Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b(559) from higher plants.

A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and ß cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ßß) homodimers and (αß) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the ß subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ßß) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

Enzymatic activities of isolated cytochrome bc1-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Homodimeric structure of cytochrome bc1, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc1-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc1 with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc1 with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives.

White beans provide more bioavailable iron than red beans: studies in poultry (Gallus gallus) and an in vitro digestion/Caco-2 model.

Iron-biofortification of crops is a strategy that alleviates iron deficiency. The common bean (Phaseolus vulgaris L.) is an attractive candidate for biofortification. However, beans are high in polyphenols that may inhibit iron absorption. In vitro studies have shown that iron bioavailability from white beans is higher than that from colored beans. In this study, our objective was to determine if white beans contain more bioavailable iron than red beans and to determine if the in vitro observations of bean-iron bioavailability would be evident in an in vivo feeding trial. We compared iron bioavailability between diets containing either white (Matterhorn) or red (Merlot) beans, which differ in polyphenol content. One-week-old chicks (Gallus gallus) were divided into four groups: 1. "WB": 40% white-bean diet; 2. "RB" :40% red-bean diet; 3. "WB+Fe": 40% white-bean diet; 4. "RB+Fe": 40% red-bean diet (51, 47, 179, and 175 ppm iron, respectively). Diets 1 and 2 had no supplemental iron; whereas 125 µg/g iron was added to diets 3 and 4. For 8 weeks, hemoglobin, feed consumption, and body weights were measured. Divalent metal transporter 1 (iron-uptake-transporter), duodenal-cytochrome-B (iron reductase), and ferroportin (iron-exporter) expressions were higher (p<0.05), villus-surface-area (tissue iron-deficiency adaptation) was greater in the "RB" group vs. other groups. Cecal microflora was similar between treatments. Hemoglobin, body-hemoglobin iron, and body weights were lower in the "RB" group vs. other groups (p<0.05). In vitro analysis showed lower ferritin formation (less bioavailable iron) in cells exposed to the "RB" diet. We conclude that the in vivo results support the in vitro observations; i. e., white beans contain more bioavailable iron than red beans.