The permeability transition and BCL-2 family proteins in apoptosis: co-conspirators or independent agents?
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Animals↗
Biomedical subjects
M Forte
Publications and source records attributed to M Forte.
The evaluation of uncertainty in low-level LSC measurements of water samples.
The uncertainty in measurements of gross alpha and beta activities in water samples by liquid scintillation counting with alpha/beta discrimination has been evaluated considering the problems typical of low-level measurements of environmental samples. The use of a pulse shape analysis device to discriminate alpha and beta events introduces a correlation between some of the input quantities, and it has to be considered. Main contributors to total uncertainty have been assessed by specifically designed experimental tests. Results have been fully examined and discussed.
Alpha Particles↗
Cardiolipin and phosphatidylglycerol are not required for the in vivo action of Bcl-2 family proteins.
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Apoptosis↗
Assessment of drinking water radioactivity content by liquid scintillation counting: set up of high sensitivity and emergency procedures.
In our institute, different procedures have been developed to measure the radioactivity content of drinking water both in normal and in emergency situations, such as those arising from accidental and terrorist events. A single radiometric technique, namely low level liquid scintillation counting (LSC), has been used. In emergency situations a gross activity screening is carried out without any sample treatment by a single and quick liquid scintillation counting. Alpha and beta activities can be measured in more than one hundred samples per day with sensitivities of a few Bq/L. Higher sensitivity gross alpha and beta, uranium and radium measurements can be performed on water samples after specific sample treatments. The sequential method proposed is designed in such a way that the same water sample can be used in all the stages, with slight modifications. This sequential procedure was applied in a survey of the Lombardia district. At first tap waters of the 13 largest towns were examined, then a more detailed monitoring was carried out in the surroundings of Milano and Lodi towns. The high sensitivity method for the determination of uranium isotopes was used to check the presence of depleted uranium in Lake Garda. Reduced equipment requirements and relative readiness of radiochemical procedures make LSC an attractive technique which can also be applied by laboratories lacking specific radiochemistry facilities and experience.
Emergencies↗
The monitoring of tap waters in Milano: planning, methods and results.
The radiometric monitoring of tap waters has been carried out, in Northern Italy, since late the 1980s as part of the activities headed by the National Surveillance Network on environmental radioactivity. Measurements were accomplished by gamma spectrometry over large samples of water that were drained in a single place and concentrated by ion exchange resin treatment. More recently a regular, periodical monitoring was started using liquid scintillation measurements. In this manner, gross alpha and beta activities, uranium, (226)Ra and (222)Rn concentrations are determined at present. Nevertheless, a single-point sampling may be not fully representative of the whole Milano aqueduct, significantly extended and fed by a number of wells. As a control, an extended monitoring of the aqueduct was planned and variations of water radioactivity in different Milano areas have been assessed.
Chromatography, Ion Exchange↗
Reliability of two calculation codes for efficiency calibrations of HPGe detectors.
This paper reports the reliability of efficiency calibrations for gamma-ray detectors using the calculation codes ANGLE and LabSOCS. For experimental verification, three HPGe detectors under various laboratory geometry configurations were used for this study. An overall comparison between experimental and calculated efficiency calibration curves is presented and comments on the various error sources affecting the final results are given. The deviations are generally below 10%, which could be acceptable for many applications.
Journal Article↗
Heterotrimeric G proteins direct two modes of asymmetric cell division in the Drosophila nervous system.
In Drosophila, distinct mechanisms orient asymmetric cell division along the apical-basal axis in neuroblasts and along the anterior-posterior axis in sensory organ precursor (SOP) cells. Here, we show that heterotrimeric G proteins are essential for asymmetric cell division in both cell types. The G protein subunit G(alpha)i localizes apically in neuroblasts and anteriorly in SOP cells before and during mitosis. Interfering with G protein function by G(alpha)i overexpression or depletion of heterotrimeric G protein complexes causes defects in spindle orientation and asymmetric localization of determinants. G(alpha)i is colocalized and associated with Pins, a protein that induces the release of the betagamma subunit and might act as a receptor-independent G protein activator. Thus, asymmetric activation of heterotrimeric G proteins by a receptor-independent mechanism may orient asymmetric cell divisions in different cell types.
Animals↗
Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.
Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.
Amino Acid Sequence↗
A mitochondrial perspective on cell death.
