bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell-Free Reconstitution of Microautophagic Vacuole Invagination and Vesicle Formation
Authors: Sattler Tanja , Mayer Andreas
Published: July 2002

Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPγS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.
Prm1p, a Pheromone-Regulated Multispanning Membrane Protein, Facilitates Plasma Membrane Fusion during Yeast Mating
Authors: Heiman Maxwell G. , Walter Peter
Published: July 2002

Cell fusion occurs throughout development, from fertilization to organogenesis. The molecular mechanisms driving plasma membrane fusion in these processes remain unknown. While yeast mating offers an excellent model system in which to study cell fusion, all genes previously shown to regulate the process act at or before cell wall breakdown; i.e., well before the two plasma membranes have come in contact. Using a new strategy in which genomic data is used to predict which genes may possess a given function, we identified PRM1, a gene that is selectively expressed during mating and that encodes a multispanning transmembrane protein. Prm1p localizes to sites of cell–cell contact where fusion occurs. In matings between Δprm1 mutants, a large fraction of cells initiate zygote formation and degrade the cell wall separating mating partners but then fail to fuse. Electron microscopic analysis reveals that the two plasma membranes in these mating pairs are tightly apposed, remaining separated only by a uniform gap of ∼8 nm. Thus, the phenotype of Δprm1 mutants defines a new step in the mating reaction in which membranes are juxtaposed, possibly through a defined adherence junction, yet remain unfused. This phenotype suggests a role for Prm1p in plasma membrane fusion.
Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium
Authors: Frey Mark R. , Clark Jennifer A. , Leontieva Olga , Uronis Joshua M. , Black Adrian R. , Black Jennifer D.
Published: July 2002

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.
Cortical Num1p Interacts with the Dynein Intermediate Chain Pac11p and Cytoplasmic Microtubules in Budding Yeast
Authors: Farkasovsky Marian , Küntzel Hans
Published: July 2002

Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the α-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.

Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p–Kar9p complex at the bud tip of early anaphase cells.
Tortoise, a Novel Mitochondrial Protein, Is Required for Directional Responses of Dictyostelium in Chemotactic Gradients
Authors: van Es Saskia , Wessels Deborah , Soll David R. , Borleis Jane , Devreotes Peter N.
Published: July 2002

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA− partially restores chemotaxis, whereas overexpression of TorA in mek1− does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA–green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.
Phosphatidylinositol 4,5-Bisphosphate Induces Actin Stress-Fiber Formation and Inhibits Membrane Ruffling in Cv1 Cells
Authors: Yamamoto Masaya , Hilgemann Donald H. , Feng Siyi , Bito Haruhiko , Ishihara Hisamitsu , Shibasaki Yoshikazu , Yin Helen L.
Published: July 2002

Phosphatidylinositol 4,5 bisphosphate (PIP2) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP2 effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase α (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP2 signaling. PIP5KI overexpression increased PIP2 and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP2 and PI4P synthesis in cells. However, Y-27632 had no effect on PIP2 synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP2 synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP2 in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP2 in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP2.
The Drosophila Protein Asp Is Involved in Microtubule Organization during Spindle Formation and Cytokinesis
Authors: Wakefield James G. , Bonaccorsi Silvia , Gatti Maurizio
Published: July 2002

Abnormal spindle (Asp) is a 220-kD microtubule-associated protein from Drosophila that has been suggested to be involved in microtubule nucleation from the centrosome. Here, we show that Asp is enriched at the poles of meiotic and mitotic spindles and localizes to the minus ends of central spindle microtubules. Localization to these structures is independent of a functional centrosome. Moreover, colchicine treatment disrupts Asp localization to the centrosome, indicating that Asp is not an integral centrosomal protein. In both meiotic and mitotic divisions of asp mutants, microtubule nucleation occurs from the centrosome, and γ-tubulin localizes correctly. However, spindle pole focusing and organization are severely affected. By examining cells that carry mutations both in asp and in asterless, a gene required for centrosome function, we have determined the role of Asp in the absence of centrosomes. Phenotypic analysis of these double mutants shows that Asp is required for the aggregation of microtubules into focused spindle poles, reinforcing the conclusion that its function at the spindle poles is independent of any putative role in microtubule nucleation. Our data also suggest that Asp has a role in the formation of the central spindle. The inability of asp mutants to correctly organize the central spindle leads to disruption of the contractile ring machinery and failure in cytokinesis.
Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin
Authors: Palmgren Sandra , Ojala Pauli J. , Wear Martin A. , Cooper John A. , Lappalainen Pekka
Published: July 2002

