Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

An antibody against a sequence of human topoisomerase II gives different immunofluorescence patterns in quiescent and proliferating nuclei of Pisum sativum L.

Immunofluorescence with an antibody against a C-terminal sequenceof human topoisomerase II has been performed on nuclei releasedfrom different tissues of Pisum sativum L. All the nuclei labelledand preincubation of the antibody with the corresponding immunogenpeptide strongly decreased the fluorescence. The labelling pattern(particularly nucleolar) was different in quiescent and proliferatingnuclei and changed during germination. In prophase nuclei, thelabelling was at the periphery of the condensing chromosomes,and in metaphase chromosomes, a characteristic labelling atpericentromeric regions was found. A computer search indicatedthat, apart from mammalian topoisomerase II, the immunogen peptidedid not match any other sequenced protein which could reasonablybe present in plant nuclei. The possible relation between theantigen recognized and topoisomerase II is discussed. Key words: Topoisomerase II, seed germination, Pisum sativum, immunofluorescence, nuclear proteins     

Lysyl oxidase interacts with hormone placental lactogen and synergistically promotes breast epithelial cell proliferation and migration

Lysyl oxidase (LOX), an extracellular amine oxidase, catalyzes the cross-linking of collagen and elastin. LOX has been also shown to play an essential role in promoting the invasive and metastatic potential of breast tumor cells. However, the LOX-interacting factors in these processes are not known. In this study, we identified placental lactogen (PL), a member of the growth hormone/prolactin hormone family, as a LOX-interacting partner using yeast two-hybrid screens. PL is normally only expressed in placental syncytiotrophoblasts, but PL genes are amplified and expressed in a high percentage of invasive ductal breast carcinomas. We confirmed LOX-PL interactions using far Western and solid phase binding assays. In activity assays, PL was not a substrate or inhibitor of LOX. We further demonstrated that PL is expressed in breast tumor epithelial cells and detected LOX-PL interactions by coimmunoprecipitation in invasive breast cancer cells. In MCF-10A normal breast epithelial cells stably expressing LOX, PL, or both, LOX had no effect on cell proliferation, PL alone increased proliferation by 49%, and coexpression of LOX and PL led to a 121% increase in cell proliferation. Unlike in tumor cells, LOX did not induce a more migratory phenotype in MCF-10A cells; nor did PL. However, their coexpression resulted in a 240% increase in cell migration, suggesting that these interactions may be highly relevant to the transition of epithelial cells toward a migratory phenotype during the development and progression of breast carcinoma and a significant role for LOX-PL interactions in epithelial cell behavior.     

DNA methylation regulates tissue-specific expression of Shank3

Tissue-specific gene expression can be controlled by epigenetic modifications such as DNA methylation. SHANK3, together with its homologues SHANK1 and SHANK2, has a central functional and structural role in excitatory synapses and is involved in the human chromosome 22q13 deletion syndrome. In this report, we show by DNA methylation analysis in lymphocytes, brain cortex, cerebellum and heart that the three SHANK genes possess several methylated CpG boxes, but only SHANK3 CpG islands are highly methylated in tissues where protein expression is low or absent and unmethylated where expression is present. SHANK3 protein expression is significantly reduced in hippocampal neurons after treatment with methionine, while HeLa cells become able to express SHANK3 after treatment with 5-Aza-2'-deoxycytidine. Altogether, these data suggest the existence of a specific epigenetic control mechanism regulating SHANK3, but not SHANK1 and SHANK2, expression.     

Quantifying the errors in animal contacts recorded by proximity loggers

Automated contact detection by means of proximity loggers permits the measurement of encounters between individuals (animal-animal contacts) and the time spent by individuals in the proximity of a focal resource of interest (animal-fixed logger contacts). The ecological inference derived from contact detection is intrinsically associated with the distance at which the contact occurred. But no proximity loggers currently exist that record this distance and therefore all distance estimations are associated with error. Here we applied a probabilistic approach to model the relationship between contact detection and inter-logger distance, and quantify the associated error, on free-ranging animals in semi-controlled settings. The probability of recording a contact declined with the distance between loggers, and this decline was steeper for weaker radio transmission powers. Even when proximity loggers were adjacent, contact detection was not guaranteed, irrespective of the radio transmission power. Accordingly, the precision and sensitivity of the system varied as a function of inter-logger distance, radio transmission power, and experimental setting (e.g., depending on animal body mass and fine-scale movements). By accounting for these relationships, we were able to estimate the probability that a detected contact occurred at a certain distance, and the probability that contacts were missed (i.e., false negatives). These calibration exercises have the potential to improve the predictability of the study and enhance the applicability of proximity loggers to key wildlife management issues such as disease transmission rates or wildlife use of landscape features and resources.     

Role for glutathione in the hyposensitivity of LPS-pretreated mice to LPS anorexia

To study the role of the redox state regulator glutathione (GSH) in bacterial lipopolysaccharide (LPS)-induced anorexia we measured total reduced GSH (trGSH) in liver, serum and brain in response to intraperitoneal (ip) lipopolysaccharide (LPS, 4 microg/mouse) injection in LPS-na?ve and LPS-pretreated (4 microg/mouse given 3 days earlier) mice. LPS reduced food intake in LPS-na?ve mice and LPS pretreatment attenuated this effect. LPS decreased trGSH at 24 hours after injection in LPS-na?ve mice but 4 days later trGSH levels were upregulated in brain and liver, and this was associated with a significant attenuation of LPS-induced anorexia. In addition, LPS increased mitochondrial GSH levels in brain and liver at 4 days after injection. Pharmacological GSH depletion with diethylmaleate and L-buthionine sulfoximine in LPS-pretreated mice ablated the hyposensitivity to the anorexic effect of LPS. Together, these findings suggest a prominent role for GSH and its intracellular repartition in LPS anorexia.     

Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance

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The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production.