The role of mitochondria as crucial participants in cell death programs is well established, yet the mechanisms responsible for the release of mitochondrial activators and the role of BCL2 family proteins in this process remain controversial. Here, we point out the limitations of current approaches used to monitor the physiological responses of mitochondria during cell death, the implications arising from modern views of mitochondrial structure, and briefly assess two proposed mechanisms for the release of mitochondrial proteins during apoptosis.
Animals↗
Genetic analysis of the Drosophila Gs(alpha) gene.
One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.
Adenylyl Cyclases↗
Determination of uranium isotopes in food and environmental samples by different techniques: a comparison.
The uranium concentration in 59 samples of bottled and tap water, mainly from northern Italy, was measured by different techniques. Results obtained by inductively coupled plasma mass spectrometry (ICP-MS), semiconductor alpha spectrometry and low level liquid scintillation counting with alpha/beta discrimination (LSC) have been compared. High resolution gamma spectrometry and semiconductor alpha spectrometry have been used to analyse uranium in a variety of organic and inorganic samples. Isotopic secular equilibrium in the 238U series may be lacking or hidden by auto-absorption phenomena, so caution should be used in evaluating gamma spectrometry data. Alpha spectrometry has also been used to ascertain the possible pollution from depleted uranium in the environment.
Food Contamination, Radioactive↗
Ectopic G-protein expression in dopamine and serotonin neurons blocks cocaine sensitization in Drosophila melanogaster.
Sensitization to repeated doses of psychostimulants is thought to be an important component underlying the addictive process in humans [1] [2] [3] [4]. In all vertebrate animal models, including humans [5], and even in fruit flies, sensitization is observed after repeated exposure to volatilized crack cocaine [6]. In vertebrates, sensitization is thought to be initiated by processes occurring in brain regions that contain dopamine cell bodies [2] [7]. Here, we show that modulated cell signaling in the Drosophila dopamine and serotonin neurons plays an essential role in cocaine sensitization. Targeted expression of either a stimulatory (Galpha(s)) or inhibitory (Galpha(i)) Galpha subunit, or tetanus toxin light chain (TNT) in dopamine and serotonin neurons of living flies blocked behavioral sensitization to repeated cocaine exposures. These flies showed alterations in their initial cocaine responsiveness that correlated with compensatory adaptations of postsynaptic receptor sensitivity. Finally, repeated drug stimulation of a nerve cord preparation that is postsynaptic to the brain amine cells failed to induce sensitization, further showing the importance of presynaptic modulation in sensitization.
Animals↗
Evaluation of two nested polymerase chain reactions for diagnosis of Pneumocystis carinii pneumonia in immunocompromised patients.
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Bronchoalveolar Lavage Fluid↗
Biochemical and genetic analysis of the mitochondrial response of yeast to BAX and BCL-X(L).
The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X(L)) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X(L) using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X(L) prevented all BAX-mediated responses. We also assessed the function of BCL-X(L) and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic beta subunit or the delta subunit of the F(0)F(1)-ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho(0)] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the beta subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X(L) protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X(L): cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.
Apoptosis↗
Modulation of the light response by cAMP in Drosophila photoreceptors.
Phototransduction in Drosophila is mediated by a G-protein-coupled phospholipase C transduction cascade in which each absorbed photon generates a discrete electrical event, the quantum bump. In whole-cell voltage-clamp recordings, cAMP, as well as its nonhydrolyzable and membrane-permeant analogs 8-bromo-cAMP (8-Br-cAMP) and dibutyryl-cAMP, slowed down the macroscopic light response by increasing quantum bump latency, without changes in bump amplitude or duration. In contrast, cGMP or 8-Br-cGMP had no effect on light response amplitude or kinetics. None of the cyclic nucleotides activated any channels in the plasma membrane. The effects of cAMP were mimicked by application of the non-specific phosphodiesterase inhibitor IBMX and the adenylyl cyclase activator forskolin; zaprinast, a specific cGMP-phosphodiesterase inhibitor, was ineffective. Bump latency was also increased by targeted expression of either an activated G(s) alpha subunit, which increased endogenous adenylyl cyclase activity, or an activated catalytic protein kinase A (PKA) subunit. The action of IBMX was blocked by pretreatment with the PKA inhibitor H-89. The effects of cAMP were abolished in mutants of the ninaC gene, suggesting this nonconventional myosin as a possible target for PKA-mediated phosphorylation. Dopamine (10 microM) and octopamine (100 microM) mimicked the effects of cAMP. These results indicate the existence of a G-protein-coupled adenylyl cyclase pathway in Drosophila photoreceptors, which modulates the phospholipase C-based phototransduction cascade.