Twinfilin is a ubiquitous actin monomer–binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the α and β subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer–sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.
Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice
Authors: Barton Elisabeth R. , Morris Linda , Musaro Antonio , Rosenthal Nadia , Sweeney H. Lee
Published: July 2002

Duchenne muscular dystrophy is an X-linked degenerative disorder of muscle caused by the absence of the protein dystrophin. A major consequence of muscular dystrophy is that the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration–regeneration and ultimately resulting in the replacement of muscle fibers with fibrotic tissue. Because insulin-like growth factor I (IGF-I) has been shown to enhance muscle regeneration and protein synthetic pathways, we asked whether high levels of muscle-specific expression of IGF-I in mdx muscle could preserve muscle function in the diseased state. In transgenic mdx mice expressing mIgf-I (mdx:mIgf+/+), we showed that muscle mass increased by at least 40% leading to similar increases in force generation in extensor digitorum longus muscles compared with those from mdx mice. Diaphragms of transgenic mdx:mIgf+/+ exhibited significant hypertrophy and hyperplasia at all ages observed. Furthermore, the IGF-I expression significantly reduced the amount of fibrosis normally observed in diaphragms from aged mdx mice. Decreased myonecrosis was also observed in diaphragms and quadriceps from mdx:mIgf+/+ mice when compared with age-matched mdx animals. Finally, signaling pathways associated with muscle regeneration and protection against apoptosis were significantly elevated. These results suggest that a combination of promoting muscle regenerative capacity and preventing muscle necrosis could be an effective treatment for the secondary symptoms caused by the primary loss of dystrophin.
A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase
Authors: Mori Hiroki , Cline Kenneth
Published: July 2002

The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.
Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels
Authors: Sawhney Ravi K. , Howard Jonathon
Published: July 2002

Aclassic model for tissue morphogenesis is the formation of ligament-like straps between explants of fibroblasts placed in collagen gels. The patterns arise from mechanical forces exerted by cells on their substrates (Harris et al., 1981). However, where do such straps come from, and how are slow local movements of cells transduced into dramatic long-distance redistributions of collagen? We embedded primary mouse skin and human periodontal ligament fibroblasts in collagen gels and measured the time course of patterning by using a novel computer algorithm to calculate anisotropy, and by tracking glass beads dispersed in the gel. As fibroblasts began to spread into their immediate environments, a coordinated rearrangement of collagen commenced throughout the gel, producing a strap on a time scale of minutes. Killing of cells afterwards resulted in a partial relaxation of the matrix strain. Surprisingly, relatively small movements of collagen molecules on the tensile axis between two pulling explants induced a much larger concomitant compression of the gel perpendicular to the axis, organizing and aligning fibers into a strap. We propose that this amplification is due to the geometry of the collagen matrix, and that analogous amplified movements may drive morphological changes in other biological meshes, both outside and inside the cell.
Impaired Inflammatory Responses in the Reverse Arthus Reaction Through Genetic Deletion of the C5a Receptor
Authors: Höpken Uta E. , Lu Bao , Gerard Norma P. , Gerard Craig
Published: July 2002

We recently demonstrated that gene-targeted disruption of the C5a anaphylatoxin receptor prevented lung injury in immune complex–mediated inflammation. In this study, we compare the effect of C5aR deficiency in immune complex–induced inflammation in the peritoneal cavity and skin with the results derived from our immune complex alveolitis model. C5aR- deficient mice exhibit decreased migration of neutrophils and decreased levels of TNF-α and interleukin 6 in the peritoneal reverse passive Arthus reaction compared to their wild-type littermates. In the reverse passive Arthus reaction in the skin the C5aR was also required for the full expression of neutrophil influx and edema formation; C5aR-deficient mice showed reduced neutrophil migration and microvascular permeability changes. In contrast to our studies in immune complex–induced lung inflammation, C5aR deficiency does not completely prevent injury in the peritoneal cavity and skin. These data indicate a dominant role for the C5aR and its ligand in the reverse passive Arthus reaction in the lung and a synergistic role together with other inflammatory mediators in immune complex–mediated peritonitis and skin injury.
Neutrophil Emigration in the Skin, Lungs, and Peritoneum: Different Requirements for CD11/CD18 Revealed by CD18-deficient Mice
Authors: Mizgerd Joseph P. , Kubo Hiroshi , Kutkoski Gregory J. , Bhagwan Sabrina D. , Scharffetter-Kochanek Karin , Beaudet Arthur L. , Doerschuk Claire M.
Published: July 2002

