Novel FMN-containing rotenone-insensitive NADH dehydrogenase from Trypanosoma brucei mitochondria: isolation and characterization

A rotenone-insensitive NADH dehydrogenase has been isolated from the mitochondria of the procyclic form of African parasite, Trypanosoma brucei. The active form of the purified enzyme appears to be a dimer consisting of two 33-kDa subunits with noncovalently bound FMN as a cofactor. Hypotonic treatment of intact mitochondria revealed that the NADH dehydrogenase is located in the inner membrane/matrix fraction facing the matrix. The treatment of mitochondria with increasing concentrations of digitonin suggested that the NADH dehydrogenase is loosely bound to the inner mitochondrial membrane. The NADH:ubiquinone reductase activity is insensitive to rotenone, flavone, or dicumarol; however, it was inhibited by diphenyl iodonium in a time- and concentration-dependent manner. Maximum inhibition by diphenyl iodonium required preincubation with NADH to reduce the flavin. More complete inhibition was obtained with the more hydrophobic electron acceptors, such as Q(1) or Q(2), as compared to the more hydrophilic ones, such as Q(0) or dichloroindophenol. Kinetic analysis of the enzyme indicated that the enzyme followed a ping-pong mechanism. The enzyme conducts a one-electron transfer and can reduce molecular oxygen forming superoxide radical.     

Lysophosphatidic acid is a bioactive mediator in ovarian cancer

Lysophosphatidic acid (LPA) is a naturally occurring phospholipid that exhibits pleiotrophic biological activities, ranging from rapid morphological changes to long-term cellular effects such as induction of gene expression and stimulation of cell proliferation and survival on a wide spectrum of cell types. LPA binds and activates distinct members of the Edg/LP subfamily of G protein-coupled receptors that link to multiple G proteins including Gi, Gq and G12/13 to elicit cellular responses. LPA plays a critical role as a general growth, survival and pro-angiogenic factor, in the regulation of physiological and pathophysiological processes in vivo and in vitro. Our previous work indicates that abnormalities in LPA metabolism and function in ovarian cancer patients may contribute to the initiation and progression of the disease. Thus, LPA could be a potential target for cancer therapy. This review summarizes evidence that implicates LPA in the pathophysiology of human ovarian cancer and likely other types of human malignancies.     

Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells

Probiotic bacteria are microorganisms that benefit the host by preventing or ameliorating disease. However, little information is known regarding the scientific rationale for using probiotics as alternative medicine. The purpose of this paper is to investigate the mechanisms of probiotic beneficial effects on intestinal cell homeostasis. We now report that one such probiotic, Lactobacillus rhamnosus GG (LGG), prevents cytokine-induced apoptosis in two different intestinal epithelial cell models. Culture of LGG with either mouse or human colon cells activates the anti-apoptotic Akt/protein kinase B. This model probiotic also inhibits activation of the pro-apoptotic p38/mitogen-activated protein kinase by tumor necrosis factor, interleukin-1alpha, or gamma-interferon. Furthermore, products recovered from LGG culture broth supernatant show concentration-dependent activation of Akt and inhibition of cytokine-induced apoptosis. These observations suggest a novel mechanism of communication between probiotic microorganisms and epithelia that increases survival of intestinal cells normally found in an environment of pro-apoptotic cytokines.     

Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are readily incorporated into phospholipids of smooth muscle cells (SMC) and endothelial cells (EC). Incorporation of EETs into intact porcine coronary arteries potentiates EC-dependent relaxation, but not vasorelaxation induced by agents that act solely on SMC. To explore the potential mechanisms responsible for this difference, porcine coronary artery SMC and EC preloaded with [3H]14,15-EET were treated with calcium ionophore A23187. Although the amount of EET incorporated into EC and SMC was similar, A23187 stimulated a five-fold increase in release of radioactivity from EC, but only a 21% increase in release from SMC. Thin layer chromatography (TLC) examination of cell lipids demonstrated that > 70% of the incorporated radioactivity was present in phosphatidylcholine (PC) in both SMC and BC. After treatment of EC PC with PLA2, TLC analysis indicated that approximately equal to 75% of radioactivity was present as free EET, and 25% of radioactivity was present as lyso-PC. Therefore, most of the 14,15-EET was esterified into the sn-2 position of PC in EC. However, in SMC, approximately equal to 70% of radioactivity was present as lyso-PC after PLA2 treatment, indicating that the EET was predominately esterified into the sn-1 position. In contrast, all of the 14,15-EET was esterified into the sn-2 position of PI in both EC and SMC. These results suggest that the preferential incorporation of 14,15-EET into the sn-1 position of PC in SMC may help to explain the greater retention of the compound in SMC, while incorporation into the sn-2 position of PC in EC may facilitate agonist-induced 14,15-EET release and potentiation of EC-dependent porcine coronary artery relaxation.     

Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes

CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.     

Spatial and temporal effects of free-air CO2 enrichment (POPFACE) on leaf growth,cell expansion,and cell production in a closed canopy of poplar

Leaf expansion in the fast-growing tree, Populus x euramericana was stimulated by elevated [CO(2)] in a closed-canopy forest plantation, exposed using a free air CO(2) enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO(2)] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0-3) and late (LPI, 6-8) stages in development. Early and late effects of elevated [CO(2)] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO(2)] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO(2)]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO(2)] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO(2)] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO(2)].     

Development of a set of expression vectors in Hansenula polymorpha

Four expression vectors based on formate dehydrogenase promoter (FMDp) and methanol oxidase promoter (MOXp) from Hansenula polymorpha were developed to express heterologous genes in Hansenula polymorpha. A secretion signal sequence of the mating factor-alpha from Saccharomyces cerevisiae was inserted in the secretory expression plasmids for efficient secretion. A modified green fluorescent protein (mGFP5) was used as the marker of expression for the first time in H. polymorpha NCYC495 (leu 1.1) to determine the expression ability of these plasmids. The mGFP5 thus expressed retained its biochemical and physiological properties, such as accumulation inside cells and efficient secretion into the culture media. These results indicated that the four integrative vectors are useful expression systems which could be directly applied for production of heterologous proteins of interests in H. polymorpha.     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)