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PubMed1996

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging.

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron-specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 microns) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.

Animals↗
PubMed2001

EXIT procedure in a twin gestation and review of the literature.

Prenatal diagnosis can show masses of the fetal neck, mouth, and face that can potentially cause respiratory distress at birth. To prevent such an emergency, the EXIT (ex utero intrapartum technique) is performed: it is the intrapartum intubation of the fetus at term while still connected to the placenta. The EXIT procedure was first performed in a case of cervical teratoma. Up to now a total of 34 cases are described, mostly cervical teratomas (13 cases), lymphangiomas (7), epignathus (3); babies' outcome has been successful in 25 of them, with one death related to the procedure. Among the reported cases we are aware of only one where EXIT was performed in a twin gestation, in which the normal twin was delivered first. In our case the normal fetus was posterior to the twin with cervical malformation, requiring us to work on the latter while the former was still in the uterus. After having safely secured the airway in twin A, twin B was prompt delivered with excellent general conditions. Our limited experience enlarges the possibility to perform this prenatal procedure even in "nonstandard" conditions, such as a twin gestation, and may prove useful to those who are going to deal with such issues.

Adult↗
PubMed2000

Successful EXIT (ex utero intrapartum treatment) procedure in a fetus diagnosed prenatally with congenital high-airway obstruction syndrome due to laryngeal atresia.

Congenital high-airway obstruction syndrome (CHAOS) is due to rare malformations and has been reported previously in only few cases. If the diagnosis can be made prenatally, the ex utero intrapartum treatment (EXIT) procedure may be life-saving. A healthy 28-year old nulli-para was referred because of isolated ascites found at gestational week 16 during routine ultrasound scan. Repeated scans showed overdistended hyperechogenic lungs with inverted diaphragm and a dilated trachea, which was interpreted as a CHAOS resulting from laryngeal atresia. The ascites eventually disappeared. An EXIT procedure was performed at 35 weeks of gestation. Anesthesia of the mother was induced with thiopental, succinylcholine and fentanyl followed by intubation, and maintained with isoflurane and nitrous oxide. A low abdominal midline incision was performed followed by a low transverse incision of the uterus. The fetal head, right arm and shoulder were delivered and intramuscular anesthesia was administered to the fetus. Immediate laryngoscopy confirmed the diagnosis and a tracheostomy was therefore performed. Surfactant was given after a few minutes of ventilation. Compliance improved and when the fetus was easy to ventilate, it was delivered. The baby is developing normally at 18 months of age. Surgical correction of the malformation will be performed after two years of age. It is concluded that some fetuses with a prenatal diagnosis of CHAOS can benefit from the EXIT procedure at delivery. This necessitates a multidisciplinary management team.

Adult↗
PubMed2004

Exit interaction effect on nascent product state distribution of O(1D)+N2O-->NO+NO.

We have determined the rotational state distributions of NO(v'=0,1,2) products produced from the reaction O(1D)+N2O. This is the first full characterization of the product rotational distribution of this reaction. The main part of each rotational distribution (up to j' approximately 80) has rotational temperature approximately 20,000 K and all these distributions are quite near to those predicted by the phase space theory (PST). This observation and previously reported vibrational distribution indicate that the most part of the energy partitioning of the reaction products is at least apparently statistical although the intermediate of this reaction is not so stable as to ensure the long lifetime. On the other hand, the distributions in the high rotational levels (j'=80-100) are found to decrease more sharply as j' increases than the PST predictions. The origin of the observed decrease of the distribution is discussed with quasiclassical trajectory (QCT) calculations on a five-dimensional ab initio potential energy surface (PES). The observed near-statistical distribution and the sharp decrease in the high-j' levels are well reproduced by a "half-collision" QCT calculation, where statistical distribution at the reaction intermediate is assumed. This agreement shows the rotation-translation interaction in the exit region has an effect of yielding small high-j' populations. However, a little bias of the calculated distribution toward lower rotational excitation than the observed one indicates that the combination of the statistical intermediate and the exit interaction on the current PES does not completely describe the real system. It is suggested that the reaction intermediate is generated with the distribution which is close to statistical but a little biased toward yielding high-j' products, and that the interaction in the exit region of the PES results in the sharp decrease in the high-j' levels.

Journal Article↗
PubMed2005

DNA damage-induced mitotic catastrophe is mediated by the Chk1-dependent mitotic exit DNA damage checkpoint.

Mitotic catastrophe is the response of mammalian cells to mitotic DNA damage. It produces tetraploid cells with a range of different nuclear morphologies from binucleated to multimicronucleated. In response to DNA damage, checkpoints are activated to delay cell cycle progression and to coordinate repair. Cells in different cell cycle phases use different mechanisms to arrest their cell cycle progression. It has remained unclear whether the termination of mitosis in a mitotic catastrophe is regulated by DNA damage checkpoints. Here, we report the presence of a mitotic exit DNA damage checkpoint in mammalian cells. This checkpoint delays mitotic exit and prevents cytokinesis and, thereby, is responsible for mitotic catastrophe. The DNA damage-induced mitotic exit delay correlates with the inhibition of Cdh1 activation and the attenuated degradation of cyclin B1. We demonstrate that the checkpoint is Chk1-dependent.