To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18−/− mutants). Peripheral blood of CD18−/− mutants contained 11-fold more neutrophils than did blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less in CD18−/− mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil emigration in CD18−/− mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18−/− mutants, but rather was greater than the WT values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate (90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18−/− mutant mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia or peritonitis.
A Novel Sialic Acid Binding Site on Factor H Mediates Serum Resistance of Sialylated Neisseria gonorrhoeae
Authors: Ram Sanjay , Sharma Ajay K. , Simpson Scott D. , Gulati Sunita , McQuillen Daniel P. , Pangburn Michael K. , Rice Peter A.
Published: July 2002

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I–mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6–10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5′-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16–20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16–20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.
Complementary Roles for Scavenger Receptor A and CD36 of Human Monocyte–derived Macrophages in Adhesion to Surfaces Coated with Oxidized Low-Density Lipoproteins and in Secretion of H2O2
Authors: Maxeiner Horst , Husemann Jens , Thomas Christian A. , Loike John D. , Khoury Joseph El , Silverstein Samuel C.
Published: July 2002

Oxidized low-density lipoprotein (oxLDL) is considered one of the principal effectors of atherogenesis. To explore mechanisms by which oxLDL affects human mononuclear phagocytes, we incubated these cells in medium containing oxLDL, acetylated LDL (acLDL), or native LDL, or on surfaces coated with these native and modified lipoproteins. The presence of soluble oxLDL, acLDL, or native LDL in the medium did not stimulate H2O2 secretion by macrophages. In contrast, macrophages adherent to surfaces coated with oxLDL secreted three- to fourfold more H2O2 than macrophages adherent to surfaces coated with acLDL or native LDL. Freshly isolated blood monocytes secreted little H2O2 regardless of the substrate on which they were plated. H2O2 secretion was maximal in cells maintained for 4–6 d in culture before plating on oxLDL-coated surfaces. Fucoidan, a known ligand of class A macrophage scavenger receptors (MSR-A), significantly reduced macrophage adhesion to surfaces coated with oxLDL or acLDL. Monoclonal antibody SMO, which blocks oxLDL binding to CD36, did not inhibit adhesion of macrophages to oxLDL-coated surfaces but markedly reduced H2O2 secretion by these cells. These studies show that MSR-A is primarily responsible for adhesion of macrophages to oxLDL-coated surfaces, that CD36 signals H2O2 secretion by macrophages adherent to these surfaces, and that substrate-bound, but not soluble, oxLDL stimulates H2O2 secretion by macrophages.
T Helper Cell Type 1–associated and Cytotoxic T Lymphocyte–mediated Tumor Immunity Is Impaired in Interleukin 4–deficient Mice
Authors: Schüler Thomas , Qin Zhihai , Ibe Sabrina , Noben-Trauth Nancy , Blankenstein Thomas
Published: July 2002

It is widely accepted that cellular immune responses are induced by CD4+ T helper 1 (Th1) cells secreting interleukin (IL)-2 and interferon (IFN)-γ. Tumor immunity is often mediated by cytotoxic T lymphocytes (CTLs) whose activation is supported by Th1 cytokines. Since IL-4 directs Th2 development and has been shown to inhibit Th1-dominated responses, we assumed that IL-4–deficient (IL-4−/−) mice would develop vigorous CTL-mediated tumor immunity compared with IL-4–competent (IL-4+/+) mice. Surprisingly, IL-4−/− mice were severely impaired to develop tumor immunity to both a mammary adenocarcinoma line and a colon carcinoma line. The lack of tumor immunity in IL-4−/− mice was associated with reduced IFN-γ production, diminished levels of tumor-reactive serum IgG2a, and undetectable CTL activity, indicating a defective Th1 response in the absence of endogenous IL-4. Anti–IL-4 monoclonal antibody blocked tumor immunity in IL-4+/+ mice when administered at the time of immunization but not at the time of challenge. Additionally, tumor immunity could be induced in IL-4−/− mice, if IL-4 was provided by gene-modified cells together with immunizing tumor cells. These results demonstrate that tumor immunity requires IL-4 in the priming phase for the generation of effector cells rather than for their maintenance and exclude secondary, developmental defects in the “knockout” strain. Together, our results demonstrate a novel and previously unanticipated role of IL-4 for the generation of Th1-associated, CTL-mediated tumor immunity.
Anti-HIV Agent Trichosanthin Enhances the Capabilities of Chemokines to Stimulate Chemotaxis and G Protein Activation, and This Is Mediated through Interaction of Trichosanthin and Chemokine Receptors
Authors: Zhao Jian , Ben Li-Hong , Wu Ya-Lan , Hu Wei , Ling Kun , Xin Shun-Mei , Nie Hui-Ling , Ma Lan , Pei Gang
Published: July 2002

