Hepatocyte Permissiveness to Plasmodium Infection Is Conveyed by a Short and Structurally Conserved Region of the CD81 Large Extracellular Domain

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria infection, and thus represents an attractive target for anti-malarial interventions. Still, the molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that the tetraspanin CD81, a known receptor for the hepatitis C virus (HCV), is required on hepatocytes for infection by sporozoites of several Plasmodium species. Here we have characterized CD81 molecular determinants required for infection of hepatocytic cells by P. yoelii sporozoites. Using CD9/CD81 chimeras, we have identified in CD81 a 21 amino acid stretch located in a domain structurally conserved in the large extracellular loop of tetraspanins, which is sufficient in an otherwise CD9 background to confer susceptibility to P. yoelii infection. By site-directed mutagenesis, we have demonstrated the key role of a solvent-exposed region around residue D137 within this domain. A mAb that requires this region for optimal binding did not block infection, in contrast to other CD81 mAbs. This study has uncovered a new functionally important region of CD81, independent of HCV E2 envelope protein binding domain, and further suggests that CD81 may not interact directly with a parasite ligand during Plasmodium infection, but instead may regulate the function of a yet unknown partner protein

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions

Generation of shRNA Transgenic Mice

RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a faster alternative to conventional knockout approaches. Here, we describe an advanced strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time- and tissue-dependent manner. Single-copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase-mediated cassette exchange and transmitted through the germline of chimaeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site-specific insertion of single-copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo

Aberrant regulation of bone trace elements in motheaten and osteopetrosis mutant mice.

Increasing numbers of genetic diseases involving bone development and models for these diseases have been identified recently. Analysis of these bone diseases have revealed that regulated action of multiple growth factors and subsequent signal transduction are essential for normal bone formation. In this paper, two murine mutant mice viable motheaten and osteopetrosis are analyzed. Mice with the recessive \u27viable motheaten\u27 mutation express a severe immunodeficiency syndrome and bone defects. Mutations at the motheaten locus were shown to be the result of aberrant splicing of the gene encoding hematopoietic cell phosphatase (Hcph). Mice homozygous for the osteopetrosis mutation develop congenital osteopetrosis due to a severe deficiency of osteoclasts. It has been recognized that bone trace element composition analysis helps to define bone-related physiological conditions. We have analyzed bone trace element composition in viable motheaten and osteopetrosis mutant animal models in this study. In order to gain insights into the effects of particular genetic defects on bone trace element composition, inductively coupled plasma atomic emissions spectrometry (ICP-AES) analysis was performed. Marked changes in bone trace element levels were found in limb bones of viable motheaten and osteopetrosis mutant mice. An assessment of these trace element spectrum in the two mutant models with respect to each genetic defects are discussed in this paper

Pravastatin induces placental growth factor (PGF) and ameliorates preeclampsia in a mouse model

Preeclampsia is a relatively common pregnancy-related disorder. Both maternal and fetal lives will be endangered if it proceeds unabated. Recently, the placenta-derived anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFLT1) and soluble endoglin (sENG), have attracted attention in the progression of preeclampsia. Here, we established a unique experimental model to test the role of sFLT1 in preeclampsia using a lentiviral vector-mediated placenta-specific expression system. The model mice showed hypertension and proteinuria during pregnancy, and the symptoms regressed after parturition. Intrauterine growth restriction was also observed. We further showed that pravastatin induced the VEGF-like angiogenic factor placental growth factor (PGF) and ameliorated the symptoms. We conclude that our experimental preeclamptic murine model phenocopies the human case, and the model identifies low-dose statins and PGF as candidates for preeclampsia treatment

Investigation of the role of TASK-2 channels in rat pulmonary arteries; pharmacological and functional studies following RNA interference procedures

1. In the present study, we investigated the ability of RNA interference technology to suppress TASK-2 potassium channel expression in human embryonic kidney (HEK293) cells stably transfected with TASK-2 cDNA and in rat isolated intact pulmonary arteries. 2. Lipofectamine-induced transfection of a specific siRNA sequence targeted against TASK-2 resulted in a dose- and time-dependent decrease in TASK-2 channel protein expression. In siRNA-transfected cells the TASK-2 peak currents were significantly smaller than in control cells at every investigated pH, while the pH sensitivity was not altered. Using scrambled siRNA as a negative control, there were no significant changes in TASK-2 protein expression or current compared to mock-transfected cells. 3. In TASK-2 siRNA-transfected small pulmonary arteries, but not in scrambled siRNA-treated vessels, myocyte resting membrane potential at pH 7.4 was significantly less negative and the hyperpolarisations in response to increasing pH from 6.4 to 8.4 were significantly smaller compared with control. 4. The application of levcromakalim (10 μM), NS1619 (33 μM) and a potassium channel inhibitor cocktail (5 mM 4-aminopyridine, 10 mM tetraethylammonium chloride, 30 μM Ba(2+) and 10 μM glibenclamide) had similar effects in control and in siRNA-transfected vessels. The TASK-1 (anandamide-sensitive) contribution to resting membrane potential was comparable in each group. Clofilium (100 μM) generated significantly smaller responses in transfected artery segments. 5. These results suggest that RNA interference techniques are effective at inhibiting TASK-2 channel expression in cultured cells and in intact vessels and that TASK-2 channels have a functional role in setting the membrane potential of pulmonary artery myocytes