BRCA1 Protein↗
PubMed1999

Proton exit from the heme-copper oxidase of Escherichia coli.

Pathways of proton entry have been identified in the proton-translocating heme-copper oxidases, but the proton exit pathway is unknown. Here we report experiments with cytochrome bo3 in Escherichia coli cells that may identify the beginning of the exit pathway. Systematic mutations of arginines 438 and 439 (R481 and R482 in the E. coli enzyme), numbering as in cytochrome aa3 from bovine heart mitochondria, which interact with the ring D propionates of the two heme groups, reveal that the D propionate of the oxygen-binding heme is involved in proton pumping; its anionic form must be stabilized in order for proton translocation to occur. This may locate the beginning of the pathway by which pumped protons exit from the enzyme structure.

Biological Transport↗
PubMed2001

A membrane protein enriched in endoplasmic reticulum exit sites interacts with COPII.

Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER). We report here that a mammalian protein (Yip1A) that is about 31% identical to S. cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport. Yip1A transcripts are ubiquitously expressed. Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart. Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region. The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex. Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites. Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus. A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport. Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24. A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction. Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.

Amino Acid Sequence↗
PubMedDate not supplied

Comprehensive schoolteachers at risk of early exit from work.

Risk of early exit from work for teachers was operationalized as high burnout scores, working part-time due to heavy burden and illness or working part-time while also receiving partial disability pension. Data were collected by mailed questionnaires in a cross-sectional study to a random sample of Norwegian comprehensive schoolteachers, response rate = 86% (N = 1860 valid cases). High age increased the risk of early exit from work, but for cynicism the age effect disappeared when sense of competence and stress were introduced in the regression model. Age had no effect for low professional efficacy. Sense of competence effected burnout, but actual competence level and the gap between actual competence and teaching obligations did not. Stress effected all measures of risk of early exit, especially exhaustion. Change as stress factor increased the exhaustion scores, and were also relevant to risk of having a part-time position, and/or partial disability pension.

Adult↗
PubMed1993

The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected.

We have characterized a new CHO cell line (12-4) derived from a parental line, TRVb-1, that expresses the human transferrin receptor. This mutant belongs to the end2 complementation group of endocytosis mutants. Like other end2 mutants, the endosomes in 12-4 cells show a partial acidification defect. These cells internalize LDL and transferrin at 70% of the rate of parental cells and externalize transferrin at 55% of the parental rate (Johnson, L. S., J. F. Presley, J. C. Park, and T. E. McGraw. J. Cell Physiol. 1993). In this report, we have used fluorescence microscopy to determine which step in receptor trafficking is affected in the mutants. Transferrin is sorted from LDL and is delivered to a peri-centriolar recycling compartment at rates similar to parental cells. However, the rate constant for exit of transferrin from the recycling compartment in mutant cells is 0.025 min-1 vs 0.062 min-1 in the parental line. We also measured the trafficking of a bulk membrane marker, 6-[N-[7-nitrobenzo-2-oxa-1,3-diazol-4-yl]-amino]hexanoyl- sphingosylphosphorylcholine (C6-NBD-SM) that labels the exofacial side of the plasma membrane. C6-NBD-SM enters the same recycling compartment as transferrin, and it exits the recycling compartment at a rate of 0.060-0.065 min-1 in both parental and 12-4 cells. We conclude that bulk membrane flow in the recycling pathway of 12-4 cells is normal, but exit of transferrin from the recycling compartment is slowed due to retention in this compartment. Thus, in the mutant cell line the recycling compartment carries out a sorting function, retaining transferrin over bulk membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed1998

A single point mutation controls the cholesterol dependence of Semliki Forest virus entry and exit.

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

Amino Acid Sequence↗
PubMed2003

The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER.

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1-1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1-1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

Cells, Cultured↗
PubMed1994

Analysis of photon beam exit dose using photon point kernels.

The Monte Carlo method is used to analyse the dose fall-off at the exit surface of a megavoltage photon beam. The convolution/superposition method of dose calculation using Monte-Carlo-generated homogeneous photon kernels is shown to be in error for exit dose calculation. Instead, photon kernels that incorporate modelling of the exit surface were generated, also using Monte Carlo, to analyse the problem, and the calculated dose fall-off using these kernels agrees well with measured data. In addition, the physics underlying the characteristics of the dose fall-off is analysed based on complete Monte Carlo modelling. Practical improvements to the convolution/superposition method are suggested.