Trichosanthin (TCS), an active protein component isolated from a traditional Chinese medicinal herb Trichosanthes kirilowii, has been shown to inhibit HIV infection and has been applied in clinical treatment of AIDS. The recent development that chemokines and chemokine receptors play important roles in HIV infection led us to investigate the possible functional interaction of TCS with chemokines and their receptors. This study demonstrated that TCS greatly enhanced both RANTES (regulated upon activation, normal T cell expressed and secreted)– and stromal cell–derived factor (SDF)-1α–stimulated chemotaxis (EC50 ≅ 1 nM) in leukocytes (THP-1, Jurkat, and peripheral blood lymphocyte cells) and activation of pertussis toxin–sensitive G proteins (EC50 ≅ 20 nM). TCS also significantly augmented chemokine-stimulated activation of chemokine receptors CCR5 and CXCR4 as well as CCR1, CCR2B, CCR3, and CCR4 transiently expressed in HEK293 cells. A mutant TCS with 4,000-fold lower ribosome-inactivating activity showed similar augmentation activity as wild-type TCS. Moreover, flow cytometry demonstrated that the specific association of TCS to the cell membranes required the presence of chemokine receptors, and laser confocal microscopy reveals that TCS was colocalized with chemokine receptors on the membranes. The results from TCS-Sepharose pull-down and TCS and chemokine receptor coimmunoprecipitation and cross-linking experiments demonstrated association of TCS with CCR5. Thus, our data clearly demonstrated that TCS synergizes activities of chemokines to stimulate chemotaxis and G protein activation, and the effects of TCS are likely to be mediated through its interaction with chemokine receptors.
Early Activation of Caspases during T Lymphocyte Stimulation Results in Selective Substrate Cleavage in Nonapoptotic Cells
Authors: Alam Antoine , Cohen Luchino Y. , Aouad Salah , Sékaly Rafick-Pierre
Published: July 2002

Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4+, CD8+, CD45RA+, and CD45RO+), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.
H2-M3–Restricted T Cells in Bacterial Infection
Authors: Kerksiek Kristen M. , Busch Dirk H. , Pilip Ingrid M. , Allen S. Elise , Pamer Eric G.
Published: July 2002

Major histocompatibility complex (MHC) class Ib molecules have been implicated in CD8+ T cell–mediated defenses against intracellular bacterial infection, but the relative importance of MHC class Ib–restricted T cells in antimicrobial immunity is unknown. In this report, we use MHC tetramers to characterize T cell responses restricted by H2-M3, an MHC class Ib molecule that selectively presents N-formyl peptides. We find that sizeable H2-M3–restricted T cell responses, occurring earlier than MHC class Ia–restricted T cell responses, are mounted after primary infection with the intracellular bacterium Listeria monocytogenes. These H2-M3–restricted T cells are cytolytic and produce interferon γ. However, after a second L. monocytogenes infection, H2-M3–restricted memory T cell responses are minor in comparison to the much larger MHC class Ia–restricted responses. This first direct characterization of an MHC class Ib–restricted T cell response indicates that CD8+ T cells responding to L. monocytogenes infection can be divided into two groups: H2-M3–restricted responses, which provide rapid and quantitatively substantial effector function during primary infections but contribute relatively little to memory responses, and MHC class Ia–restricted responses, which expand later during primary infection but form memory T cells that respond rapidly and dramatically in response to subsequent infections by the same pathogen.
The Toll-like Receptor Protein Rp105 Regulates Lipopolysaccharide Signaling in B Cells
Authors: Ogata Hirotaka , Su I-hsin , Miyake Kensuke , Nagai Yoshinori , Akashi Sachiko , Mecklenbräuker Ingrid , Rajewsky Klaus , Kimoto Masao , Tarakhovsky Alexander
Published: July 2002

