bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Imaging of Mitochondria in Saponin-permeabilized Mice Muscle Fibers
Authors: Kuznetsov Andrey V. , Mayboroda Oleg , Kunz Dagmar , Winkler Kirstin , Schubert Walter , Kunz Wolfram S.
Published: July 2002

Confocal laser-scanning and digital fluorescence imaging microscopy were used to quantify the mitochondrial autofluorescence changes of NAD(P)H and flavoproteins in unfixed saponin-permeabilized myofibers from mice quadriceps muscle tissue. Addition of mitochondrial substrates, ADP, or cyanide led to redox state changes of the mitochondrial NAD system. These changes were detected by ratio imaging of the autofluorescence intensities of fluorescent flavoproteins and NAD(P)H, showing inverse fluorescence behavior. The flavoprotein signal was colocalized with the potentiometric mitochondria-specific dye dimethylaminostyryl pyridyl methyl iodide (DASPMI), or with MitoTracker™ Green FM, a constitutive marker for mitochondria. Within individual myofibers we detected topological mitochondrial subsets with distinct flavoprotein autofluorescence levels, equally responding to induced rate changes of the oxidative phosphorylation. The flavoprotein autofluorescence levels of these subsets differed by a factor of four. This heterogeneity was substantiated by flow-cytometric analysis of flavoprotein and DASPMI fluorescence changes of individual mitochondria isolated from mice skeletal muscle. Our data provide direct evidence that mitochondria in single myofibers are distinct subsets at the level of an intrinsic fluorescent marker of the mitochondrial NAD–redox system. Under the present experimental conditions these subsets show similar functional responses.
E1B 19K Inhibits Fas-mediated Apoptosis through FADD-dependent Sequestration of FLICE
Authors: Perez Denise , White Eileen
Published: July 2002

E1B 19K, the adenovirus Bcl-2 homologue, is a potent inhibitor of apoptosis induced by various stimuli including Fas and tumor necrosis factor-α. Fas and TNFR-1 belong to a family of cytokine-activated receptors that share key components in their signaling pathways, Fas-associating protein with death domain (FADD) and FADD-like interleukin-1β–converting enzyme (FLICE), to induce an apoptotic response. We demonstrate here that E1B 19K and Bcl-xL are able to inhibit apoptosis induced by FADD, but not FLICE. Surprisingly, apoptosis was abrogated by E1B 19K and Bcl-xL when FADD and FLICE were coexpressed. Immunofluorescence studies demonstrated that FADD expression produced large insoluble death effector filaments that may represent oligomerized FADD. E1B 19K expression disrupted FADD filament formation causing FADD and FLICE to relocalize to membrane and cytoskeletal structures where E1B 19K is normally localized. E1B 19K, however, does not detectably bind to FADD, nor does it inhibit FADD and FLICE from being recruited to the death-inducing signaling complex (DISC) when Fas is stimulated. Thus, E1B 19K may inhibit Fas-mediated cell death downstream of FADD recruitment of FLICE but upstream of FLICE activation by disrupting FADD oligomerization and sequestering an essential component of the DISC.
R-Ras Signals through Specific Integrin α Cytoplasmic Domains to Promote Migration and Invasion of Breast Epithelial Cells
Authors: Keely Patricia J. , Rusyn Elena V. , Cox Adrienne D. , Parise Leslie V.
Published: July 2002

Specificity and modulation of integrin function have important consequences for cellular responses to the extracellular matrix, including differentiation and transformation. The Ras-related GTPase, R-Ras, modulates integrin affinity, but little is known of the signaling pathways and biological functions downstream of R-Ras. Here we show that stable expression of activated R-Ras or the closely related TC21 (R-Ras 2) induced integrin-mediated migration and invasion of breast epithelial cells through collagen and disrupted differentiation into tubule structures, whereas dominant negative R-Ras had opposite effects. These results imply novel roles for R-Ras and TC21 in promoting a transformed phenotype and in the basal migration and polarization of these cells. Importantly, R-Ras induced an increase in cellular adhesion and migration on collagen but not fibronectin, suggesting that R-Ras signals to specific integrins. This was further supported by experiments in which R-Ras enhanced the migration of cells expressing integrin chimeras containing the α2, but not the α5, cytoplasmic domain. In addition, a transdominant inhibition previously noted only between integrin β cytoplasmic domains was observed for the α2 cytoplasmic domain; α2β1-mediated migration was inhibited by the expression of excess α2 but not α5 cytoplasmic domain-containing chimeras, suggesting the existence of limiting factors that bind the integrin α subunit. Using pharmacological inhibitors, we found that R-Ras induced migration on collagen through a combination of phosphatidylinositol 3-kinase and protein kinase C, but not MAPK, which is distinct from the other Ras family members, Rac, Cdc42, and N- and K-Ras. Thus, R-Ras communicates with specific integrin α cytoplasmic domains through a unique combination of signaling pathways to promote cell migration and invasion.
Localization in the Nucleolus and Coiled Bodies of Protein Subunits of the Ribonucleoprotein Ribonuclease P
Authors: Jarrous Nayef , Wolenski Joseph S. , Wesolowski Donna , Lee Christopher , Altman Sidney
Published: July 2002