Algorithms↗
PubMed2002

Structure of the Golgi and distribution of reporter molecules at 20 degrees C reveals the complexity of the exit compartments.

Incubating cells at 20 degrees C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20 degrees C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with "cisternal" domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.

Animals↗
PubMed1996

Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.

Animals↗
PubMed2004

Genomic analysis of stationary-phase and exit in Saccharomyces cerevisiae: gene expression and identification of novel essential genes.

Most cells on earth exist in a quiescent state. In yeast, quiescence is induced by carbon starvation, and exit occurs when a carbon source becomes available. To understand how cells survive in, and exit from this state, mRNA abundance was examined using oligonucleotide-based microarrays and quantitative reverse transcription-polymerase chain reaction. Cells in stationary-phase cultures exhibited a coordinated response within 5-10 min of refeeding. Levels of >1800 mRNAs increased dramatically (>or=64-fold), and a smaller group of stationary-phase mRNAs decreased in abundance. Motif analysis of sequences upstream of genes clustered by VxInsight identified an overrepresentation of Rap1p and BUF (RPA) binding sites in genes whose mRNA levels rapidly increased during exit. Examination of 95 strains carrying deletions in stationary-phase genes induced identified 32 genes essential for survival in stationary-phase at 37 degrees C. Analysis of these genes suggests that mitochondrial function is critical for entry into stationary-phase and that posttranslational modifications and protection from oxidative stress become important later. The phylogenetic conservation of stationary-phase genes, and our findings that two-thirds of the essential stationary-phase genes have human homologues and of these, many have human homologues that are disease related, demonstrate that yeast is a bona fide model system for studying the quiescent state of eukaryotic cells.

Base Sequence↗
PubMed2004

Conformational defects slow Golgi exit, block oligomerization, and reduce raft affinity of caveolin-1 mutant proteins.

Caveolin-1, a structural protein of caveolae, is cleared unusually slowly from the Golgi apparatus during biosynthetic transport. Furthermore, several caveolin-1 mutant proteins accumulate in the Golgi apparatus. We examined this behavior further in this mutant study. Golgi accumulation probably resulted from loss of Golgi exit information, not exposure of cryptic retention signals, because several deletion mutants accumulated in the Golgi apparatus. Alterations throughout the protein caused Golgi accumulation. Thus, most probably acted indirectly, by affecting overall conformation, rather than by disrupting specific Golgi exit motifs. Consistent with this idea, almost all the Golgi-localized mutant proteins failed to oligomerize normally (even with an intact oligomerization domain), and they showed reduced raft affinity in an in vitro detergent-insolubility assay. A few mutant proteins formed unstable oligomers that migrated unusually slowly on blue native gels. Only one mutant protein, which lacked the first half of the N-terminal hydrophilic domain, accumulated in the Golgi apparatus despite normal oligomerization and raft association. These results suggested that transport of caveolin-1 through the Golgi apparatus is unusually difficult. The conformation of caveolin-1 may be optimized to overcome this difficulty, but remain very sensitive to mutation. Disrupting conformation can coordinately affect oligomerization, raft affinity, and Golgi exit of caveolin-1.

Amino Acid Sequence↗
PubMed2005

Phosphatase 2A negatively regulates mitotic exit in Saccharomyces cerevisiae.

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

Cell Cycle Proteins↗
PubMed2005

The mitotic exit network Mob1p-Dbf2p kinase complex localizes to the nucleus and regulates passenger protein localization.

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved signaling network that coordinates CDK inactivation, cytokinesis and G1 gene transcription. The MEN Cdc14p phosphatase is sequestered in the nucleolus and transiently released in early anaphase and telophase. Cdc14p mediates mitotic exit by dephosphorylating Cdk1p substrates and promoting Cdk1p inactivation. Cdc14p also regulates the localization of chromosomal passenger proteins, which redistribute from kinetochores to the mitotic spindle during anaphase. Here we present evidence that the MEN protein kinase complex Mob1p-Dbf2p localizes to mitotic nuclei and partially colocalizes with Cdc14p and kinetochore proteins. Chromatin immunoprecipitation (ChIP) experiments reveal that Mob1p, Dbf2p, and Cdc14p associate with centromere DNA and require the centromere binding protein Ndc10p for this association. We establish that Mob1p is essential for maintaining the localization of Aurora, INCENP, and Survivin chromosomal passenger proteins on anaphase spindles, whereas Cdc14p and the Mob1p-Dbf2p-activating kinase Cdc15p are required for establishing passenger protein localization on the spindle. Moreover, Mob1p, but not Cdc15p, is required for dissociating Aurora from the kinetochore region. These findings reveal kinetochores as sites for MEN signaling and implicate MEN in coordinating chromosome segregation and/or spindle integrity with mitotic exit and cytokinesis via regulation of chromosome passenger proteins.

Cell Cycle↗