The susceptibility to infections induced by Gram-negative bacteria is largely determined by innate immune responses to bacteria cell wall lipopolysaccharide (LPS). The stimulation of B cells by LPS enhances their antigen-presenting capacity and is accompanied by B cell proliferation and secretion of large quantities of LPS-neutralizing antibodies. Similar to macrophages and neutrophils, the LPS-induced activation of B cells is dependent on Toll-like receptor (TLR)4. Here, we demonstrate that the responses of B cells to LPS are also regulated by another TLR protein, RP105, which is predominantly expressed on mature B cells in mice and humans. The analysis of mice homozygous for the null mutation in the RP105 gene revealed impaired proliferative and humoral immune responses of RP105-deficient B cells to LPS. Using originally LPS-unresponsive Ba/F3 cells expressing exogenous TLR4 and RP105, we demonstrate the functional cooperation between TLR4 and RP105 in LPS-induced nuclear factor κB activation. These data suggest the existence of the TLR4–RP105 signaling module in the LPS-induced B cell activation.
Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors
Authors: Scharfstein Julio , Schmitz Veronica , Morandi Veronica , Capella Marcia M. A. , Lima Ana Paula C. A. , Morrot Alexandre , Juliano Luiz , Müller-Esterl Werner
Published: July 2002

The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease.
Identification of a Novel Prostaglandin F2α Synthase in Trypanosoma brucei
Authors: Kubata Bruno Kilunga , Duszenko Michael , Kabututu Zakayi , Rawer Marc , Szallies Alexander , Fujimori Ko , Inui Takashi , Nozaki Tomoyoshi , Yamashita Kouwa , Horii Toshihiro , Urade Yoshihiro , Hayaishi Osamu
Published: July 2002

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH2. Among all PGs synthesized, PGF2α was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF2α synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF2α in the physiology of the parasite and the pathogenesis of African trypanosomiasis.
Separation of Notch1 Promoted Lineage Commitment and Expansion/Transformation in Developing T Cells
Authors: Allman David , Karnell Fredrick G. , Punt Jennifer A. , Bakkour Sonia , Xu Lanwei , Myung Peggy , Koretzky Gary A. , Pui John C. , Aster Jon C. , Pear Warren S.
Published: July 2002

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4+CD8+ double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2−/−) or Src homology 2 domain–containing leukocyte protein of 76 kD (SLP-76)−/− mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2−/− progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3ε and pre-Tα mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2−/− mice with a TCRβ transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell–specific signals associated with development of DP thymocytes.
Cell Swelling Activates the K+ Conductance and Inhibits the Cl− Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium
Authors: Torres Ruben J. , Subramanyam Muthangi , Altenberg Guillermo A. , Reuss Luis
Published: July 2002

Necturus gallbladder epithelial cells bathed in 10 mM HCO3/1% CO2 display sizable basolateral membrane conductances for Cl− (GClb) and K + (GKb). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K +/Cl− selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca2+]i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca2+ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (Vcs) produced a significant increase in the change in Vcs elicited by lowering basolateral solution [Cl−], whereas hyperpolarization of Vcs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases GKb and decreases G Clb. These two effects appear to be linked, i.e., the increase in G Kb produces membrane hyperpolarization, which in turn reduces G Clb. ( b) Hyperosmotic shrinkage has the opposite effects on GKb and G Clb. ( c) Cell swelling causes a transient increase in [Ca2+]i, but this response may not be necessary for the increase in GKb during cell swelling.
Functional Reconstitution of a Prokaryotic K+ Channel
Authors: Heginbotham Lise , Kolmakova-Partensky Ludmila , Miller Christopher
Published: July 2002

SliK, a K+ channel encoded by the Streptomyces KcsA gene, was expressed, purified, and reconstituted in liposomes. A concentrative 86Rb+ flux assay was used to assess the ion transport properties of SliK. SliK-mediated ionic flux shows strong selectivity for K+ over Na+ and is inhibited by micromolar concentrations of Ba2+, mirroring the basic permeation characteristic of eukaryotic K+ channels studied by electrophysiological methods. 86Rb+ uptake kinetics and equilibrium measurements also demonstrate that the purified protein is fully active.
Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding
Authors: Isenring Paul , Jacoby Steven C. , Chang John , Forbush Bliss
Published: July 2002

The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.
Parallel integer relation detection: Techniques and applications
Authors: Bailey David , Broadhurst David
Published: July 2002