The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors.
Parameters That Specify the Timing of Cytokinesis
Authors: Shuster Charles B. , Burgess David R.
Published: July 2002

One model for the timing of cytokinesis is based on findings that p34cdc2 can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595–605), and this inhibition is proposed to delay cytokinesis until p34cdc2 activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373–1386). Among these kinases and substrates is p34cdc2 and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34cdc2 influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [32P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.
Debcl, a Proapoptotic Bcl-2 Homologue, Is a Component of the Drosophila melanogaster Cell Death Machinery
Authors: Colussi Paul A. , Quinn Leonie M. , Huang David C.S. , Coombe Michelle , Read Stuart H. , Richardson Helena , Kumar Sharad
Published: July 2002

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.
Two Distinct Regions in a Yeast Myosin-V Tail Domain Are Required for the Movement of Different Cargoes
Authors: Catlett Natalie L. , Duex Jason E. , Tang Fusheng , Weisman Lois S.
Published: July 2002

The Saccharomyces cerevisiae myosin-V, Myo2p, is essential for polarized growth, most likely through transport of secretory vesicles to the developing bud. Myo2p is also required for vacuole movement, a process not essential for growth. The globular region of the myosin-V COOH-terminal tail domain is proposed to bind cargo. Through random mutagenesis of this globular tail, we isolated six new single point mutants defective in vacuole inheritance, but not polarized growth. These point mutations cluster to four amino acids in an 11-amino acid span, suggesting that this region is important for vacuole movement. In addition, through characterization of myo2-ΔAflII, a deletion of amino acids 1,459–1,491, we identified a second region of the globular tail specifically required for polarized growth. Whereas this mutant does not support growth, it complements the vacuole inheritance defect in myo2-2 (G1248D) cells. Moreover, overexpression of the myo2-ΔAflII globular tail interferes with vacuole movement, but not polarized growth. These data indicate that this second region is dispensable for vacuole movement. The identification of these distinct subdomains in the cargo-binding domain suggests how myosin-Vs can move multiple cargoes. Moreover, these studies suggest that the vacuole receptor for Myo2p differs from the receptor for the essential cargo.
The Yeast Class V Myosins, Myo2p and Myo4p, Are Nonprocessive Actin-Based Motors
Authors: Reck-Peterson Samara L. , Tyska Matthew J. , Novick Peter J. , Mooseker Mark S.
Published: July 2002

The motor properties of the two yeast class V myosins, Myo2p and Myo4p, were examined using in vitro motility assays. Both myosins are active motors with maximum velocities of 4.5 μm/s for Myo2p and 1.1 μm/s for Myo4p. Myo2p motility is Ca2+ insensitive. Both myosins have properties of a nonprocessive motor, unlike chick myosin-Va (M5a), which behaves as a processive motor when assayed under identical conditions. Additional support for the idea that Myo2p is a nonprocessive motor comes from actin cosedimentation assays, which show that Myo2p has a low affinity for F-actin in the presence of ATP and Ca2+, unlike chick brain M5a. These studies suggest that if Myo2p functions in organelle transport, at least five molecules of Myo2p must be present per organelle to promote directed movement.
Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex
Authors: Li Xinmin , Rivas Marcos P. , Fang Min , Marchena Jennifer , Mehrotra Bharat , Chaudhary Anu , Feng Li , Prestwich Glenn D. , Bankaitis Vytas A.
Published: July 2002

