bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclosporin A Decreases Human Macrophage Interleukin‐6 Synthesis at Post‐Transcriptional Level
Authors: García Juan E. Losa , López Ana M. Rodríguez , De Cabo María Rosa Martín , Rodríguez Fernando Mateos , Losada Jesús Pérez , Sarmiento Rogelio González , López Antonio Jiménez , Arellano José Luis Pérez
Published: July 2002

In addition to its well‐established effect on T cells, cyclosporin A (CsA) also inhibits inflammatory cytokine production by macrophages. However, little is known about the mechanism of action of CsA on macrophage cytokine production. We measured the effect of CsA on basal and phorbol‐myristate‐acetate (PMA)‐stimulated production of interleukin‐6 using the human monocyte cell line U937 differentiated with dimethylsulfoxide (DMSO). Interleukin‐6 levels were measured in supernatant and cell lysates using specific enzyme‐linked immunosorbent assays. We found that CsA decreases not only IL‐6 release but also cytokine synthesis. The concentration of CsA used did not affect either cell viability or proliferation. Three possibilities may be advanced to explain the CsA‐due decrease in IL‐6 production by macrophages: (a) inhibition of the synthesis of an early common regulatory protein, (b) inhibition of cytokine gene transcription, or (c) modulation of post‐transcriptional events. The first possibility was tested by measuring the effect of cycloheximide on the experimental system during the first 3 hours of culture. Although cycloheximide decreased total cytokine synthesis, the pattern of cytokine modulation by CsA persisted. These data suggest that CsA‐mediated macrophage cytokine inhibition is not mediated by an early common regulatory protein. To further explore the inhibition mechanism, we measured IL‐6 mRNA levels by Northern blot. IL‐6 mRNA levels were unaffected by CsA both in resting and PMA‐stimulated cells. We conclude that in human macrophages CsA diminishes IL‐6 production at post‐transcriptional level.
Stimulant Psychosis: Symptom Profile and Acute Clinical Course
Authors: Harris Debra , Batki Steven L.
Published: July 2002

Nineteen patients seen at a psychiatric emergency service with amphetamine‐ or cocaine‐induced psychotic disorder were assessed with structured interviews, chart review, and blood and urine testing. All had a predominance of positive symptoms from the Positive and Negative Syndrome Scale (PANSS). However, some subjects had substantial Negative Scale scores (26%), bizarre delusions (95%), and Schneiderian hallucinations (63%), mimicking a broad range of schizophrenic symptoms. Several PANSS scores were correlated with treatment intensity: Positive score with seclusion hours, General Psychopathology and Negative scores with hospitalization length, and General Psycho‐pathology score with neuroleptic dose. Presenting symptoms may help in treatment planning.
Personality Dimensions and Substance Misuse: Relationships in Adolescents, Mothers and Fathers
Authors: Gabel Stewart , Stallings Michael C. , Schmitz Stephanie , Young Susan E. , Fulker David W.
Published: July 2002

This research addressed the question of whether relationships exist between personality dimensions, antisocial behavior, and alcohol or other substance misuse (AOSM) in adolescents and in their fathers and mothers, who often also have histories of AOSM. One hundred male adolescents (mean age 15.8 years) entering a residential treatment center for youths with AOSM, their mothers (n = 88, mean age 39.4 years), their fathers (n = 36, mean age 44.9 years), and community controls (n = 100 adolescents, mean age 16.5 years; n = 96 mothers, mean age 43.8 years; n = 87 fathers, mean age 45.9 years) were recruited. All participants completed a personality questionnaire and were interviewed on several measures, including structured interviews for psychopathology and substance misuse. The findings indicated that novelty seeking (NS), one of the personality dimensions, was significantly correlated with substance misuse in adolescent probands, adolescent controls, and proband fathers and mothers, but not in control fathers and mothers. Regression analyses that included conduct disorder (CD) or antisocial personality disorder (APD) symptoms indicated that both NS and CD or APD symptoms made significant contributions to the prediction of substance misuse in treatment group probands and in their fathers and mothers. The findings further suggest that NS and antisocial behaviors contribute independently to substance misuse in severely impaired adolescents and their fathers, but not in their mothers.
Prenatal Oral (Gavage) Developmental Toxicity Study of Decabromodiphenyl Oxide in Rats
Authors: Hardy M. L. , Schroeder R. , Biesemeier J. , Manor O.
Published: July 2002

