase in the expression levels of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes respectively while treatment with deferoxamine and diminazene aceturate also showed differential expressions of the transferrin receptor, ribonucleotide reductase and cyclin 2 genes. The data suggests that gallic acid possibly exerts its effect on T. brucei via iron chelation leading to structural and morphological changes and arrest of the cell cycle. These together provide information on the cell biology of the parasite under iron starved conditions and provide leads into alternative therapeutic approaches in the treatment of African trypanosomiasis