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.
CD80 Costimulation Is Essential for the Induction of Airway Eosinophilia
Authors: Harris Nicola , Peach Robert , Naemura Joe , Linsley Peter S. , Le Gros Graham , Ronchese Franca
Published: July 2002

CD80 and CD86 (B7-1 and B7-2) are the ligands on antigen-presenting cells (APCs) which bind CD28 and deliver the costimulatory signals necessary for T cell activation. The reasons for the existence of two CD28 binding molecules are not well understood. We created a mutant version of CTLA4-Ig that could selectively bind CD80 and block CD28-CD80 interaction but leave CD28-CD86 binding intact. CD80 blockade prevented antigen-induced accumulation of eosinophils and lymphocytes in the lung of immunized mice, but did not block antigen induced systemic blood eosinophilia or IgE antibody production. No preferential expression of CD80 could be demonstrated on a population of lung APC consisting mainly of macrophages. These results indicate that CD80 costimulation is not necessary for the induction of Th2 immune responses but rather for the maintenance or amplification of lung inflammatory responses.
Paracrine Transfer of Mouse Mammary Tumor Virus Superantigen
Authors: Delcourt Marc , Thibodeau Jacques , Denis Francois , Sekaly Rafick-Pierre
Published: July 2002

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II− to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive Vβs. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II− murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific Vβs led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II–specific mAbs. Moreover, injection of vSAG7+ class II− cells in mice led to expansion of Vβ6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II− and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the α1 domain of HLA-DR.
Highly Biased CDR3 Usage in Restricted Sets of β Chain Variable Regions During Viral Superantigen 9 Response
Authors: Ciurli Cristina , Posnett David N. , Sékaly Rafick-Pierre , Denis François
Published: July 2002

Superantigens encoded by the mouse mammary tumor virus can stimulate a large proportion of T cells through interaction with germline-encoded regions of the T cell receptor β chain like the hypervariable region 4 (HV4) loop. However, several lines of evidence suggest that somatically generated determinants in the CDR3 region might influence superantigen responses. We stimulated T cells from donors differing at the BV6S7 allele with vSAG9 to assess the nature and structure of the T cell receptor in amplified T cells and to evaluate the contribution of non-HV4 elements in vSAG recognition. This report demonstrates that vSAG9 stimulation caused the expansion of TCR BV6-expressing T cells, although to varying degrees depending on the BV6 subfamily. The BV6S7 subfamily was preferentially expanded in all donors, but in donors homozygous for the BV6S7*2 allele, a significant number of BV6S5 T cells were amplified and showed a highly biased β chain junctional region (BJ) and CDR3 usage. As CDR3 regions are involved in major histocompatibility complex (MHC)–peptide interaction, such a selection is highly suggestive of an intimate MHC–TCR interaction and would imply that the topology of the MHC-vSAG-TCR complex is similar to the one occurring during conventional antigen recognition.
Ca2+ Signaling Modulates Cytolytic T Lymphocyte Effector Functions
Authors: Esser Mark T. , Haverstick Doris M. , Fuller Claudette L. , Gullo Charles A. , Braciale Vivian Lam
Published: July 2002

Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)–mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8+ CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions.
Complementation of Lymphotoxin α Knockout Mice with Tumor Necrosis Factor–expressing Transgenes Rectifies Defective Splenic Structure and Function
Authors: Alexopoulou Lena , Pasparakis Manolis , Kollias George
Published: July 2002

Lymphotoxin (LT)α knockout mice, as well as double LTα/tumor necrosis factor (TNF) knockout mice, show a severe splenic disorganization with nonsegregating T/B cell zones and complete absence of primary B cell follicles, follicular dendritic cell (FDC) networks, and germinal centers. In contrast, as shown previously and confirmed in this study, LTβ-deficient mice show much more conserved T/B cell areas and a reduced but preserved capacity to form germinal centers and FDC networks. We show here that similar to the splenic phenotype of LTβ-deficient mice, complementation of LTα knockout mice with TNF-expressing transgenes leads to a p55 TNF receptor–dependent restoration of B/T cell zone segregation and a partial preservation of primary B cell follicles, FDC networks, and germinal centers. Notably, upon lipopolysaccharide challenge, LTα knockout mice fail to produce physiological levels of TNF both in peritoneal macrophage supernatants and in their serum, indicating a coinciding deficiency in TNF expression. These findings suggest that defective TNF expression contributes to the complex phenotype of the LTα knockout mice, and uncover a predominant role for TNF and its p55 TNF receptor in supporting, even in the absence of LTα, the development and maintenance of splenic B cell follicles, FDC networks, and germinal centers.
Neu Differentiation Factor (NDF), a Dominant Oncogene, Causes Apoptosis In Vitro and In Vivo
Authors: Grimm Stefan , Weinstein Edward J. , Krane Ian M. , Leder Philip
Published: July 2002

