RanGTP-mediated nuclear export of karyopherin alpha involves its interaction with the nucleoporin Nup153

Using binding assays, we discovered an interaction between karyopherin alpha2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin beta1, termed beta1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear karyopherin alpha from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin alpha antibodies, we found that karyopherin alpha export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin alpha was inhibited by peptides representing the interacting domains of Nup153 and karyopherin alpha2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, beta1*, although it inhibited import, did not inhibit export of karyopherin alpha. Hence, karyopherin alpha import into and export from nuclei are asymmetric processes.     

A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.     

Nup84, a novel nucleoporin that is associated with CAN/Nup214 on the cytoplasmic face of the nuclear pore complex

The short filaments extending from the cytoplasmic face of nuclear pore complexes are thought to contain docking sites for nuclear import substrates. One component of these filaments is the large O-linked glycoprotein CAN/Nup214. Immunoprecipitation studies carried out under nondenaturing conditions, and using a variety of antibodies, reveal a novel nonglycosylated nucleoporin, Nup84, that is tightly associated with CAN/Nup214. Consistent with such an association, Nup84 is found to be exposed on the cytoplasmic face of the nuclear pore complex. cDNA sequence analyses indicate that Nup84 contains neither the GLFG nor the XFXFG repeats that are a characteristic of a number of other nuclear pore complex proteins. Secondary structure predictions, however, suggest that Nup84 contains a coiled-coil COOH-terminal domain, a conclusion supported by the observation of significant sequence similarity between this region of the molecule and various members of the tropomyosin family. Mutagenesis and expression studies indicate that the putative coiled-coil domain is required for association with the cytoplasmic face of the nuclear pore complex, whereas it is the NH2-terminal region of Nup84 that contains the site of interaction with CAN/Nup214. These findings suggest a model in which Nup84 may function in the attachment of CAN/Nup214 to the central framework of the nuclear pore complex. In this way, Nup84 could play a central role in the organization of the interface between the pore complex and the cytoplasm.     

Mas6p can be cross-linked to an arrested precursor and interacts with other proteins during mitochondrial protein import

Mas6p is an integral membrane protein of the yeast mitochondrial inner membrane, which is essential for mitochondrial protein import (1). To determine whether Mas6p is directly involved in recognizing precursors or translocating them across the inner membrane, we asked if Mas6p was in close proximity to precursor proteins being imported into mitochondria. We report here that Mas6p can be chemically cross-linked to an imported protein arrested in transit through the mitochondrial inner membrane. Antiserum to Mas6p specifically immunoprecipitates one of several different mitochondrial proteins that are cross-linked to blocked precursors. Our results strongly suggest that Mas6p physically interacts with precursors during their translocation into the matrix. In addition, at least two other mitochondrial proteins that are each cross-linked to arrested precursors can be coimmunoprecipitated along with Mas6p under non-denaturing conditions. These observations provide evidence for a complex of proteins including Mas6p, each of which interacts with mitochondrial precursors during import.     

Apoptosis of central and peripheral neurons can be prevented with cyclin-dependent kinase/mitogen-activated protein kinase inhibitors

Previous studies have indicated that certain members of the cyclin-dependent kinase/mitogen-activated protein kinase superfamily are involved in apoptosis of neuronal cells. Here, we have examined programmed cell death induced by withdrawal of neurotrophic support from CNS (rat retinal) and PNS (chick sympathetic, sensory, and ciliary) neurons. All four neuron types were equally rescued by the purine analogues olomoucine and roscovitine. Olomoucine inhibits multiple cyclin-dependent and mitogen-activated protein kinases with similar potency. Roscovitine is a more selective cyclin-dependent kinase inhibitor; but, so is butyrolactone I, which did not prevent retinal ganglion cell death. The specific p38MAPK inhibitor SB-203580 did not prevent apoptosis in retinal ganglion cells. Death of these cells in the absence of neurotrophic factors was accompanied by morphological changes indicative of apoptosis, including nuclear condensation and fragmentation. Treatment with olomoucine or roscovitine not only prevented these apoptotic changes in retinal ganglion cells but also blocked neurite outgrowth. The survival-promoting activity of olomoucine correlated with its in vitro IC50 for c-Jun N-terminal kinase-1 and its potency to repress c-jun induction in live PC12 cells. Roscovitine was more potent in rescuing neurons than in inhibiting Jun kinase. Thus, the antiapoptotic action of roscovitine might be due to inhibition of additional kinases.     

