Plant cells integrate signals from external sources and from organelles to regulate gene expression, referred to as anterograde and retrograde signalling respectively. Functional characterisation of the promoter of alternative oxidase AOX1a from Arabidopsis thaliana, a marker for mitochondrial retrograde response, was carried out by testing the ability of the AOX1a promoter to drive expression of the reporter gene beta-glucuronidase (GUS). This approach identified a strong repressor element, designated the B element, that was necessary for an increased promoter activity in response to the mitochondrial complex I inhibitor rotenone. This element overlaps with a previously identified potential binding site for the transcription factor abcisic acid insensitive 4 (ABI4). AOX1a promoter activity was fully de-repressed in abi4 mutants, and was unresponsive to rotenone. Furthermore, deletion of the B element of the AOX1a promoter resulted in increased GUS staining activity compared to the wild type promoter in transgenic plants. Binding of the ABI4 transcription factor to this region of the AOX1a promoter was demonstrated by electromobility shift and yeast 1-hybrid assays. Analysis of transcript abundance for AOX1a in abi4 mutant lines revealed significantly increased levels of AOX1a mRNA that could not be further induced by rotenone, consistent with the role of ABI4 as a repressor that is de-repressed in response to rotenone. These results show that ABI4 plays a central role in mediating mitochondrial retrograde signals to induce the expression of AOX1a. Furthermore, they provide a molecular link between mitochondrial and chloroplast retrograde signalling, as ABI4 has been previously shown to act downstream of at least two chloroplast retrograde signalling pathways.

Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-2(-/-) mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with E1A and Ras oncogenes, caspase-2(-/-) MEFs grew significantly faster than caspase-2(+/+) MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse Emu-Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-2(-/-) mice. The caspase-2(-/-) cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-2(-/-) MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following gamma-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein.

We aimed to analyze the outcomes and cost-effectiveness of gamma knife radiosurgery (GKRS) and whole brain radiotherapy (WBRT) for multiple metastatic brain tumors. Over a period of 5 years, 156 patients with multiple metastatic brain tumors were enrolled and freely assigned by the referring doctors to either gamma knife radiosurgery (GKRS, Group A, n=56), or to whole brain radiotherapy (WBRT, Group B, n=100). The follow-up time was set at 1200 days (3.3 years) post-treatment. The number of tumors, patient age, extent of systemic disease and Karnofsky performance scale (KPS) score, were recorded and recursive partitioning analysis used. The outcomes analyzed were: mortality, survival time, neurological complications, post-treatment KPS score, quality-adjusted life years (QALY), and cost-effectiveness. A paired t-test was used for statistical analysis. Mortality rates for patients receiving GKRS and WBRT were 81.1% and 93.0%, respectively (p=0.05). The mortality rate was lower for GKRS (74.4%) than for WBRT (97.1%) in patients with initial KPS70 (p=0.02). The mortality rate was also significantly lower for GKRS (78.9%) than WBRT (95.5%) in patients with 2-5 tumors (p<0.05). Post-treatment KPS score (mean +/- standard deviation [s.d.] was higher for patients receiving GKRS (73.8+/-13.2) than for those receiving WBRT (45.5+/-26.0), p<0.01. The median survival time for GKRS and WBRT was 9.5 months and 8.3 months, respectively, p=0.72. The mean (+/- s.d.) QALY was 0.76+/-0.23 for GKRS and 0.59+/-0.18 for WBRT, respectively (p<0.05). The cost-effectiveness per unit of QALY was better for the GKRS treatment (US$10,381/QALY) than in the WBRT treatment (US$17,622/QALY), p<0.05. The cost-effectiveness per KPS score was also higher for the GKRS treatment (US$139/KPS score) than for WBRT (US$229/KPS score), p<0.01. Thus, the mortality rate for multiple metastatic brain tumors treated by GKRS is significantly better with a good initial KPS score and when the tumor number is 2-5. GKRS results in a better post-treatment KPS score, QALY, and higher cost-effectiveness than WBRT for treating multiple metastatic brain tumors.

