Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.     

Interactions of 5-deazapteridine derivatives with Mycobacterium tuberculosis and with human dihydrofolate reductases

There are major differences between the structures of human dihydrofolate reductase (hDHFR) and Mycobacterium tuberculosis dihydrofolate reductase (mtDHFR). These differences may allow us to design more selective mtDHFR inhibitors. In this paper we study the reactions of six different compounds derived from 5-deazapteridine with human and bacterial enzymes. Results suggest that the addition of hydrophobic groups to the aminophenyl ring would increase mtDHFR-inhibitor affinity and selectivity.     

ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.     

Larvicidal activity of essential oils from Brazilian plants against Aedes aegypti L

Aedes aegypti L. is the major vector of dengue fever, an endemic disease in Brazil. In an effort to find effective and affordable ways to control this mosquito, the larvicidal activities of essential oils from nine plants widely found in the Northeast of Brazil were analyzed by measurement of their LC50. The essential oils were extracted by steam distillation and their chemical composition determined by GL-chromatography coupled to mass spectroscopy. The essential oils from Cymbopogon citratus and Lippia sidoides, reported in the literature to have larvicidal properties against A. aegypti, were used for activity comparison. The results show that Ocimum americanum and Ocimum gratissimum have LC50 of 67 ppm and 60 ppm respectively, compared to 63 ppm for L. sidoides and 69 ppm for C. citratus. These results suggest a potential utilization of the essential oil of these two Ocimum species for the control of A. aegypti.     

Response characteristics of the mitochondrial DNA genome in developmental health and disease

This review focuses on mitochondrial biology in mammalian development; specifically, the dynamics of information transfer from nucleus to mitochondrion in the regulation of mitochondrial DNA genomic expression, and the reverse signaling of mitochondrion to nucleus as an adaptive response to the environment. Data from recent studies suggest that the capacity of embryonic cells to react to oxygenation involves a tradeoff between factors that influence prenatal growth/development and postnatal growth/function. For example, mitochondrial DNA replication and metabolic set points in nematodes may be determined by mitochondrial activity early in life. The mitochondrial drug PK11195, a ligand of the peripheral benzodiazepine receptor, has antiteratogenic and antidisease action in several developmental contexts in mice. Protein malnutrition during early life in rats can program mitochondrial DNA levels in adult tissues and, in humans, epidemiological data suggest an association between impaired fetal growth and insulin resistance. Taken together, these findings raise the provocative hypothesis that environmental programming of mitochondrial status during early life may be linked with diseases that manifest during adulthood. Genetic defects that affect mitochondrial function may involve the mitochondrial DNA genome directly (maternal inheritance) or indirectly (Mendelian inheritance) through nuclear-coded mitochondrial proteins. In a growing number of cases, the depletion of, or deletion in, mitochondrial DNA is seen to be secondary to mutation of key nuclear-coded mitochondrial proteins that affect mitochondrial DNA replication, expression, or stability. These defects of intergenomic regulation may disrupt the normal cross-talk or structural compartmentation of signals that ultimately regulate mitochondrial DNA integrity and copy number, leading to depletion of mitochondrial DNA.     

Unbound form of tomato inhibitor-II reveals interdomain flexibility and conformational variability in the reactive site loops

The Potato II (Pot II) family of proteinase inhibitors plays important roles in the constitutive and inducible defense of plants against predation by a wide range of pests. The structural basis of inhibition by a multidomain Pot II family inhibitor was revealed recently by the structure of the ternary complex between the two-headed tomato inhibitor-II (TI-II) and two molecules of subtilisin Carlsberg. Here we report the 2.15-A resolution crystal structure of the unbound form of TI-II that reveals significant conformational flexibility in the absence of bound proteinase molecules. The four independent copies of unbound TI-II in the asymmetric unit of the unit cell display a range of different conformations when compared with the bound form of the inhibitor, most strikingly in the orientations of the inhibitory domains and in the conformations of the reactive site loops. One of the two linker segments (residues 74 to 79) between the two domains as well as the adjacent beta-strand in Domain I (residues 80-85) is well ordered in all four copies of the unbound inhibitor, even though this region appeared to be disordered in the structure of the ternary complex. Conformational flexibility seen in the reactive site loops of unbound TI-II suggests a mechanism by which the inhibitor can balance the need for tight binding with the need for broad inhibitory function.     

Structure of arterivirus nsp4. The smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole

Arteriviruses are enveloped, positive-stranded RNA viruses and include pathogens of major economic concern to the swine- and horse-breeding industries. The arterivirus replicase gene encodes two large precursor polyproteins that are processed by the viral main proteinase nonstructural protein 4 (nsp4). The three-dimensional structure of the 21-kDa nsp4 from the arterivirus prototype equine arteritis virus has been determined to 2.0 A resolution. Nsp4 adopts the smallest known chymotrypsin-like fold with a canonical catalytic triad of Ser-120, His-39, and Asp-65, as well as a novel alpha/beta C-terminal extension domain that may play a role in mediating protein-protein interactions. In different copies of nsp4 in the asymmetric unit, the oxyanion hole adopts either a collapsed inactive conformation or the standard active conformation, which may be a novel way of regulating proteolytic activity.     

First report of Lappetascaris lutjani Rasheed, 1965 (Nematoda, Ascaridoidea, Anisakidae) parasitizing Trachipterus arawatae (Pisces, Lampridiformes) on the Atlantic coast of Brazil

New host and geographical records are reported for the nematode Lappetascaris lutjani Rasheed, 1965, parasitizing the marine fish Trachipterus arawatae Clark, 1881 in Brazilian waters. Morphometric data and illustrations of the parasites are included.     

Cytotoxic,hypoglycemic activity and phytochemical analysis of Rubus imperialis (Rosaceae)

Rubus imperialis, Artemia salina, 3-O-methylellagic-4'-O-alpha-rhamnose Acid Screening of different extracts, fractions and compounds from Rubus imperialis Chum. Schl. (Rosaceae) has been conduced using the brine shrimp microwell cytotoxicity assay. Three parts of the plant (methanolic extract from leaves, roots and stems), three fractions from roots (hexane, ethyl acetate and butanol) and three isolated compounds (niga-ichigoside F1, 23-hydroxytormentic acid, ellagic acid derivative) were tested. The most promising material (LC50 <1000 microg/ml) were the methanolic extract and ethyl acetate fraction from roots. However, there was little correlation observed in the degree of toxicities observed between the isolated compounds. On the other hand, the cytotoxicity and in vivo assays confirmed the hypoglycemic activity of methanolic extract and validated the Brazilian popular use of R. imperialis as an antidiabetic agent.     

Xanthine oxidase and aldehyde oxidase: a simple procedure for the simultaneous purification from rat liver

Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.