A metalloprotease activity from C6 glioma cells inactivates the myelin-associated neurite growth inhibitors and can be neutralized by antibodies

Glioblastoma cells infiltrate brain tissue and migrate preferentially along white matter fibre tracts, an environment that is highly inhibitory to the migration of astrocytes and the growth of neurites because of the presence of specific inhibitory proteins. In vitro, spreading and migration of rat C6 glioma cells on a CNS (central nervous system) myelin substrate is correlated with and dependent on the presence of a metalloprotease. This membrane-bound metalloendoprotease exhibits a blocker profile different from known proteases. Pretreatment of CNS myelin or of a highly purified CNS myelin component, the inhibitory protein bNI-220, with C6 metalloproteolytic activity converts these non-permissive substrates into permissive environments for astrocytes and fibroblasts, indicating that this C6 cell-derived metalloprotease may inactivate myelin-associated inhibitory proteins. Antibodies were raised in chicken against fractions enriched in metalloproteolytic activity; these antibodies were able to inhibit specifically spreading and migration of C6 glioma cells on a CNS myelin substrate, as well as the invasion of C6 cells into adult rat optic nerve explants in vitro. These results suggest a crucial involvement of a membrane-bound metalloprotease in the mechanisms of C6 glioma migration and infiltration of brain tissue by proteolytic inactivation of the neurite growth inhibitory proteins present in CNS myelin.     

U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.     

Assembly of MHC class I molecules with biosynthesized endoplasmic reticulum-targeted peptides is inefficient in insect cells and can be enhanced by protease inhibitors

To study the requirements for assembly of MHC class I molecules with antigenic peptides in the endoplasmic reticulum (ER), we studied Ag processing in insect cells. Insects lack a class I recognition system, and their cells therefore provide a "blank slate" for identifying the proteins that have evolved to facilitate assembly of class I molecules in vertebrate cells. H-2Kb heavy chain, mouse beta 2-microglobulin, and an ER-targeted version of a peptide corresponding to Ova(257-264) were expressed in insect cells using recombinant vaccinia viruses. Cell surface expression of Kb-OVA(257-264) complexes was quantitated using a recently described complex-specific mAb (25-D1.16). Relative to TAP-deficient human cells, insect cells expressed comparable levels of native, peptide-receptive cell surface Kb molecules, but generated cell surface Kb-OVA(257-264) complexes at least 20-fold less efficiently from ER-targeted peptides. The inefficient assembly of Kb-OVA(257-264) complexes in the ER of insect cells cannot be attributed solely to a requirement for human tapasin, since first, human cells lacking tapasin expressed endogenously synthesized Kb-OVA(257-264) complexes at levels comparable to tapasin-expressing cells, and second, vaccinia virus-mediated expression of human tapasin in insect cells did not detectably enhance the expression of Kb-OVA(257-264) complexes. The assembly of Kb-OVA(257-264) complexes could be greatly enhanced in insect but not human cells by a nonproteasomal protease inhibitor. These findings indicate that insect cells lack one or more factors required for the efficient assembly of class I-peptide complexes in vertebrate cells and are consistent with the idea that the missing component acts to protect antigenic peptides or their immediate precursors from degradation.     

CDw78--a determinant on a major histocompatibility complex class II subpopulation that can be induced to associate with the cytoskeleton

In the present study we demonstrate that CDw78 monoclonal antibody (mAb) recognizes a distinct subpopulation of major histocompatibility complex (MHC) class II molecules. We show that the CDw78 epitope is present on less than 10% of the total number of MHC class II molecules expressed on different cells, is not linked to a single isotype, and exhibits a characteristic expression pattern in tonsils. While mAb against MHC class II (DR, DP and DQ) stained the majority of cells both in the mantle zone and in germinal centers, the CDw78 staining was more heterogeneous with the strongest reactivity and the highest number of positive cells in the mantle zone and in the light centrocyte-rich part of the germinal centers. Antibodies to this MHC class II subpopulation (e.g. FN1) induced association with the cytoskeleton and a subsequent capping in more than 90% of peripheral blood B cells. In contrast, mAb against MHC class II (DR, DP and DQ) did not induce association with the cytoskeleton and only 10-20% of B cells were induced to cap, suggesting that CDw78 defines a population of MHC class II molecules functionally different from the majority of these antigens. Scatchard plot analysis indicates that FN1 mAb is of relatively low affinity (Ka = 1.5 x 10(8) M(-1)) and monovalent Fab fragments fail to bind to the cell surface with measurable affinity. Our data seen in the context of the ability of FN1 to co-stimulate B cells with a suboptimal dose of anti-mu suggest that CDw78 mAb might recognize a functional important subpopulation of MHC class II molecules so far not described. It seems likely that this subpopulation represents dimerized or aggregated MHC class II molecules that can selectively bind this low-affinity mAb.     