Neu differentiation factor (NDF, also called neuregulin) is a potent inducer of epithelial cell proliferation and has been shown to induce mammary carcinomas in transgenic mice. Notwithstanding this proliferative effect, we have shown that a novel isoform of NDF can induce apoptosis when overexpressed. Here we report that this property also extends to other NDF isoforms and that the cytoplasmic portion of NDF is largely responsible for the apoptotic effect, whereas the proliferative activity is likely to depend upon the secreted version of NDF. In accordance with these contradictory properties, we find that tumors induced by NDF display extensive apoptosis in vivo. NDF is therefore an oncogene whose deregulation can induce transformation as well as apoptosis.
Defective Interleukin (IL)-18–mediated Natural Killer and T Helper Cell Type 1 Responses in IL-1 Receptor–associated Kinase (IRAK)-deficient Mice
Authors: Kanakaraj Palanisamy , Ngo Karen , Wu Ying , Angulo Ana , Ghazal Peter , Harris Crafford A. , Siekierka John J. , Peterson Per A. , Fung-Leung Wai-Ping
Published: July 2002

Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor–associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-κB. The role of IRAK in IL-18–induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-κB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-γ mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-γ production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-γ production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18–induced signaling and function.
The Stimulation of Low-Affinity, Nontolerized Clones by Heteroclitic Antigen Analogues Causes the Breaking of Tolerance Established to an Immunodominant T Cell Epitope
Authors: Wang Rongfang , Wang-Zhu Yiran , Gabaglia Claudia Raja , Kimachi Kazuhiko , Grey Howard M.
Published: July 2002

H-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-Ek–restricted epitope from moth cytochrome c (MCC) 88–103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88–103 analogues that are heteroclitic antigens as assessed on representative MCC 88–103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88–103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vβ3+/Vα11+, none of the hybridomas derived from tolerance-broken animals expressed Vβ3, although they were all Vα11+. Also, the Vβ complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88–103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vβ3+/Vα11+ transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.
The HIV-1 Viral Protein R Induces Apoptosis via a Direct Effect on the Mitochondrial Permeability Transition Pore
Authors: Jacotot Etienne , Ravagnan Luigi , Loeffler Markus , Ferri Karine F. , Vieira Helena L.A. , Zamzami Naoufal , Costantini Paola , Druillennec Sabine , Hoebeke Johan , Briand Jean Paul , Irinopoulou Theano , Daugas Eric , Susin Santos A. , Cointe Denis , Xie Zhi Hua , Reed John C. , Roques Bernard P. , Kroemer Guido
Published: July 2002

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨm), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.
Activated Akt Protects the Lung from Oxidant-Induced Injury and Delays Death of Mice
Authors: Lu Yunbiao , Parkyn Lisa , Otterbein Leo E. , Kureishi Yasuko , Walsh Kenneth , Ray Anuradha , Ray Prabir
Published: July 2002

Oxidant-induced injury to the lung causes extensive damage to lung epithelial cells. Impaired protection and repair of the lung epithelium can result in death. The serine-threonine kinase Akt has been implicated in inhibiting cell death induced by different stimuli including growth factor withdrawal, cell cycle discordance, DNA damage, and loss of cell adhesion in different cell types. However, the in vivo relevance of this prosurvival pathway has not been explored. Here we show that a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death of mice. This is the first demonstration of the in vivo protective function of Akt in the context of oxidant-induced lung injury.
Monovalent Cation Permeation through the Connexin40 Gap Junction Channel
Authors: Beblo Dolores A. , Veenstra Richard D.
Published: July 2002

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γj in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γj are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl− (0.19), glutamate− (0.04), and NO3− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.
Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo
Authors: Oberwinkler Johannes , Stavenga Doekele G.
Published: July 2002

