Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Reelin is a positional signal for the lamination of dentate granule cells

Reelin is required for the proper positioning of neurons in the cerebral cortex. In the reeler mutant lacking reelin, the granule cells of the dentate gyrus fail to form a regular, densely packed cell layer. Recent evidence suggests that this defect is due to the malformation of radial glial processes required for granule cell migration. Here, we show that recombinant reelin in the medium significantly increases the length of GFAP-positive radial glial fibers in slice cultures of reeler hippocampus, but does not rescue either radial glial fiber orientation or granule cell lamination. However, rescue of radial glial fiber orientation and granule cell lamination was achieved when reelin was present in the normotopic position provided by wild-type co-culture, an effect that is blocked by the CR-50 antibody against reelin. These results indicate a dual function of reelin in the dentate gyrus, as a differentiation factor for radial glial cells and as a positional cue for radial fiber orientation and granule cell migration.     

Mycoplasma haemocanis infection--a kennel disease?

Mycoplasma haemocanis (formerly Haemobartonella canis) is a red blood cell parasite that causes disease mainly in immunosuppressed and splenectomized dogs. Clinical outbreak of the disease resulted in failure of a large experimental project. We aimed to identify whether M. haemocanis has increased prevalence in kennel-raised dogs. In a prospective study, we compared the prevalence of M. haemocanis in whole blood (anti-coagulated by use of EDTA) collected from pet dogs (University of Illinois, Urbana Champaign, Ill.; n = 60) with that in blood from dogs raised in three distinct kennels in western Europe (WE; n = 23), eastern Europe (EE; n = 20), and North America (NA; n = 20). Screening included antibody testing and microscopy of blood smears. The presence of M. haemocanis was identified using a polymerase chain reaction (PCR) assay for specific DNA of the organism. None of the pet dogs (0%) was test positive for M. haemocanis DNA. Mycoplasma haemocanis was found in dogs tested at all of the kennels. Infection rate in the three kennels was 30, 35, and 87%, respectively (all P < 0.001 versus control, chi2-test). Latent infection with M. haemocanis was not a single observation in kennel-raised dogs. Prevalence may be higher than that in a pet dog population. The potential exists for these latent infections to adversely affect or confound research results.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

The calcium sensor protein visinin-like protein-1 modulates the surface expression and agonist sensitivity of the alpha 4beta 2 nicotinic acetylcholine receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.     

Reelin and ApoE receptors cooperate to enhance hippocampal synaptic plasticity and learning

Two apolipoprotein E (apoE) receptors, the very low density lipoprotein (VLDL) receptor and apoE receptor 2 (apoER2), are also receptors for Reelin, a signaling protein that regulates neuronal migration during brain development. In the adult brain, Reelin is expressed by GABA-ergic interneurons, suggesting a potential function as a modulator of neurotransmission. ApoE receptors have been indirectly implicated in memory and neurodegenerative disorders because their ligand, apoE, is genetically associated with Alzheimer disease. We have used knockout mice to investigate the role of Reelin and its receptors in cognition and synaptic plasticity. Mice lacking either the VLDL receptor or the apoER2 show contextual fear conditioning deficits. VLDL receptor-deficient mice also have a moderate defect in long term potentiation (LTP), and apoER2 knockouts have a pronounced one. The perfusion of mouse hippocampal slices with Reelin has no effect on baseline synaptic transmission but significantly enhances LTP in area CA1. This Reelin-dependent augmentation of LTP is abolished in VLDL receptor and apoER2 knockout mice. Our results reveal a role for Reelin in controlling synaptic plasticity in the adult brain and suggest that both of its receptors are necessary for Reelin-dependent enhancement of synaptic transmission in the hippocampus. Thus, the impairment of apoE receptor-dependent neuromodulation may contribute to cognitive impairment and synaptic loss in Alzheimer disease.     

Brevican isoforms associate with neural membranes

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.     

Frequentist estimation of coalescence times from nucleotide sequence data using a tree-based partition

This article proposes a method of estimating the time to the most recent common ancestor (TMRCA) of a sample of DNA sequences. The method is based on the molecular clock hypothesis, but avoids assumptions about population structure. Simulations show that in a wide range of situations, the point estimate has small bias and the confidence interval has at least the nominal coverage probability. We discuss conditions that can lead to biased estimates. Performance of this estimator is compared with existing methods based on the coalescence theory. The method is applied to sequences of Y chromosomes and mtDNAs to estimate the coalescent times of human male and female populations.     

Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis

BACKGROUND: Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis. MATERIALS AND METHODS: To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies. RESULTS: FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation. CONCLUSION: Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.