tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.     

Prevention and correction mechanisms behind anaphase synchrony: implications for the genesis of aneuploidy

The perpetuation of the species' genomic identity strongly depends on the accurate maintenance of chromosome number through countless cell generations. The synchronous entry and progression of all chromosomes through anaphase is fundamental for the quality of mitosis and is guaranteed by error prevention and correction mechanisms that ultimately certify the bipolar attachment of chromosomes to the mitotic spindle, the uniform distribution of forces amongst different chromosomes, and the simultaneity of sister-chromatid separation. The existence of a kinetochore-attachment checkpoint (KAC; also known as spindle-assembly checkpoint) ensures a delay in anaphase onset if any kinetochore remains unattached or devoid of a proper complement of microtubules. The stochastic nature of microtubule-kinetochore interactions predisposes the mitotic process to mistakes, but different molecular players cooperate by detecting and releasing incorrect attachments and thus delaying checkpoint satisfaction. Conversely, correct microtubule-kinetochore interactions become selectively stabilized. Once anaphase onset is triggered, the segregation velocities achieved by each chromosome should be similar, so that none of the chromosomes is lagged behind. This reflects the uniformity of forces acting on the different chromosomes and relies on a conspicuous mitotic spindle property known as microtubule poleward flux. Importantly, not all incorrect attachments are detected and resolved prior to anaphase leading to asynchronous chromosome segregation, but several mechanisms are in place to prevent aneuploidy. One of these mechanisms relies on anaphase spindle forces and another, known as the NoCut checkpoint, delays cell cleavage during cytokinesis until chromosomes can free the spindle mid-region. In this review we discuss how these different mechanisms act in concert to ensure the fidelity of the mitotic process.     

Intron-mediated gusA expression in tritordeum and wheat resulting from particle bombardment

The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.     

Differential distribution of vanilloid receptors in the primary sensory neurons projecting to the dorsal skin and muscles

We examined transient receptor potential (TRP) V1 and TRPV2 expression in calcitonin gene-related peptide (CGRP) positive (+) primary sensory neurons projecting to the skin and skeletal muscles of the rat dorsum. Among the dorsal root ganglia at the levels from C2 to Th1, 34.9% of neurons projecting to the skin were positive for CGRP, and 32.6% or 21.6% of neurons projecting to the trapezius muscle or the longissimus muscle were positive for CGRP. Of the small CGRP+ neurons projecting to the skin, 53.5% were positive for TRPV1, 11.6% were positive for TRPV2. Of the small CGRP+ neurons projecting to the trapezius or the longissimus, 53.1 or 53.2% were positive for TRPV1, 8.8 or 8.3% were positive for TRPV2, respectively. In the periphery, 29.3% of CGRP+ nerve fibers were positive for TRPV1 in the skin, whereas 65.0 or 59.8% were positive in the trapezius or the longissimus. Therefore, the present study showed that the percentage of CGRP+ neurons projecting to the trapezius is higher than that to the longissimus, and that the co-localization percentage of CGRP and TRPV1 on the sensory nerves was also higher in the trapezius than in the longissimus and the skin.     

Cyclic and acyclic oxorhenium(V)-peptide conjugates as new ligands of the human cyclophilin hCyp-18

Peptide metalloconstructs display interesting conformations, activities, and resistance to proteolysis. However, introduction of a metal core close to the residues that interact with the protein might strongly affect the binding. We investigated the effects of a coordinated oxorhenium core on the binding of model peptides to cyclophilin hCyp-18, a protein implicated in important biological processes and several diseases. For this purpose, we synthesized a series of linear metalloconstructs bearing an oxorhenium(V) core (ReO3+), as well as a peptide cyclized through oxorhenium(V) coordination. All these peptides contain an Ala-Pro-Xaa-pNA moiety (Xaa = Cys derivative) and are anticipated to bind simultaneously to the S1-S1' and S2'-S3' subsites of hCyp-18. Therefore, the metal core is coordinated to both the cysteine residue and exogenous or endogenous NS2 tridentate systems. Cyclization of the peptide through metal coordination did not affect the affinity whereas bimolecular oxorhenium metalloconstructs bind hCyp-18 with a slightly better affinity than the corresponding nonmetalated peptide. Peptide labeling with a 99mTcO3+ core was also carried out successfully.     

Arabidopsis thaliana chromosome III restores fertility in a cytoplasmic male-sterile Brassica napus line with A. thaliana mitochondrial DNA

Somatic Brassica napus (+) Arabidopsis thaliana hybrids with a cytoplasmic male sterility (CMS)-inducing cytoplasm were screened for fertility-restored plants. One line was selected and recurrently backcrossed with the maintainer line, B. napus, resulting in fertile/sterile segregating populations. Restriction fragment length polymorphism mapping showed the co-segregation of A. thaliana chromosome (chr) III markers with the fertility trait. As it was not possible to stabilise the fertility trait via selfings, a dihaploidisation strategy was assessed. Ninety haploid plants were regenerated and analysed with numerous simple sequence length polymorphism (SSLP) markers. Markers covering both arms of A. thaliana chr III were present in two plants, whereas no A. thaliana DNA could be detected in the other plants. Following colchicine-induced chromosome doubling only these two plants with A. thaliana DNA produced fertile offspring. In one of the two lines, however, the A. thaliana-specific DNA markers and fertility were lost in subsequent generations. The other line remained fertile after repeated selfings. Using genomic in situ hybridisation (GISH) we were able to demonstrate that this latter line possessed a disomic addition of the A. thaliana chromosome. The restored line was comparable to the maintainer line with respect to flower morphology, but the petals and stamens were slightly reduced in size. The homeotic conversion of stamens to pistil-like structures, which is typical for the CMS line, was reversed, and stamens with a normal appearance with viable pollen appeared. Flowering time was as in the CMS line—in both lines it was delayed in comparison to the maintainer line. The introgressed chromosome also contributes to several pleiotropic effects, such as reduced leaf crinkling and shorter stems. The ability to restore fertility through the introgression of nuclear genes from the main cytoplasmic donor species indicates that the CMS trait in this system mainly is due to B. napus/A. thaliana alloplasmic incompatibility and not mitochondrial DNA rearrangements. Further exploitation of the material is discussed.Communicated by C. Möllers     

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.     

Production of soluble P-selectin by platelets and endothelial cells

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.