In this study we analysed transcript abundance and promoters of genes encoding mitochondrial proteins to identify signalling pathways that regulate stress-induced gene expression. We used Arabidopsis alternative oxidase AOX1a, external NAD(P)H-dehydrogenase NDB2 and two additional highly stress-responsive genes, At2g21640 and BCS1. As a starting point, the promoter region of AOX1a was analysed and functional analysis identified ten cis-acting regulatory elements (CAREs), which played a role in response to treatment with H2O2, rotenone or both. Six of these elements were also functional in the NDB2 promoter. The promoter region of At2g21640, previously defined as a hallmark of oxidative stress, shared two functional CAREs with AOX1a and was responsive to treatment with H2O2 but not rotenone. Microarray analysis further supported that signalling pathways induced by H2O2 and rotenone are not identical. The promoter of BCS1 was not responsive to H2O2 or rotenone, but highly responsive to salicylic acid (SA), while the promoters of AOX1a and NDB2 were unresponsive to SA. Analysis of transcript abundance of these genes in a variety of defence signalling mutants confirmed that BCS1 expression is regulated in a different manner compared to AOX1a, NDB2 and At2g21640. These mutants also revealed a pathway, associated with programmed cell death, that regulated AOX1a in a manner distinct from the other genes. Thus at least three distinctive pathways regulate mitochondrial stress response at a transcriptional level, an SA dependent pathway represented by BCS1, a second pathway that represents a convergence point for signals generated by H2O2 and rotenone on multiple CAREs, some of which are shared between responsive genes, and a third pathway that acts via EDS1 and PAD4 regulating only AOX1a. Furthermore, post-transcriptional regulation accounts for changes in transcript abundance by SA treatment for some genes.

OBJECTIVES:: To develop a modified Intracerebral Hemorrhage Score (MICH) score to determine optimal cut-offs for conservative treatment vs surgical intervention for basal ganglia hemorrhage and to predict outcomes. DESIGN:: Prospective randomized trial. SETTING:: A 1720-bed medical center affiliated with a university. PATIENTS:: In all, 226 patients with basal ganglia hemorrhage who presented at our hospital from 2001-2005. INTERVENTIONS:: Group A (n = 113) underwent endoscopic surgery; group B (n = 113) underwent conservative treatment. Score differences on the Glasgow Outcome Scale and 1-yr Barthel Index were analyzed by chi-square test and Student's t-tests. Cut-offs for MICH scoring were evaluated using receiver operating characteristic curves for calculating the Youden Index. The treatment odds ratio (OR) was analyzed by univariate, multivariate, and multiple logistic regressions. MEASUREMENTS AND MAIN RESULTS:: The optimal cut-off point for mortality was a MICH score >/=3 in which the Youden Index is 0.66 (sensitivity, 76.3%; specificity, 89.8%; area under the receiver operating characteristic curve, 0.897). The positive and negative predictive values were 81.8% and 86.3%, respectively. The treatment OR for surgical treatment was 6.87 (95% confidence interval [CI], 3.13-14.5) at MICH scores >/=3. The best cut-off for good functional outcomes (Glasgow Outcome Scale >/=4 or Barthel index >/=55) was MICH >/=2. Conservative treatment achieved a better mean Barthel Index at MICH = 0 or 1 than surgical treatment, p < 0.01. At MICH scores = 3 or 4, 6-month mortality for conservative treatment was higher than for surgical treatment, p < 0.01 and p = 0.04, respectively. At MICH scores of 5, all patients died. CONCLUSIONS:: MICH scoring provides a simple, reliable system for treatment decisions regarding basal ganglia hemorrhage and may accurately predict functional outcomes. Conservative treatment is recommended for basal ganglia ICH patients with low MICH scores (0, 1) to preserve neurologic function. Surgery is recommended for patients with a midlevel MICH score to obtain better functional outcomes (MICH = 2) and to reduce mortality (MICH = 3 or 4). At MICH scores = 5, there are no indications for surgery.

Treatment of alternative oxidase 1a mutant plants (aox1a) with moderate light under drought conditions resulted in a phenotypic difference compared to Col-0, evidenced by a 10-fold increase in the accumulation of anthocyanins in leaves, alterations in photosynthetic efficiency, increased O2(-) and reduced root growth at the early stages of seedling growth. Analysis of metabolite profiles revealed significant changes upon treatment in aox1a plants typical of combined stress treatments and these were less pronounced or absent in Col-0 plants. These changes were accompanied by alteration in the abundance of a variety of transcripts during the stress treatment, providing a molecular fingerprint for the stress-induced phenotype of aox1a plants. Transcripts encoding proteins involved in the synthesis of anthocyanins, transcription factors, chloroplastic and mitochondrial components, cell wall synthesis, sucrose and starch metabolism changed indicating that effects were not confined to mitochondria where the AOX1a protein is located. Microarray and QRT-PCR analysis revealed that transcripts typically induced upon stress treatment or involved in anti-oxidant defence systems, especially chloroplast located anti-oxidant defence components, had altered basal levels in untreated aox1a plants, suggesting a significant change in the basal equilibrium of signalling pathways that regulate these components. Taken together, these results indicate aox1a plants have a greatly altered stress response even when mitochondria or the mitochondrial electron transport chain are not the primary target of the stress and that AOX1a plays a broad role in determining the normal REDOX balance in the cell.

