Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells

The microsomal enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and the low density lipoprotein (LDL) receptor pathway carry out a key role on cholesterol homeostasis in eucaryotic cells. The HMG-CoA reductase is sensitive to oxidative inactivation and to phosphorylation by many kinases that are able to inactivate the protein and increase its susceptibility to proteolysis. We previously demonstrated that a calf thymus Cu,Zn SOD affects cholesterol metabolism. This protein binds with rat hepatocyte cell membrane by a specific surface membrane receptor. The involvement of Cu,Zn SOD in cholesterol metabolism is confirmed further by the presence of this antioxidant enzyme in circulating serum lipoproteins. We studied the effect of native human Cu,Zn SOD, metal-free SOD (apo SOD), and SOD-inactivated with hydrogen peroxide on cholesterol metabolism in human hepatocarcinoma HepG2 cells. Results showed that all forms of SODs used, at the concentration of 150 ng/ml, are able to affect cholesterol metabolism decreasing both HMG-CoA reductase activity and its protein levels; this inhibitory effect is accompanied by reduced cholesterol synthesis measured as [14C]acetate incorporation into [14C]cholesterol and by an increased [125I]LDL binding to HepG2 cells. Furthermore, the inhibitory effect of Cu,Zn SOD on cholesterol synthesis was completely abolished when the cells were incubated with Cu,Zn SOD in the presence of bisindoilmaleimide (BDM), an inhibitor of protein kinase C (PKC); moreover, we demonstrated that Cu,Zn SOD as well as apo SOD was able to increase PKC activity. Overall, data demonstrate that Cu,Zn SOD affects cholesterol metabolism independently from its dismutase activity and its metal content and that the inhibitory action on cholesterol synthesis is mediated by an activation of protein kinase C.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

5'-Nucleotidases: specific assays for five different enzymes in cell extracts

Several mammalian 5'-nucleotidases (5-NTs), attached to membranes or present in the cytosol or in mitochondria, remove the phosphate from ribo- and deoxyribonucleotides with different specificities for the sugar and base moieties. Some enzymes probably participate in signaling functions by producing adenosine from AMP. A more general function may be to prevent overproduction of deoxyribonucleotides. 5-NTs may affect the pharmacological activity of nucleoside analogs and also be involved in their mitochondrial toxicity. Here we describe for five cloned 5-NT specific assays that largely rely on new inhibitors for some of the enzymes. The assays can be used to quantitate each enzyme in crude cell extracts. To ascertain their validity we applied each assay to extracts from genetically modified cells that overproduce separately each of the five enzymes. The methodology should be useful in further studies of the physiological function of 5-NTs and their influence on the clinical use of nucleoside analogs.     

Respiratory function in cybrid cell lines carrying European mtDNA haplogroups: implications for Leber's hereditary optic neuropathy

The possibility that some combinations of mtDNA polymorphisms, previously associated with Leber's hereditary optic neuropathy (LHON), may affect mitochondrial respiratory function was tested in osteosarcoma-derived transmitochondrial cytoplasmic hybrids (cybrids). In this cellular system, in the presence of the same nuclear background, different exogenous mtDNAs are used to repopulate a parental cell line previously devoid of its original mtDNA. No detectable differences in multiple parameters exploring respiratory function were observed when mtDNAs belonging to European haplogroups X, H, T and J were used. Different possible explanations for the previously established association between haplogroup J and LHON 11778/ND4 and 14484/ND6 pathogenic mutations are discussed, including the unconventional proposal that mtDNA haplogroup J may exert a protective rather than detrimental effect.     

Recent findings and technological advances in phosphoproteomics for cells and tissues

Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.     

Genetic analysis of Soil-Borne Cereal Mosaic Virus response in durum wheat: evidence for the role of the major quantitative trait locus QSbm.ubo-2BS and of minor quantitative trait loci

Genetic analysis of Soil-Borne Cereal Mosaic Virus (SBCMV) resistance in durum wheat was carried out using a population of 180 recombinant inbred lines (RILs) obtained from Simeto (susceptible) × Levante (resistant). The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection in two growing seasons and genotyped with simple sequence repeat (SSR) and Diversity Arrays Technology^? markers. Transgressive segregation was observed for disease reaction as estimated by symptom severity scores and virus concentration in leaves. Heritability of the disease response was high, with h ² values consistently above 80%. A major quantitative trait locus (QTL) (QSbm.ubo-2BS) in the distal telomeric region of chromosome 2BS accounted for 60–70% of the phenotypic variation for symptom severity, 40–55% for virus concentration and 15–30% for grain yield. The favorable allele was contributed by Levante. Seven additional QTL influenced SBCMV resistance, with the low-susceptibility allele contributed by Levante at five QTL and by Simeto at the remaining two. The meta-QTL analysis carried out using the data from two mapping populations (Simeto × Levante and Meridiano × Claudio) suggests that in both populations SBCMV resistance is likely controlled by QSbm.ubo-2BS. Our results confine QSbm.ubo-2BS to a c. 2-cM-wide interval flanked by SSR markers that are already being used for marker-assisted selection.