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 μM, buffering becomes minimal.
Mechanism of Generation of Spontaneous Miniature Outward Currents (SMOCs) in Retinal Amacrine Cells
Authors: Mitra Pratip , Slaughter Malcolm M.
Published: July 2002

A subtype of retinal amacrine cells displayed a distinctive array of K+ currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of −60 to −40 mV. Depolarizations above approximately −40 mV were associated with the disappearance of SMOCs and the appearance of transient (Ito) and sustained (Iso) outward K+ currents. Ito appeared at about −40 mV and its apparent magnitude was biphasic with voltage, whereas Iso appeared near −30 mV and increased linearly. SMOCs, Ito, and a component of Iso were Ca2+ dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of −20 mV and above. Their mean amplitude was Nernstian with respect to [K+]ext and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca2+-free solutions, indicative of BK-type Ca2+-activated K+ currents. Dihydropyridine Ca2+ channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca2+ permeation through the kainic acid receptor had no effect. Blockade of organelle Ca2+ channels by ryanodine, or intracellular Ca2+ store depletion with caffeine, eradicated SMOCs. Internal Ca2+ chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca2+ influx through L-type Ca2+ channels and its subsequent amplification by Ca2+-induced Ca2+ release via the ryanodine receptor leads to a localized elevation of internal Ca2+. This amplified Ca2+ signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.
An asymmetric Putnam–Fuglede theorem for unbounded operators
Authors: Stochel Jan
Published: July 2002

The intertwining relations between cosubnormal and closed hyponormal (resp. cohyponormal and closed subnormal) operators are studied. In particular, an asymmetric Putnam–Fuglede theorem for unbounded operators is proved.
Operator versions of the Kantorovich inequality
Authors: Spain P.
Published: July 2002

The Operator Kantorovich Inequality
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−

r
2

)

u
∗

(

a
∗

a
)

u
≤

R
2

(

u
∗

a
∗

u
)
(

u
∗

a
u
)

(R^2 - r^2) \, u^* (a^* a) \, u \le R^2 \, (u^* a^* u) (u^* a u)

\]
holds for a wide class of operators

a
a

on a Hilbert space

H

\mathcal {H}

and all operators

u
:

K

→

H

u: \mathcal {K}\to \mathcal {H}

for which

[
a
]

u

[a] \, u

is a partial isometry,

[
a
]

[a]

being the range projection of

a
.

a.
A remark on commuting operator exponentials
Authors: Wermuth Edgar
Published: July 2002

In a previous paper the author proved that for square matrices with algebraic entries exp

(
A
)

(A)

exp

(
B
)
=

(B)=

exp

(
B
)

(B)

exp

(
A
)

(A)

if and only if

A
B
=
B
A

AB=BA

. This result is extended here to bounded operators on an arbitrary Banach space.
Constructing free subgroups of integral group ring units
Authors: Marciniak Zbigniew , Sehgal Sudarshan
Published: July 2002

Let

G
G

be an arbitrary group. It is proved that if

Z

G

\mathbb {Z}G

contains a bicyclic unit

u
≠
1

u\ne 1

, then

⟨
u
,

u
∗

⟩

\langle u,u^*\rangle

is a nonabelian free subgroup of invertible elements.
Unique continuation on the boundary for Dini domains
Authors: Kukavica Igor , Nyström Kaj
Published: July 2002

We show that the normal derivative of a harmonic function which vanishes on an open subset of the boundary of a Dini domain cannot vanish on a subset of positive surface measure.
Invariant manifolds of hypercyclic vectors for the real scalar case
Authors: Bès Juan
Published: July 2002

We show that every hypercyclic operator on a real locally convex vector space admits a dense invariant linear manifold of hypercyclic vectors.
On multiplicities in polynomial system solving
Authors: Marinari M. , Möller H. , Mora T.
Published: July 2002

This paper deals with the description of the solutions of zero dimensional systems of polynomial equations. Based on different models for describing solutions, we consider suitable representations of a multiple root, or more precisely suitable descriptions of the primary component of the system at a root. We analyse the complexity of finding the representations and of algorithms which perform transformations between the different representations.
Lévy group action and invariant measures on 𝛽{ℕ}
Authors: Blümlinger Martin
Published: July 2002

For

f
∈

ℓ

∞

(

N

)

f\in \ell ^{\infty }( \mathbb {N})

let

T
f

Tf

be defined by

T
f
(
n
)
=

1
n

∑

i
=
1

n

f
(
i
)

