Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA–protein complexes into nuclei. Import of VirD2–ssDNA–VirE2 complexes was fast and efficient, and was shown to depended on importin α, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein–DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

β3 integrins mediate the cellular entry of hantaviruses that cause respiratory failure

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to β3 integrins and by the β3-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and β1-specific antibodies but not by β3-specific antibodies or vitronectin. Transfection with recombinant αIIbβ3 or αvβ3 integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that αIIbβ3 and αvβ3 integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, αIIbβ3-integrin mutants. Hence, NY-1 and SNV entry is independent of β3 integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.

Calx, a Na-Ca exchanger gene of Drosophila melanogaster

We have cloned Calx, a gene that encodes a Na-Ca exchanger of Drosophila melanogaster. Calx encodes two repeated motifs, Calx-α and Calx-β, that overlap domains required for exchanger activity and regulation. Calx has multiple transcripts in adults, including at least one expressed in the retina. The Calx genomic locus comprises ≥35 kb between the Atpα and rudimentary-like genes in chromosomal region 93B. In Xenopus oocytes, microinjected Calx cRNA induces calcium uptake like that of its homolog, the 3Na+-1Ca2+ exchanger of mammalian heart. Implications of Calx-α motifs for the mechanism of Na-Ca exchange are discussed.

Ceramide-induced inhibition of T lymphocyte voltage-gated potassium channel is mediated by tyrosine kinases

The n-type K+ channel (n-K+, Kv1.3) in lymphocytes has been recently implicated in the regulation of Fas-induced programmed cell death. Here, we demonstrate that ceramide, a lipid metabolite synthesized upon Fas receptor ligation, inhibits n-K+ channel activity and induces a tyrosine phosphorylation of the Kv1.3 protein in Jurkat T lymphocytes. Tyrosine phosphorylation of the n-K+ channel correlated with an activation of the Src-like tyrosine kinase p56lck upon cellular treatment with the ceramide analog C6-ceramide. Because genetic deficiency of p56lck or inhibition of Src-like tyrosine kinases by herbimycin A prevented ceramide-mediated n-K+ channel inhibition and tyrosine phosphorylation, we propose a ceramide-initiated activation of p56lck resulting in tyrosine phosphorylation and inhibition of the n-K+ channel protein.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

A relationship between behavior, neurotrophin expression, and new neuron survival

The high vocal center (HVC) controls song production in songbirds and sends a projection to the robust nucleus of the archistriatum (RA) of the descending vocal pathway. HVC receives new neurons in adulthood. Most of the new neurons project to RA and replace other neurons of the same kind. We show here that singing enhances mRNA and protein expression of brain-derived neurotrophic factor (BDNF) in the HVC of adult male canaries, Serinus canaria. The increased BDNF expression is proportional to the number of songs produced per unit time. Singing-induced BDNF expression in HVC occurs mainly in the RA-projecting neurons. Neuronal survival was compared among birds that did or did not sing during days 31–38 after BrdUrd injection. Survival of new HVC neurons is greater in the singing birds than in the nonsinging birds. A positive causal link between pathway use, neurotrophin expression, and new neuron survival may be common among systems that recruit new neurons in adulthood.

High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia

Mitochondria are confronted with low oxygen levels in the microenvironment within tissues; yet, isolated mitochondria are routinely studied under air-saturated conditions that are effectively hyperoxic, increase oxidative stress, and may impair mitochondrial function. Under hypoxia, on the other hand, respiration and ATP supply are restricted. Under these conditions of oxygen limitation, any compromise in the coupling of oxidative phosphorylation to oxygen consumption could accentuate ATP depletion, leading to metabolic failure. To address this issue, we have developed the approach of oxygen-injection microcalorimetry and ADP-injection respirometry for evaluating mitochondrial function at limiting oxygen supply. Whereas phosphorylation efficiency drops during ADP limitation at high oxygen levels, we show here that oxidative phosphorylation is more efficient at low oxygen than at air saturation, as indicated by higher ratios of ADP flux to total oxygen flux at identical submaximal rates of ATP synthesis. At low oxygen, the proton leak and uncoupled respiration are depressed, thus reducing maintenance energy expenditure. This indicates the importance of low intracellular oxygen levels in avoiding oxidative stress and protecting bioenergetic efficiency.

Huntingtin aggregation monitored by dynamic light scattering

An initial stage of fibrillogenesis in solutions of glutathione S-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-l-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesis in vitro.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (Jahraus et al., 1998). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (Defacque et al., 2000a), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.