FSBA modifies both alpha- and beta-subunits of F1 specifically and can be bound together with AXP at the same alpha-subunit

Binding of 1 mole 5'-fluorosulfonylbenzoyladenosine (FSBA) per mol F1 induces about 50% inhibition of ATPase activity and 80% inhibition of ITPase activity. The binding of additional ligand results in a further inhibition of both activities. Maximally 5 mol/mol F1, causing complete inhibition of activity, can be bound. Using radioactive FSBA more label is found on alpha-subunits than on beta-subunits under the usual buffer conditions. The modified amino acids are alpha-Tyr300, alpha-Tyr244 and beta-Tyr368. Binding of FSBA, at least up to 3 mol/mol F1, does not result in loss of bound ADP, whether the starting enzyme contains 2, 3 or 4 bound nucleotides. Added adenine nucleotides compete with FSBA only for binding that results in modification of beta-subunits, shifting the alpha/beta ratio of bound label to higher values. It is concluded that the alpha-subunits contain two hydrophobic pockets for the binding of nucleoside moieties, with a different orientation relative to the P-loop. One pocket contains alpha-Tyr244 and alpha-Tyr300, the other beta-Tyr368. Since, however, in the binding of adenine nucleotide di- or triphosphates the P-loop is involved, only one of these ligands can bind per subunit. The previously not understood binding characteristics of several substrate analogues have now become interpretable on the assumption that also the structurally homologous beta-subunits contain 2 pockets where nucleoside moieties can bind. The kinetic effects of FSBA binding indicate that the first FSBA binds at the regulatory site that has a high affinity for ADP and pyrophosphate. Binding of pyrophosphate at this high-affinity regulatory site increases the Vmax of the enzyme, while binding at a second regulatory site, a low-affinity site, increases the rate of binding of FSBA with a factor of about 3. Binding of bicarbonate at this latter site is responsible for the disappearance of the apparent negative cooperativity of the ATPase activity.     

Upsetting experiences for the therapist in-session: How they can be dealt with and what they are good for.

Several major schools of psychotherapy require a genuine emotional presence from therapists in-session, as well as a nondefensive awareness of their emotional responses to the client. The present article addresses the part played by upsetting experiences in the context of these requirements. In order to identify relevant issues for a discussion of the literature, grounded theory analysis is presented of a series of conversations with four psychotherapists about their experience with in-session distress. The participants reported client confrontation and rejection evoked a variety of negative emotions ranging from helplessness to anger. Besides traditional professional resources like supervision and personal therapy, they discussed several coping strategies that focused on emotions. One strategy that stood out was to analyze how the emotional impact occurred in order to clarify client issues. This strategy was reported to yield information that improved therapeutic effectiveness in the case at hand, but it also was an effective method for overcoming therapist distress and a means of promoting broader professional growth. These observations suggest issues for integrative psychotherapy research into emotional risk taken by therapists and its payoff. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Continuing the debate on empty follicle syndrome: can it be associated with normal bioavailability of beta-human chorionic gonadotrophin on the day of oocyte recovery?

This paper describes our experience with four ovarian stimulation in-vitro fertilization (IVF) cycles in which we failed to retrieve oocytes despite normal bioavailability of beta-human chorionic gonadotrophin (beta-HCG) in patients' blood 35 h after HCG administration. In three cases, the oocyte recovery procedure was interrupted, a second dose of HCG was administered and 24 h later mature oocytes were collected from two of the patients. In the first case, the three metaphase II oocytes collected fertilized after intracytoplasmic sperm injection (ICSI) and two cleaved grade three embryos were transferred but pregnancy did not ensue. In the second case, six out of eight metaphase II oocytes fertilized and cleaved following ICSI, leading to transfer of one grade two and two grade three embryos. This resulted in a clinical pregnancy which at the time of this report is ongoing. A similar rescue protocol was used for the third case who had empty follicle syndrome (EFS) in her previous treatment cycle but only cumulus-corona complexes were aspirated. Five additional patients who had EFS before instituting pregnancy diagnostic test screening have had further treatment cycles in which oocytes were collected but pregnancy did not ensue. We conclude that normal bioavailability of beta-HCG on the day of oocyte recovery does not exclude the diagnosis of EFS. EFS does not predict a reduced fertility potential in future cycles, although it may recur due to a biological abnormality in the availability of mature oocytes that are retrievable. In such patients, oocyte donation may offer the chance of achieving a pregnancy.     