Decabromodiphenyl oxide (DBDPO) is a highly effective flame retardant that is primarily used in electrical and electronic equipment with a secondary, but important, application in upholstery textiles. DBDPO, the second largest volume brominated flame retardant in use today, has undergone a wide range of toxicology tests in mammalian species with the results indicating a no-adverse-effect level of ∼1000 mg/kg/day in oral repeated-dose studies. An oral prenatal developmental toxicity study of the commercial DBDPO product (97% purity) was performed under current EPA OPPTS and OECD guidelines. Female Sprague-Dawley rats (25 mated females/group) received 0, 100, 300 or 1000 mg DBPDO/kg/day via gavage in corn oil during gestation days 0 through 19. All females survived until scheduled sacrifice. No clinical signs of toxicity were observed. Pregnancy rates in the control and treated groups ranged from 96% to 100% and provided 23 or more litters in each group for evaluation on gestation day 20. No effect of treatment was seen in maternal gestational parameters (body weight, body weight gain, and food consumption), uterine implantation data, liver weight, or necropsy findings. Likewise, no effect of treatment was seen in fetal body weights, fetal sex distribution, or during the fetal external, visceral, or skeletal examinations. The NOEL (noobservable-effect level) for maternal and developmental toxicity was 1000 mg DBPDO/kg/day, the highest dose level administered on gestation days 0 to 19.
Endocrine-Modulating Substances in the Environment: The Wildlife Connection
Authors: Solomon Keith R.
Published: July 2002

Historical observations, first publicized in Rachel Carson's Silent Spring, demonstrated biological effects of persistent, bioaccumulative pollutants on wildlife. These effects included disruption of reproduction and, in some situations, responses mediated through the endocrine system. The substances that caused these effects were mainly highly chlorinated halocarbon compounds, such as DDT (and metabolites), other organochlorine pesticides, polychlori-nated biphenyls, poly chlorinated dibenzo-p-dioxins, and poly chlorinated dibenzofurans. In contrast, responses of fish to industrial discharges and pulp mill effluents have implicated more water-soluble compounds. Characterizations of wildlife exposures require consideration of temporal and spatial factors that they exacerbate or ameliorate responses. Likewise, effects of endocrine-modulating substances m ay appear at subsequent stages of development, not at the time of exposure. Consistent with the declines in environmental concentrations of persistent, bioaccumulative substances, populations of several wild bird species, including bald eagles, cormorants, herring gulls, and Caspian terns, have increased, recovering from declines noted in previously polluted areas during the 1960s and 1970s.
Neuropharmacology and therapeutic potential of cannabinoids
Authors: Pertwee Roger G.
Published: July 2002

Mammalian tissues contain at least two types of cannabinoid receptor, CB 1, found mainly on neurones and CB 2, found mainly in immune cells. Endogenous ligands for these receptors have also been identified. These endocannabinoids and their receptors constitute the endogenous cannabinoid system. Two cannabinoid receptor agonists, Δ9‐tetrahydrocannabinol and nabilone, are used clinically as anti‐emetics or to boost appetite. Additional therapeutic uses of cannabinoids may include the suppression of some multiple sclerosis and spinal injury symptoms, the management of pain, bronchial asthma and glaucoma, and the prevention of neurotoxicity. There are also potential clinical applications for CB 1 receptor antagonists, in the management of acute schizophrenia and cognitive/memory dysfunctions and as appetite suppressants. Future research is likely to be directed at characterizing the endogenous cannabinoid system more completely, at obtaining more conclusive clinical data about cannabinoids with regard to both beneficial and adverse effects, at developing improved cannabinoid formulations and modes of administration for use in the clinic and at devising clinical strategies for separating out the sought‐after effects of CB 1 receptor agonists from their psychotropic and other unwanted effects.
Neuroprotective Action of Cycloheximide Involves Induction of Bcl-2 and Antioxidant Pathways
Authors: Furukawa Katsutoshi , Estus Steven , Fu Weiming , Mark Robert J. , Mattson Mark P.
Published: July 2002

The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of “killer” proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid β-peptide was increased in cultures pretreated with CHX at concentrations of 50–500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20–40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription–PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/ Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.
The First 35 Amino Acids and Fatty Acylation Sites Determine the Molecular Targeting of Endothelial Nitric Oxide Synthase into the Golgi Region of Cells: A Green Fluorescent Protein Study
Authors: Liu Jianwei , Hughes Thomas E. , Sessa William C.
Published: July 2002

Catalytically active endothelial nitric oxide synthase (eNOS) is located on the Golgi complex and in the caveolae of endothelial cells (EC). Mislocalization of eNOS caused by mutation of the N-myristoylation or cysteine palmitoylation sites impairs production of stimulated nitric oxide (NO), suggesting that intracellular targeting is critical for optimal NO production. To investigate the molecular determinants of eNOS targeting in EC, we constructed eNOS–green fluorescent protein (GFP) chimeras to study its localization in living and fixed cells. The full-length eNOS–GFP fusion colocalized with a Golgi marker, mannosidase II, and retained catalytic activity compared to wild-type (WT) eNOS, suggesting that the GFP tag does not interfere with eNOS localization or function. Experiments with different size amino-terminal fusion partners coupled to GFP demonstrated that the first 35 amino acids of eNOS are sufficient to target GFP into the Golgi region of NIH 3T3 cells. Additionally, the unique (Gly-Leu)5 repeat located between the palmitoylation sites (Cys-15 and -26) of eNOS is necessary for its palmitoylation and thus localization, but not for N-myristoylation, membrane association, and NOS activity. The palmitoylation-deficient mutants displayed a more diffuse fluorescence pattern than did WT eNOS–GFP, but still were associated with intracellular membranes. Biochemical studies also showed that the palmitoylation-deficient mutants are associated with membranes as tightly as WT eNOS. Mutation of the N-myristoylation site Gly-2 (abolishing both N-myristoylation and palmitoylation) caused the GFP fusion protein to distribute throughout the cell as GFP alone, consistent with its primarily cytosolic nature in biochemical studies. Therefore, eNOS targets into the Golgi region of NIH 3T3 cells via the first 35 amino acids, including N-myristoylation and palmitoylation sites, and its overall membrane association requires N-myristoylation but not cysteine palmitoylation. These results suggest a novel role for fatty acylation in the specific compartmentalization of eNOS and most likely, for other dually acylated proteins, to the Golgi complex.
Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle
Authors: Coopersmith Craig M. , Chandrasekaran Chitra , McNevin M. Shane , Gordon Jeffrey I.
Published: July 2002

