Discrete mutations in the presequence of potato formate dehydrogenase inhibit the in vivo targeting of GFP fusions into mitochondria

Most mitochondrial proteins are encoded by the nucleus, translated in the cytosol, and imported. Mitochondrial precursors generally contain their targeting information in a cleavable N-terminal presequence, which is rich in hydroxylated and positively charged residues and can form amphiphilic alpha-helices. We report the in vivo targeting of green fluorescent protein (GFP) by the FDH presequence, as well as several truncated or mutated variants. Some of these mutations modify the amphiphilicity of the predicted alpha-helix. The removal of the first two residues abolishes import and some single amino acid mutations strongly inhibit import. Such strong effects on import had not been observed in similar studies on other plant mitochondrial presequences, suggesting that the FDH presequence is a particularly good model for functional studies.     

Mitochondrial localization of Smad5 in a human chondrogenic cell line

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily and regulate the formation of cartilage and bone tissues as well as other key events during development. TGF-beta superfamily signaling is mediated intracellularly by Smad proteins, some of which can translocate into the cell nucleus and influence gene expression. Although much progress has been made in understanding how TGF-beta superfamily signaling regulates expression of target genes, little formal proof has been presented regarding the intracellular distribution of the Smad proteins before their entry into the nucleus. In the literature, non-nuclear Smad proteins are generally referred to as cytoplasmic. Using confocal microscopy, we here show for the first time that immunofluorescent labeling of Smad5, one of the Smad proteins associated with BMP signaling, colocalizes with the mitochondrion-specific probe MitoTracker, demonstrating a mitochondrial distribution of Smad5 in non-stimulated chondroprogenitor cells.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions

Cold preservation results in cell death via iron-dependent formation of reactive oxygen species, leading to apoptosis during rewarming. We aimed to study cold-induced damage (i.e., injury as a consequence of hypothermia itself and not cold ischemia) in proximal tubular cells (PTC) in various preservation solutions presently applied and to clarify the role of mitochondria in this injury. Primary cultures of rat PTC were incubated at 4 degrees C for 24 h in culture medium, UW, Euro-Collins or HTK solution with and without the iron chelator desferal and rewarmed at 37 degrees C in culture medium. Cell damage, morphology, and apoptosis were studied and mitochondrial membrane potential was assessed by fluorescence microscopy. Cold incubation of PTC in culture medium followed by rewarming caused marked cell damage compared to warm incubation alone (LDH release 39+/-10% vs. 1.6+/-0.3%). Cold-induced damage was aggravated in all preservation solutions (LDH release 85+/-2% for UW; similar in Euro-Collins and HTK). After rewarming, cells showed features suggestive for apoptosis. Desferal prevented cell injury in all solutions (e.g., 8+/-2% for UW). Mitochondrial membrane potential was lost during rewarming and this loss could also be inhibited by desferal. Trifluoperazine, which is known to inhibit mitochondrial permeability transition (MPT), was able to prevent cold-induced injury (LDH 85+/-5% vs. 12+/-2%). We conclude that cold-induced injury occurs in PTC and is aggravated by UW, Euro-Collins, and HTK solution. Iron-dependent MPT is suggested to play a role in this damage. Strategies to prevent cold-induced injury should aim at reducing the availability of "free" iron.     

Generation and maintenance of synchrony in Saccharomyces cerevisiae continuous culture

Cultures of Saccharomyces cerevisiae grown continuously produce an autonomous oscillation in many metabolic outputs. The most conveniently measured variable, i.e., dissolved oxygen concentration, oscillates with a period of 40-55 min. Previously we have identified two compounds capable of resetting phase, acetaldehyde and hydrogen sulfide. The phase-response curves constructed for acetaldehyde show a strong (Type 0) response at 3.0 mM and a weak (Type 1) response at 1.0 mM. Ammonium sulfide phase-response curves (pulse injected at 1.0 microM and 3.0 microM) revealed that sulfide is only an effective perturbation agent when endogenous sulfide concentrations are at a maximum. Also only Type 1 phase responses were observed. When the phase-response curve for sulfite (at 3.0 M) was constructed, phase responses were at a maximum at 60 degrees, indicating the possible involvement of sulfite in cell synchronization. It is concluded that endogenously produced acetaldehyde and sulfite tune the oscillation of mitochondrial energization state whereas sulfide mediates population synchrony.     

Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil

By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.     

A microcalorimetric study of the effect of La3+ on mitochondria isolated from Star-Cross 288 chicken liver tissue cells

The thermogenic curves of the metabolism of mitochondria isolated from the liver of chicken Star-Cross 288 and the effect of La³⁺ on it were studied by using an LKB-2277 BioActivity Monitor, ampoule method, at 37°C. After isolation from the chicken liver tissue, mitochondria still have metabolic activity and can live for a long time by using the stored nutrients. From the thermogenic curves, we obtained the thermokinetic equations under different condition. The kinetic study shows that La³⁺ has changed the metabolism completely.     

Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion

Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.     

Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D-loop region sequences, we designed four species-specific primers (for products 130-161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large-scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.