Tf(n)=\frac {1}{n}\sum _{i=1}^{n}f(i)

. We investigate permutations

g
g

of

N

\mathbb {N}

, which satisfy

T
f
(
n
)
−
T

f

g

(
n
)
→
0

Tf(n)-Tf_{g}(n)\to 0

as

n
→
∞

n\to \infty

with

f

g

(
n
)
=
f
(
g
n
)

f_{g}(n)=f(gn)

for

f
∈

ℓ

∞

(

N

)

f\in \ell ^{\infty }( \mathbb {N})

(i.e.

g
g

is in the Lévy group

G

)

\mathcal {G})

, or for

f
f

in the subspace of Cesàro-summable sequences. Our main interest are

G

\mathcal {G}

-invariant means on

ℓ

∞

(

N

)

\ell ^{\infty }( \mathbb {N})

or equivalently

G

\mathcal {G}

-invariant probability measures on

β

N

\beta \mathbb {N}

. We show that the adjoint

T

∗

T^{*}

of

T
T

maps measures supported in

β

N

∖

N

\beta \mathbb {N} \setminus \mathbb {N}

onto a weak*-dense subset of the space of

G

\mathcal {G}

-invariant measures. We investigate the dynamical system

(

G

,
β

N

)

( \mathcal {G}, \beta \mathbb {N})

and show that the support set of invariant measures on

β

N

\beta \mathbb {N}

is the closure of the set of almost periodic points and the set of non-topologically transitive points in

β

N

∖

N

\beta \mathbb {N}\setminus \mathbb {N}

. Finally we consider measures which are invariant under

T

∗

T^{*}

.
On convergence of numerical schemes for hyperbolic conservation laws with stiff source terms
Authors: Chalabi Abdallah
Published: July 2002

We deal in this study with the convergence of a class of numerical schemes for scalar conservation laws including stiff source terms. We suppose that the source term is dissipative but it is not necessarily a Lipschitzian function. The convergence of the approximate solution towards the entropy solution is established for first and second order accurate MUSCL and for splitting semi-implicit methods.
Every odd perfect number has a prime factor which exceeds 10⁶
Authors: Hagis Peter , Cohen Graeme
Published: July 2002

It is proved here that every odd perfect number is divisible by a prime greater than

10

6

10^{6}

.
The Hom-spaces between projective functors
Authors: Backelin Erik
Published: July 2002

The category of projective functors on a block of the category

O

(

g

)

\mathcal O(\mathfrak g)

of Bernstein, Gelfand and Gelfand, over a complex semisimple Lie algebra

g

\mathfrak g

, embeds to a corresponding block of the category

O

(

g

×

g

)

\mathcal O(\mathfrak g \times \mathfrak g)

. In this paper we give a nice description of the object

V
V

in

O

(

g

×

g

)

\mathcal O(\mathfrak g \times \mathfrak g)

corresponding to the identity functor; we show that

V
V

is isomorphic to the module of invariants, under the diagonal action of the center

Z

\mathcal Z

of the universal enveloping algebra of

g

\mathfrak g

, in the so-called anti-dominant projective. As an application we use Soergel’s theory about modules over the coinvariant algebra

C
C

, of the Weyl group, to describe the space of homomorphisms of two projective functors

T
T

and

T
′

T’

. We show that there exists a natural

C
C

-bimodule structure on

Hom

{
Functors
}

⁡
(
T
,

T
′

)

\operatorname {Hom}_{\{\operatorname {Functors}\}}(T, T’)

such that this space becomes free as a left (and right)

C
C

-module and that evaluation induces a canonical isomorphism
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="k circled-times Subscript upper C Baseline upper H o m Subscript StartSet upper F u n c t o r s EndSet Baseline left-parenthesis upper T comma upper T Superscript prime Baseline right-parenthesis approximately-equals upper H o m Subscript script upper O left-parenthesis German g right-parenthesis Baseline left-parenthesis upper T left-parenthesis upper M Subscript e Baseline right-parenthesis comma upper T prime left-parenthesis upper M Subscript e Baseline right-parenthesis right-parenthesis">

k

⊗
C

Hom

{
Functors
}

⁡
(
T
,

T
′

)
≅

Hom

O

(

g

)

