Accumulation of DNA damage in the organs of mice deficient in gamma-glutamyltranspeptidase

We have used a differential alkaline single cell gel electrophoresis assay of DNA ("omet assay" at pH 13 and 12.3) to evaluate DNA damage as a function of age in mice with an inherited defect in gluthathione (GSH) metabolism. The mice are homozygous null for gamma-glutamyltranspeptidase (GGT), the enzyme responsible for initiating the catabolism of GSH, and paradoxically have reduced levels of GSH and cysteine in many organs. We found an accumulation of DNA damage in lung, liver and kidney in these mice as a function of age. The largest differences were in assays run at pH 13, suggesting that the accumulation of apurinic/apryrimidinic (AP) sites and oxidative damage of DNA was largely responsible. In contrast, little if any accumulation of these lesions was detected in wild-type mice. Although these findings do not allow a precise analysis of the molecular basis of damage accumulation in GGT-deficient mice, they implicate low GSH and cysteine levels as a cause of accumulative DNA damage in the intact mammal.     

Tumor cells present MHC class II-restricted nuclear and mitochondrial antigens and are the predominant antigen presenting cells in vivo

MHC class II-restricted tumor Ags presented by class II(+) tumor cells identified to date are derived from proteins expressed in the cytoplasm or plasma membrane of tumor cells. It is unclear whether MHC class II(+) tumor cells present class II-restricted epitopes derived from other intracellular compartments, such as nuclei and/or mitochondria, and whether class II(+) tumor cells directly present Ag in vivo. To address these questions, a model Ag, hen egg lysozyme, was targeted to various subcellular compartments of mouse sarcoma cells, and the resulting cells were tested for presentation of three lysozyme epitopes in vitro and for presentation of nuclear Ag in vivo. In in vitro studies, Ags localized to all tested compartments (nuclei, cytoplasm, mitochondria, and endoplasmic reticulum) are presented in the absence invariant chain and H-2M. Coexpression of invariant chain and H-2M inhibit presentation of some, but not all, of the epitopes. In vivo studies demonstrate that class II(+) tumor cells, and not host-derived cells, are the predominant APC for class II-restricted nuclear Ags. Because class II(+) tumor cells are effective APC in vivo and probably present novel tumor Ag epitopes not presented by host-derived APC, their inclusion in cancer vaccines may enhance activation of tumor-reactive CD4(+) T cells.     

Disruption of reproductive activity of Coptotermes formosanus (Isoptera: Rhinotermitidae) primary reproductives by three chitin synthesis inhibitors

Effects of the chitin synthesis inhibitors (CSIs) diflubenzuron, hexaflumuron, and lufenuron on the Formosan subterranean termite, Coptotermes formosanus Shiraki, primary reproductives were studied in the laboratory. Incipient colonies were established by collecting and pairing C. formosanus alates and placing them in dishes containing an artificial diet. Three groups of 56 colonies each were fed with a diet containing 10 ppm of one of each of the CSIs and were compared with a control group fed with an untreated diet. All eggs oviposited by treated young queens failed to hatch at the end of 6 mo. Estimated queen fecundity was significantly lower in the lufenuron treatment compared with the control group. Fecundity of hexaflumuron-treated queens did not differ significantly from that of queens from the control group and the other treatments. Adult mortality was significantly higher in the diflubenzuron and lufenuron treatments than in the control group at the end of 6 mo., but not in the hexaflumuron treatment. All the pairs died within 8 mo. in the diflubenzuron and lufenuron treatments, even after treatment was suspended at the end of 6 mo. Mortality in the hexaflumuron treatment was significantly higher than in the control group by the end of 9 mo. The three CSIs tested eliminated reproduction in C. formosanus by preventing egg hatching and induced adult mortality. Possible mechanisms by which CSIs induce termite adult death are discussed.     