Separate and combined effects of methylphenidate and behavior modification on boys with attention deficit-hyperactivity disorder in the classroom

This study evaluated the separate and combined effects of behavior modification and 2 doses of methylphenidate (MPH; 0.3 and 0.6 mg/kg) compared with baseline (no behavior modification and a placebo) on the classroom behavior and academic performance of 31 ADHD (attention deficit-hyperactivity disorder) boys attending a summer treatment program. Results revealed significant effects of both interventions, with the mean effect size of medication being more than twice as great as that of behavior modification. Relatively small incremental value was gained by the higher dose of medication or the addition of behavior modification, compared with the effects of the low dose of MPH. In contrast, the addition of either dose of MPH resulted in improvement beyond the effects of behavior modification alone. These group effects reflected those obtained in analyses of individual differences. Furthermore, comparisons of individual responsiveness showed that boys who responded to one treatment also responded to the other.     

The immunosenescent phenotype in mice and humans can be defined by alterations in the natural immunity reversal by immunomodulation with oral AM3

The reactivities of monocyte/macrophages and natural killer (NK) cells (natural immunity) were evaluated following the administration of the biological response modifier AM3. The lower number of macrophages and NK cells in middle-aged mice (MAM) compared to young adult mice (YAM) were significantly elevated following AM3 treatment to equal or greater than YAM values. Both macrophage and NK cell cytotoxicity peaked at two days following AM3 treatment and remained elevated over control values for up to 8 days following a four days treatment regimen by the oral route. Of particular interest was the clinical effect of AM3 treatment in chronic bronchitis (CB) patients and various aged volunteers. In middle-aged patients with chronic bronchitis (MACBpts) AM3 treatment resulted in significant increases in the number of monocytes as well as their phagocytic and chemotactic activity. Differential NK cell cytotoxicities were observed in MACBpts compared to middle-aged healthy adults (MAHA) and young healthy adults (YHA). Cytotoxicity in YHA was 2-fold higher than MAHA and 5-fold higher than MACBpts. The depressed number of NK cells in MACBpts was reversed following the AM3 treatment to near NK cell levels in YHA. These observations help to explain how AM3 aids in the restoration of natural cellular immunity and its possible application as an adjuvant to bacterial & viral vaccines as well as in the treatment CB.     

Calibration and diagnosticity of confidence in eyewitness identification: Comments on what can be inferred from the low confidence–accuracy correlation.

The relationship between eyewitness confidence and accuracy as measured by the ψ point-biserial correlation has been described as poor or even nonexistent in the literature on lineup identifications. In this article, 3 arguments are made. First, a low point-biserial correlation is compatible with good or even perfect calibration (realism) of confidence, and the correlation provides no information about whether witnesses over- or underestimate the probability of a correct identification. Second, point-biserial correlations provide almost no information about whether confidence is diagnostic in the sense that it should be taken into account by the court when evaluating eyewitness identifications. Third, useful information is provided by calibration analysis and computation of diagnosticity indices. These arguments are illustrated with data from an experiment with photo-confrontations that rely on photo material used by the Swedish Police and where foils were selected by experienced police officers in the manner of routine investigations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus in cultured Spodoptera frugiperda cells

The effects of inhibitors of nucleic acid and protein synthesis on the replication of Spodoptera frugiperda nuclear polyhedrosis virus have been determined. Two inhibitors of protein synthesis-cycloheximide and puromycin-were irreversible inhibitors of virus multiplication. Three inhibitors of nucleic acid synthesis-actinomycin D, cytosine arabinoside and camptothecin- prevented virus multiplication; only camptothecin was reversible. Rifampicin had no effect on virus multiplication.     

Primary gamma delta cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation

The division of CD4+ alpha beta T cells into Th1 and Th2 subsets has become an established and important paradigm. The respective activities of these subsets appear to have profound effects on the course of infectious and autoimmune diseases. It is believed that specific programs of differentiation induce the commitment of an uncommitted Th0 precursor cell to Th1 or Th2. A component of these programs is hypothesized to be the nature of MHC-peptide antigen presentation to the alpha beta T cell. It has heretofore remained uncertain whether a Th1/Th2 classification likewise defines, at the clonal level, gamma delta T cells. Such cells do not, as a general rule, express either CD4 or CD8 alpha beta, and they do not commonly recognize peptide-MHC. In this report, gamma delta cell clones are described that conform strikingly to the Th1/Th2 classification, both by cytokine expression and by functional activities of the clones in vitro and in vivo. Provocatively, both the gamma delta cell clones and primary gamma delta cells in vivo showed a strong association of the Th2 phenotype with CD4 expression. These results are discussed with regard to the immunoregulatory role that is increasingly emerging for gamma delta cells.