⁡
(
T
(

M
e

)
,

T
′

(

M
e

)
)

k \otimes _C \operatorname {Hom}_{\{\operatorname {Functors}\}} (T, T’) \cong \operatorname {Hom}_{\mathcal O(\mathfrak g)}(T(M_e), T’(M_e))

, where

M
e

M_e

denotes the dominant Verma module in the block and

k
k

is the complex numbers.
Essential Role of MCM Proteins in Premeiotic DNA Replication
Authors: Lindner Karola , Gregán Juraj , Montgomery Stuart , Kearsey Stephen E.
Published: July 2002

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2–7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2–7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2–7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.
PHYSEAN: PHYsical SEquence ANalysis for the identification of
protein domains on the basis of physical and chemical properties of
amino acids
Authors: Ladunga Istvan
Published: July 2002

Motivation: PHYSEAN predicts protein classes with highly variable sequences on the basis of their physical, chemical and biological characteristics such as diverse hydrophobicity, structural propensity and steric properties. These characteristics, calculated from multiple positions in a sequence, may be conserved even between sequences that fail to produce alignments at any acceptable level of statistical significance. PHYSEAN complements methods that require sequence alignments (BLAST, FASTA, dynamic programming) by adding less residue- and position-specific physicochemical information on the protein or the domain.

Results: We predict proteins or their domains like signal peptides using physical, chemical, geometric, and biological properties of the 20 amino acids. This comprehensive set of properties may cover the diagnostic functional and structural aspects of a domain or a protein class. We automatically select and weight a subset of properties so as to discriminate between, e.g., signal peptides and amino-termini of cytosolic proteins with the lowest number of incorrect predictions. This optimal selection of properties and their weights significantly decreases the number of incorrect predictions as compared to any single property or any combination of unweighted properties. Weights have been optimized by high-performance linear programming models that systematically find the optimal solution from among an astronomic number of property/weight combinations. PHYSEAN’s performance is demonstrated by highly accurate predictions of signal peptides (the vehicles for protein transport across membranes) and their cleavage sites. The results indicate reliable predictions are possible even in the lack of sequence conservation using an automated physical and chemical analysis of proteins.

Availability: The source code for the prediction program will be available for collaborators.

Contact: Steve_Ladunga@sbphrd.com
Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis
Authors: Molinari Marta , Mercurio Ciro , Dominguez Jorge , Goubin Francoise , Draetta Giulio F
Published: July 2002

The Cdc25 A phosphatase is required for the G1–S transition of the cell cycle and is overexpressed in human cancers. We found that it is ubiquitylated and rapidly degraded by the proteasome and that its levels increase from G1 until mitosis. By treating cells with the DNA synthesis inhibitor hydroxyurea, Cdc25 A rapidly decreased in abundance, and this was accompanied by an increase in Cdk2 phosphotyrosine content and a decrease in Cdk2 kinase activity. Cdc25 A overexpression altered the ability of cells to arrest in the presence of hydroxyurea, and caused them to undergo premature chromosome condensation. Cdc25 A overexpression could render tumor cells less sensitive to DNA replication checkpoints, thereby contributing to their genomic instability.
Systematic subcellular localization of novel proteins identified by large‐scale cDNA sequencing
Authors: Simpson Jeremy C , Wellenreuther Ruth , Poustka Annemarie , Pepperkok Rainer , Wiemann Stefan
Published: July 2002

As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N‐ and C‐terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of >100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For >80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.
Generating patterns from fields of cells
Authors: Sanson Bénédicte
Published: July 2002

In Drosophila, a cascade of maternal, gap, pair‐rule and segment polarity genes subdivides the antero/posterior axis of the embryo into repeating segmental stripes. This review summarizes what happens next, i.e. how an intrasegmental pattern is generated and controls the differentiation of specific cell types in the epidermis. Within each segment, cells secreting the signalling molecules Wingless (the homologue of vertebrate Wnt‐1) and Hedgehog are found in narrow stripes on both sides of the parasegmental boundary. The Wingless and Hedgehog organizing activities help to establish two more stripes per segment that localize ligands for the Epidermal Growth Factor and the Notch signalling pathways, respectively. These four signals then act at short range and in concert to control epidermal differentiation at the single cell level across the segment. This example from Drosophila provides a paradigm for how organizers generate precise patterns, and ultimately different cell types, in a naïve field of cells.