Enhanced production of lactococcin 972 in chemostat cultures

Lactococcus lactis subsp. lactis IPLA972 is a wild lactococcal strain suitable as a single starter in the manufacture of dairy products. This strain synthesizes lactococcin 972 (Lcn972), a unique bacteriocin that blocks septum formation. In this work, we report on the conditions to optimize biomass and Lcn972 production. In batch cultures, pH 6.8 was found to be optimum for bacteriocin synthesis and both glucose and lactose supported Lcn972 production. The inhibitory activity improved up to eight-fold with increasing carbohydrate concentration. In chemostat cultures, steady states were achieved even at dilution rates higher than _max, due to the strong wall growth. Lcn972 behaved as a true primary metabolite, as it was maximally produced when the cells were actively growing. Bacteriocin yields were improved up to ten-fold in chemostat cultures compared with those achieved in batch.     

Evaluation of the presence of a thapsigargin-sensitive calcium store in trypanosomatids using Trypanosoma evansi as a model

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.     

Determination of Trichuris skrjabini by sequencing of the ITS1-5.8S-ITS2 segment of the ribosomal DNA: comparative molecular study of different species of trichurids

Adults of Trichuris skrjahini have been isolated from the cecum of caprine hosts (Capra hircus), Trichuris ovis and Trichuris globulosa from Ovis aries (sheep) and C. hircus (goats), and Trichuris leporis from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced by polymerase chain reaction (PCR) techniques. The ITS1 of T. skrjabini, T. ovis, T. globulosa, and T. leporis was 495, 757, 757, and 536 nucleotides in length, respectively, and had G + C contents of 59.6, 58.7, 58.7, and 60.8%, respectively. Intraindividual variation was detected in the ITSI sequences of the 4 species. Furthermore, the 5.8S sequences of T. skrjabini, T. ovis, T. globulosa, and T. leporis were compared. A total of 157, 152, 153, and 157 nucleotides in length was observed in the 5.8S sequences of these 4 species, respectively. There were no sequence differences of ITS1 and 5.8S products between T. ovis and T. globulosa. Nevertheless, clear differences were detected between the ITS1 sequences of T. skrjabini, T. ovis, T. leporis, Trichuris muris, and T. arvicolae. The ITS2 fragment from the rDNA of T. skrjabini was sequenced. A comparative study of the ITS2 sequence of T. skrjabini with the previously published ITS2 sequence data of T. ovis, T. leporis, T. muris, and T. arvicolae suggested that the combined use of sequence data from both spacers would be useful in the molecular characterization of trichurid parasites.     

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3

N-glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 A resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcalpha3Galbeta4Glcbeta. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.     

Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro

Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome. One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2. VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.     

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 +/- 3.1 mg a.i./m2 in walls and 11.9 +/- 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 +/- 6.9 mg a.i./m2 and 34.7 +/- 10.4 mg a.i./m2 in walls and 28.8 +/- 19.2 mg a.i./m2 and 24.9 +/- 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.     

Tryptophan substitutions reveal the role of nicotinic acetylcholine receptor alpha-TM3 domain in channel gating: differences between Torpedo and muscle-type AChR

A recent tryptophan scanning of the alpha-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [Guzman, G., Santiago, J., Ricardo, A., Martí-Arbona, R., Rojas, L., Lasalde-Dominicci, J. (2003) Biochemistry 42, 12243-12250]. Here we extend the study of the alpha-TM3 domain to the muscle-type AChR by examining functional consequences of single tryptophan substitutions at five conserved positions (alphaM282, alphaF284, alphaV285, alphaA287, and alphaI290) homologous to the alpha-TM3 positions that were recently characterized in the Torpedo AChR. Similarly to the Torpedo AChR, mutations alphaM282W and alphaV285W, which are presumed to face the interior of the protein, did not exhibit functional channel activity. Nevertheless, significant expression levels of these mutants were observed at the oocyte surface. In contrast to the Torpedo AChR, in the muscle-type AChR, tryptophan substitution at positions F284, A287, and I290 produces a significant increase in normalized macroscopic response. Single-channel recordings at low ACh concentration revealed that the increase in AChR sensitivity for the F284W, A287W, and I290W is due to an increase in the mean open duration. These results suggest that tryptophan substitution directly affects channel gating, primarily the channel closing rate. Our results suggest that residues facing the interior of the protein (i.e., alphaM282 and alphaV285) may similarly affect channel gating in Torpedo and muscle-type AChR. However, equivalent mutations (i.e., F284W and I290W) presumably facing the lipid environment display a very different functional response between these two AChR species.