Let

{

x
1

,

x
2

,
⋯
,

x
n

}

\{x_1, x_2, \cdots , x_n\}

be a vector of real numbers. An integer relation algorithm is a computational scheme to find the

n
n

integers

a
k

a_k

, if they exist, such that

a
1

x
1

+

a
2

x
2

+
⋯
+

a
n

x
n

=
0

a_1 x_1 + a_2 x_2 + \cdots + a_n x_n= 0

. In the past few years, integer relation algorithms have been utilized to discover new results in mathematics and physics. Existing programs for this purpose require very large amounts of computer time, due in part to the requirement for multiprecision arithmetic, yet are poorly suited for parallel processing. This paper presents a new integer relation algorithm designed for parallel computer systems, but as a bonus it also gives superior results on single processor systems. Single- and multi-level implementations of this algorithm are described, together with performance results on a parallel computer system. Several applications of these programs are discussed, including some new results in mathematical number theory, quantum field theory and chaos theory.
Fast algorithms for discrete polynomial transforms
Authors: Potts Daniel , Steidl Gabriele , Tasche Manfred
Published: July 2002

Consider the Vandermonde-like matrix

P

:=
(

P
k

(
cos
⁡

j
π

N

)

)

j
,
k
=
0

N

{\mathbf {P}}:=(P_k(\cos \frac {j\pi }{N}))_{j,k=0}^N

, where the polynomials

P
k

P_k

satisfy a three-term recurrence relation. If

P
k

P_k

are the Chebyshev polynomials

T
k

T_k

, then

P

{\mathbf {P}}

coincides with

C

N
+
1

:=
(
cos
⁡

j
k
π

N

)

j
,
k
=
0

N

{\mathbf {C}}_{N+1}:= (\cos \frac {jk\pi }{N})_{j,k=0}^N

. This paper presents a new fast algorithm for the computation of the matrix-vector product

P
a

{\mathbf {Pa}}

in

O
(
N

log
2

⁡
N
)

O(N \log ^2N)

arithmetical operations. The algorithm divides into a fast transform which replaces

P
a

{\mathbf {Pa}}

with

C

N
+
1

a
~

{\mathbf {C}}_{N+1} {\mathbf {\tilde a}}

and a subsequent fast cosine transform. The first and central part of the algorithm is realized by a straightforward cascade summation based on properties of associated polynomials and by fast polynomial multiplications. Numerical tests demonstrate that our fast polynomial transform realizes

P
a

{\mathbf {Pa}}

with almost the same precision as the Clenshaw algorithm, but is much faster for

N
≥
128

N\ge 128

.
GeneDn: for high-level expression design of heterologous genes in a prokaryotic system.
Authors: Ju L W , Xing L H , Hong P W , Jin W J
Published: July 2002

RESULTS: Based on the mathematical model of high-level expression of heterologous genes in prokaryotic vector pBV220, we developed a program GeneDn for high-level expression design of natural and synthetic genes. AVAILIBILITY: The program is written in Turbo Pascal 7.0. The source code and related material are available upon request. CONTACT: wujj@nic.bmi.ac.cn
Prediction of transcriptional regulatory sites in the complete genome sequence of Escherichia coli K-12.
Authors: Thieffry D , Salgado H , Huerta A M , Collado-Vides J
Published: July 2002

MOTIVATION: As one of the best-characterized free-living organisms, Escherichia coli and its recently completed genomic sequence offer a special opportunity to exploit systematically the variety of regulatory data available in the literature in order to make a comprehensive set of regulatory predictions in the whole genome. RESULTS: The complete genome sequence of E.coli was analyzed for the binding of transcriptional regulators upstream of coding sequences. The biological information contained in RegulonDB (Huerta, A.M. et al., Nucleic Acids Res.,26,55-60, 1998) for 56 different transcriptional proteins was the support to implement a stringent strategy combining string search and weight matrices. We estimate that our search included representatives of 15-25% of the total number of regulatory binding proteins in E.coli. This search was performed on the set of 4288 putative regulatory regions, each 450 bp long. Within the regions with predicted sites, 89% are regulated by one protein and 81% involve only one site. These numbers are reasonably consistent with the distribution of experimental regulatory sites. Regulatory sites are found in 603 regions corresponding to 16% of operon regions and 10% of intra-operonic regions. Additional evidence gives stronger support to some of these predictions, including the position of the site, biological consistency with the function of the downstream gene, as well as genetic evidence for the regulatory interaction. The predictions described here were incorporated into the map presented in the paper describing the complete E.coli genome (Blattner,F.R. et al., Science, 277, 1453-1461, 1997). AVAILABILITY: The complete set of predictions in GenBank format is available at the url: http://www. cifn.unam.mx/Computational_Biology/E.coli-predictions CONTACT: ecoli-reg@cifn.unam.mx, collado@cifn.unam.mx