We study the anomalous conductance plateau around G=0.7(2e;{2}/h) and the zero bias anomaly in ballistic hole quantum wires with respect to in-plane magnetic fields applied parallel B_{ parallel} and perpendicular B_{ perpendicular} to the quantum wire. As seen in electron quantum wires, the magnetic fields shift the 0.7 structure down to G=0.5(2e;{2}/h) and simultaneously quench the zero bias anomaly. However, these effects are strongly dependent on the orientation of the magnetic field, owing to the highly anisotropic effective Landé g-factor g;{*} in hole quantum wires. Our results highlight the fundamental role that spin plays in both the 0.7 structure and zero bias anomaly.

Caspase-2 is one of the most conserved caspases, yet its biological function remains a matter of controversy. In the present article we analysed mouse embryonic fibroblasts (MEFs) from caspase-2 knockout mice for their sensitivity to various apoptosis inducing agents. We found that cell death induced by drugs that disrupt cytoskeleton is significantly inhibited in Casp2(-/-) MEFs. These drugs included zoledronic acid, vincristine, cytochalasin D and paclitaxel. We demonstrate that MEFs lacking Casp2 show clonogenic survival following drug treatment, whereas all Casp2(+/+) MEFs die, indicating that caspase-2 is required for apoptosis induced by cytoskeletal disruption. We further found that caspase-2 mediates apoptosis via Piddosome, Bid and Bax activation, and cytochrome c release. In the absence of caspase-2, Bid and Bax activation, and cytochrome c release are significantly delayed following drug treatment. Our data provide strong support for a context-dependent function of caspase-2 in apoptosis.Oncogene advance online publication, 14 January 2008; doi:10.1038/sj.onc.1211005.

The role of plant mitochondrial outer membrane proteins in the process of preprotein import was investigated, as some of the principal components characterized in yeast have been shown to be absent or evolutionarily distinct in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit translocase of the outer membrane complex. Simultaneous inactivation of two of the three TOM20 genes changed the rate of import for some precursor proteins, revealing limited isoform subfunctionalization. Inactivation of all three TOM20 genes resulted in severely reduced rates of import for some but not all precursor proteins. The outer membrane protein METAXIN was characterized to play a role in the import of mitochondrial precursor proteins and likely plays a role in the assembly of beta-barrel proteins into the outer membrane. An outer mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity to a chloroplast import receptor was shown to interact with a variety of precursor proteins. All three proteins have domains exposed to the cytosol and interacted with a variety of precursor proteins, as determined by pull-down and yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted in protein abundance changes in the others, suggesting functional redundancy. Thus, it is proposed that all three components directly interact with precursor proteins to participate in early stages of mitochondrial protein import.

The IL-1-related molecules, IL-1 and IL-18, can promote Th2 cytokine production by IgE/antigen-FcepsilonRI-stimulated mouse mast cells. Another IL-1-related molecule, IL-33, was identified recently as a ligand for T1/ST2. Although mouse mast cells constitutively express ST2, the effects of IL-33 on mast cell function are poorly understood. We found that IL-33, but not IL-1beta or IL-18, induced IL-13 and IL-6 production by mouse bone marrow-derived, cultured mast cells (BMCMCs) independently of IgE. In BMCMCs incubated with the potently cytokinergic SPE-7 IgE without specific antigen, IL-33, IL-1beta, and IL-18 each promoted IL-13 and IL-6 production, but the effects of IL-33 were more potent than those of IL-1beta or IL-18. IL-33 promoted cytokine production via a MyD88-dependent but Toll/IL-1R domain-containing adaptor-inducing IFN-beta-independent pathway. By contrast, IL-33 neither induced nor enhanced mast cell degranulation. At 200 ng/ml, IL-33 prolonged mast cell survival in the absence of IgE and impaired survival in the presence of SPE-7 IgE, whereas at 100 ng/ml, IL-33 had no effect on mast cell survival in the absence of IgE and reduced mast cell survival in the presence of IgE. These observations suggest potential roles for IL-33 in mast cell- and Th2 cytokine-associated immune responses and disorders.