We have investigated the interaction between monomers of the dimeric yeast cytochrome bc 1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c 1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ϳ200 mV and in a bc 1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c 1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc 1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc 1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b H hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinolcytochrome c reductase activity of the bc 1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc 1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc 1 complex dimer.The cytochrome bc 1 complex transfers electrons from ubiquinol to cytochrome c by the protonmotive Q cycle mechanism (1) in which there are two substrate-binding sites where ubiquinol is oxidized (center P) and ubiquinone is re-reduced (center N). Crystal structures of the bc 1 complexes obtained from various sources (2-4) show a dimeric structure in which the two ironsulfur protein subunits span both monomers in an intertwined arrangement. Crystal structures from the yeast bc 1 complex show ubiquinone at center N of only one monomer and cytochrome c also in only one monomer, suggesting a functional asymmetry in the dimer (5).Some center P inhibitors have been shown to completely block bc 1 complex activity upon binding to only half of the dimeric complex (6), suggesting anti-cooperative interaction between the ubiquinol oxidation sites in the dimer. Antimycin, a center N inhibitor, binds to only one center N of the dimeric enzyme in a mutant where the iron-sulfur cluster cannot be inserted into the Rieske protein, suggesting conformational interaction between centers P and N of different monomers (7). In addition, it has long been observed that titration of the cytochrome c reductase activity of the enzyme with antimycin yields non-linear curves (8, 9), which is unexpected for a tightly bound inhibitor. This anomalous behavior has been attributed to rapid mobility of the inhibitor between the two center N sites in the dimer (10), although an alternate explanation could be equilibration of electrons between the cytochro...

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b L within the cytochrome bc 1 complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc 1 complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c 1 reduction by ϳ10-fold but had no effect upon the rate of cytochrome c 1 reduction in bc 1 complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c 1 , reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b H and b L , exert negative control on the rate of reduction of cytochrome c 1 and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b H and b L to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.Although the protonmotive Q 1 cycle mechanism of the cytochrome bc 1 complex is generally understood (1-3), the mechanism of ubiquinol oxidation at center P has not been fully elucidated. With the determination of the crystal structure of the cytochrome bc 1 complex (4 -5), a more extensive examination of the structure-function relationships of the Q cycle mechanism is possible.It is generally accepted that the mechanism of ubiquinol oxidation at center P involves a divergent oxidation in which the iron-sulfur protein oxidizes ubiquinol to semiquinone and the semiquinone reduces cytochrome b L (1, 3). It is unclear, however, whether the oxidation of ubiquinol occurs through semiquinone in a sequential mechanism or whether ubiquinol is oxidized by the iron-sulfur protein and cytochrome b L in a concerted reaction. Earlier experiments suggested the presence of a transient semiquinone at center P (6), consistent with a sequential mechanism, although recent experiments suggest otherwise (7).There have been two proposals for concerted reaction mechanisms at center P. Link (8) proposed a "proton-gated affinity change" mechanism in which stabilization of ubisemiquinone by anti-ferromagnetic coupling to the reduced iron-sulfur protein raises the potential of the iron-sulfur cluster such that the cluster cannot be oxidized by cytochrome c 1 until the semiquinone is oxidized. Jü nemann...

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Ilicicolin H is an antibiotic isolated from the "imperfect" fungus Cylindrocladium iliciola strain MFC-870. Ilicicolin inhibits mitochondrial respiration by inhibiting the cytochrome bc 1 complex. In order to identify the site of ilicicolin action within the bc 1 complex we have characterized the effects of ilicicolin on the cytochrome

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6), we found that 70% of STAT6 mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6 mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6 mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear β-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11bLy6CCCR2 monocytes and CD11bLy6CLy6G granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment..

Colitis-associated colorectal cancer (CAC) is one of the most common cancers and is closely related to chronic or deregulated inflammation. Helminthic infections can modulate inflammatory responses in some diseases, but their immunomodulatory role during cancer development remains completely unknown. We have analyzed the role of Taenia crassiceps-induced anti-inflammatory response in determining the outcome of CAC. We show that extraintestinal T. crassiceps infection in CAC mice inhibited colonic inflammatory responses and tumor formation and prevented goblet cell loss. There was also increased expression of IL-4 and alternatively activated macrophages markers in colonic tissue and negative immunomodulation of pro-inflammatory cytokine expression. In addition, T. crassiceps infection prevented the upregulation of β-catenin and CXCR2 expression observed in the CAC mice, which are both markers associated with CAC-tumorigenesis, and reduced the numbers of circulating and colonic CD11b+Ly6ChiCCR2+ monocytes. Thus, immunomodulatory activities induced by helminth infections may have a role in the progression of CAC.

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CRC is associated with inflammatory responses in which intestinal pathology is caused by different cell populations including a T cell dysregulation that concludes in an imbalance between activated T (Tact) and regulatory T (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. A tumor-promoting function for Treg cells has been suggested in CRC, but the kinetics of Treg cells during CRC development are poorly known. Therefore, using a mouse model of colitis-associated colon cancer (CAC) induced by azoxymethane and dextran sodium sulfate, we observed the dynamic and differential kinetics of Treg cells in blood, spleen and mesenteric lymph nodes (MLNs) as CAC progresses, highlighting a significant reduction in Treg cells in blood and spleen during early CAC development, whereas increasing percentages of Treg cells were detected in late stages in MLNs. Interestingly, when Treg cells were decreased, Tact cells were increased and vice versa. Treg cells from late stages of CAC displayed an activated phenotype by expressing PD1, CD127 and Tim-3, suggesting an increased suppressive capacity. Suppression assays showed that T-CD4+ and T-CD8+ cells were suppressed more efficiently by MLN Treg cells from CAC animals. Finally, an antibody-mediated reduction in Treg cells during early CAC development resulted in a better prognostic value, because animals showed a reduction in tumor progression associated with an increased percentage of activated CD4+CD25+Foxp3- and CD8+CD25+ T cells in MLNs, suggesting that Treg cells suppress T cell activation at early steps during CAC development.

The complete genome sequence of Gloeobacter violaceus [Nakamura et al. (2003a, b) DNA Res 10: [37][38][39][40][41][42][43][44][45] allows us to understand better the structure of the phycobilisomes (PBS) of this cyanobacterium. Genomic analysis revealed peculiarities in these PBS: the presence of genes for two multidomain linker proteins, a core membrane linker with four repetitive sequences (REP domains), the absence of rod core linkers, two sets of phycocyanin (PC) a and b subunits, two copies of a rod PC associated linker (CpcC), and two rod cap associated linkers (CpcD). Also, there is one ferredoxin-NADP + oxidoreductase with only two domains. The PBS proteins were investigated by gel electrophoresis, amino acid sequencing and peptide mass fingerprinting (PMF). The two unique multidomain linkers contain three REP domains with high similarity and these were found to be in tandem and were separated by dissimilar Arms. One of these, with a mass of 81 kDa, is found in heavy PBS fragments rich in PC. We propose that it links six PC hexamers in two parallel rows in the rods. The other unique linker has a mass of 91 kDa and is easily released from the heavy fragments of PBS. We propose that this links the rods to the core. The presence of these multidomain linkers could explain the bundle shaped rods of the PBS. The presence of 4 REP domains in the core membrane linker protein (129 kDa) was established by PMF. This core linker may hold together 16 AP trimers of the pentacylindrical core, or alternatively, a tetracylindrical core of the PBS of G. violaceus.