Reduction of peroxides and dinitrobenzenes by Mycobacterium tuberculosis thioredoxin and thioredoxin reductase

The thioredoxin (Trx) and thioredoxin reductase (TR) of Mycobacterium tuberculosis have been expressed in Escherichia coli and shown to reduce peroxides and dinitrobenzenes. The reduction of H2O2 requires both Trx and TR and is more efficient under anaerobic than aerobic conditions. In contrast, cumene hydroperoxide is reduced to cumyl alcohol and acetophenone in a process that requires NADPH and TR but not Trx. Cumene hydroperoxide reduction is partially inhibited by chelation of trace metals in the medium. The reduction of cumene hydroperoxide by TR is more effective under anaerobic than aerobic conditions due to a competing oxidase reaction in which electrons are transferred from TR to O2. Under anaerobic conditions, dinitrobenzenes also serve as electron acceptors and are reduced by TR to nitroanilines, but the enzyme does not reduce mononitrobenzenes or mononitroimidazoles such as metronidazole. The reductive activity of the Trx-TR system may modify the antioxidant defenses of M. tuberculosis.     

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Retinoid-related molecules require caspase 9 for the effective release of Smac and the rapid induction of apoptosis

Certain retinoid-related molecules (RRMs) with agonist or antagonist activities have been described to induce apoptosis in a variety of cancer cell lines and show promise for the treatment of cancer. Similar to other chemotherapeutic drugs, these retinoid analogs have been suggested to induce apoptosis through the intrinsic pathway, which requires the release of cytochrome c from the mitochondria for the effective activation of caspase 9. Expression of a catalytically inactive form of caspase 9, which functions as a dominant negative mutant, inhibits the induction of DEVDase activity and nuclear fragmentation by selective RRMs. Whereas the RRMs could induce the release of cytochrome c in the absence of caspase 9 activity, the later is necessary for the effective release of Smac/Diablo from the mitochondria. Furthermore, overexpression of Bcl-2 or Bcl-X(L) also inhibits RRM-induced apoptosis. We demonstrate that activation of caspase 2 by the agonist MX2870-1 requires caspase 9 activity and is inhibited by Bcl-2 overexpression. In contrast, the antagonist MX781 induces cleavage of procaspase 2 upstream of mitochondria and independently of caspase 9. Thus, two retinoid analogs with unique characteristics activate two distinct apical caspases (2 or 9) to initiate apoptosis. In addition to caspase-mediated cell death, sustained exposure to the RRMs can also lead to loss of cell viability in cells lacking caspase 9 activity or in cells stimulated in the presence of the caspase inhibitor Z-VAD-fmk. Moreover, MX2870-1 and MX781 produce cell cycle arrest independently of caspase activity and the retinoid receptors.     

Chromosomal localization of telomeric sequences in three species of Akodon (Rodentia, Sigmodontinae)

The distribution of the vertebrate telomeric sequence T2AG3 in three species of the rodent genus Akodon was examined by FISH with a peptide nucleic acid probe. In addition to the expected telomeric hybridization, non-telomeric signals were observed in the three species. In A. dolores, centromeric signals were visible in two of the four biarmed autosome pairs featuring Robertsonian polymorphism, indicating the retention of at least part of the telomeric sequences during the fusion process, and an interstitial signal of lower intensity was observed in the short arm of another. In A. boliviensis, a strong signal was observed near the centromeric end of the first chromosome pair. The first pair of A. azarae (homologous to the first pair of A. boliviensis) showed a similar but markedly amplified signal, and a subcentromeric signal in the X chromosome corresponding to a heterochromatic region; additionally, interstitial signals of lower intensity were present in one to four chromosomes in the majority of cells examined.     

Genomic Structure and Functional Analysis of Promoter Region of Somatolactin Gene of Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone–prolactin family and is produced in the intermediate lobe of teleosts. The SL gene was isolated from a sea bream genomic library and found to be composed of 5 exons distributed within a 9-kb length of DNA. Sequence analysis of the proximal promoter region showed the presence of a classical TATA box located 59 bp upstream from the initial start ATG codon, 5 consensus sequences corresponding to the Pit-1 binding element, and a putative CREB site. In CHO cells cotransfected with the DNA from 2 plasmids, one encoding sea bream Pit-1 under Rous sarcoma virus long terminal repeat regulation and one encoding the SL promoter driving the expression of luciferase, Pit-1 was found to enhance the expression of luciferase. Only one Pit-1 binding site was necessary for enhancement. Analysis by immunoblots of in vitro culture of pituitaries of Sparus aurata showed that several agents, including estradiol, verapamil, and phorbol myristate acetate, had different inhibitory effects on SL and growth hormone released to the culture medium.     

Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats

Background  

In the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy.     

Crystal structures of epothilone D-bound,epothilone B-bound,and substrate-free forms of cytochrome P450epoK

Epothilones are potential anticancer drugs that stabilize microtubules by binding to tubulin in a manner similar to paclitaxel. Cytochrome P450epoK (P450epoK), a heme containing monooxygenase involved in epothilone biosynthesis in the myxobacterium Sorangium cellulosum, catalyzes the epoxidation of epothilones C and D into epothilones A and B, respectively. The 2.10-, 1.93-, and 2.65-A crystal structures reported here for the epothilone D-bound, epothilone B-bound, and substrate-free forms, respectively, are the first crystal structures of an epothilone-binding protein. Although the substrate for P450epoK is the largest of a P450 whose x-ray structure is known, the structural changes along with substrate binding or product release are very minor and the overall fold is similar to other P450s. The epothilones are positioned with the macrolide ring roughly perpendicular to the heme plane and I helix, and the thiazole moiety provides key interactions that very likely are critical in determining substrate specificity. Interestingly, there are strong parallels between the epothilone/P450epoK and paclitaxel/tubulin interactions. Based on structural similarities, a plausible epothilone tubulin-binding mode is proposed.     

Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity

Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.