Animals↗
Mapping of the human Voltage-Dependent Anion Channel isoforms 1 and 2 reconsidered.
Eukaryotic porins or VDACs (Voltage-Dependent Anion-selective Channels) are integral membrane proteins forming large hydrophilic pores. Three functioning genes for VDAC isoforms have been detected in mouse and the corresponding cDNAs are known also in humans. Tissue-specific VDAC isoform 1 (HVDAC1) deficiency in human skeletal muscle is responsible of a rare mitochondrial encephalomyopathy, fatal in childhood. Since coding sequences are not affected in the patient, we focused our interest in the gene structure. HVDAC1 and 2 have been previously mapped at chromosomes Xq13-21 and 21, respectively. Screening of an human chromosome X cosmid library resulted only in the isolation of processed pseudogenes, finely mapped at Xq22 and Xp11.2. Here, we report the mapping of HVDAC1 to chromosome 5q31 and HVDAC2 to chromosome 10q22 by FISH. Exon/intron probes, designed on the basis of the mouse gene structures, were obtained by long extension PCR amplification using the whole genomic DNA as a template. The sequence of the probe extremities clearly pointed to a genuine VDAC genomic sequence. Human and mouse regions where VDAC 1 and 2 genes were mapped are known to be synthetic, thus reinforcing the mapping of the human homologues.
Chromosome Mapping↗
Mutations in the voltage-dependent anion channel of the mitochondrial outer membrane cause a dominant nonlethal growth impairment.
Point mutations at K234 and K236 in the yeast voltage-dependent anion channel 1 (VDAC1) of the mitochondrial outer membrane have been shown to markedly impair the membrane insertion of this protein (Smith et al., 1995; Angeles et al., 1998). Mutants of this type were expressed in vivo in a strain of yeast with a disruption in the VDAC1 gene. Expression of the various VDAC1 forms was under the control of a Gall promoter. Wild-type VDAC1 expression fully complemented the slow growth phenotype caused by the disruption. VDAC1 mutants in which K234 and K236 were replaced by arginine, glutamate, or glutamine caused a more severe negative effect on growth. This effect appeared to be dominant since the mutant VDAC1 forms suppressed growth in a yeast strain that retained its VDAC1 gene. This apparent dominant negative effect on growth did not seem to be specific for any stage of the cell cycle. However, the growth defect was not lethal as the affected cells still could accumulate the vital stain, FUN1. Expression of a mutant in which K234 had been replaced by glutamate had more serious negative growth effects than did a similar mutation at K236. Expression of delta71-116 VDAC1 complemented the VDAC1 disruption; however, expression of the same deletion mutant in which the lysines corresponding to K234 and K236 were mutated to glutamate severely impaired growth. These results have shown that a deficiency of lysine at positions 234 and 236 in VDAC I causes a nonlethal growth defect that is more severe than deletion of 45 amino acids from VDACI or disruption of the VDAC1 gene. They also indicate that there is a hierarchy in the importance of these lysines with mutations at K234 being the more serious.
Gene Expression Regulation, Fungal↗
The topology of VDAC as probed by biotin modification.
The outer membrane of mitochondria contains channels called VDAC (mitochondrial porin), which are formed by a single 30-kDa protein. Cysteine residues introduced by site-directed mutagenesis at sites throughout Neurospora crassa VDAC (naturally devoid of cysteine) were specifically biotinylated prior to reconstitution into planar phospholipid membranes. From previous studies, binding of streptavidin to single biotinylated sites results in one of two effects: reduced single-channel conductance without blockage of voltage gating (type 1) or locking of the channels in a closed conformation (type 2). All sites react with streptavidin only from one side of the membrane. Here, we extend this approach to VDAC molecules containing two cysteines and determine the location of each biotinylated residue with respect to the other within the membrane. When a combination of a type 1 and a type 2 site was used, each site could be observed to react with streptavidin. Two sets of sites located on opposite surfaces of the membrane were identified, thereby establishing the transmembrane topology of VDAC. A revised folding pattern for VDAC, consisting of 1 alpha helix and 13 beta strands, is proposed by combining these results with previously obtained information on which sites are lining the aqueous pore.
Amino Acid Sequence↗