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ∼30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 × TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108→ Lys107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 × TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a TAg with a COOH-terminal truncation. The dysplastic epithelium of K-rasVal12 × TAgWt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enterocytes with a proliferative abnormality, K-rasVal12 facilitates progression to a dysplastic phenotype; (b) by diminishing cell survival on the villus, the oncoprotein may impede further progression; and (c) additional mutations may be needed to suppress this proapoptotic response to K-rasVal12.
SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex
Authors: Matunis Michael J. , Wu Jian , Blobel Günter
Published: July 2002

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.
Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone
Authors: Wendel Mikael , Sommarin Yngve , Heinegård Dick
Published: July 2002

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride–containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mr of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin αvβ3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.
The a-Kinase–Anchoring Protein Akap95 Is a Multivalent Protein with a Key Role in Chromatin Condensation at Mitosis
Authors: Collas Philippe , Le Guellec Katherine , Taskén Kjetil
Published: July 2002

Protein kinase A (PKA) and the nuclear A-kinase–anchoring protein AKAP95 have previously been shown to localize in separate compartments in interphase but associate at mitosis. We demonstrate here a role for the mitotic AKAP95–PKA complex. In HeLa cells, AKAP95 is associated with the nuclear matrix in interphase and redistributes mostly into a chromatin fraction at mitosis. In a cytosolic extract derived from mitotic cells, AKAP95 recruits the RIIα regulatory subunit of PKA onto chromatin. Intranuclear immunoblocking of AKAP95 inhibits chromosome condensation at mitosis and in mitotic extract in a PKA-independent manner. Immunodepletion of AKAP95 from the extract or immunoblocking of AKAP95 at metaphase induces premature chromatin decondensation. Condensation is restored in vitro by a recombinant AKAP95 fragment comprising the 306–carboxy-terminal amino acids of the protein. Maintenance of condensed chromatin requires PKA binding to chromatin-associated AKAP95 and cAMP signaling through PKA. Chromatin-associated AKAP95 interacts with Eg7, the human homologue of Xenopus pEg7, a component of the 13S condensin complex. Moreover, immunoblocking nuclear AKAP95 inhibits the recruitment of Eg7 to chromatin in vitro. We propose that AKAP95 is a multivalent molecule that in addition to anchoring a cAMP/PKA–signaling complex onto chromosomes, plays a role in regulating chromosome structure at mitosis.
Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Authors: Ao Wanyuan , Pilgrim Dave
Published: July 2002

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B–dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.
Stimulation of Fascin Spikes by Thrombospondin-1 Is Mediated by the Gtpases Rac and Cdc42
Authors: Adams Josephine Clare , Schwartz Martin Alexander
Published: July 2002

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.
Pds5p Is an Essential Chromosomal Protein Required for Both Sister Chromatid Cohesion and Condensation in Saccharomyces cerevisiae
Authors: Hartman Theresa , Stead Kristen , Koshland Douglas , Guacci Vincent
Published: July 2002

The PDS5 gene (precocious dissociation of sisters) was identified in a genetic screen designed to identify genes important for chromosome structure. PDS5 is an essential gene and homologues are found from yeast to humans. Pds5p function is important for viability from S phase through mitosis and localizes to chromosomes during this cell cycle window, which encompasses the times when sister chromatid cohesion exists. Pds5p is required to maintain cohesion at centromere proximal and distal sequences. These properties are identical to those of the four cohesion complex members Mcd1p/Scc1p, Smc1p, Smc3p, and Scc3p/Irr1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47–57; Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cell. 91:35–45; Toth, A., R. Ciosk, F. Uhlmann, M. Galova, A. Schleiffer, and K. Nasmyth. 1999. Genes Dev. 13:307–319). Pds5p binds to centromeric and arm sequences bound by Mcd1p. Furthermore, Pds5p localization to chromosomes is dependent on Mcd1p. Thus, Pds5p, like the cohesin complex members, is a component of the molecular glue that mediates sister chromatid cohesion. However, Mcd1p localization to chromosomes is independent of Pds5p, which may reflect differences in their roles in cohesion. Finally, Pds5p is required for condensation as well as cohesion, which confirms the link between these processes revealed through analysis of Mcd1p (Guacci, V., D. Koshland, and A. Strunnikov. 1997. Cell. 91:47–57). Therefore, the link between cohesion and condensation is a general property of yeast chromosomes.
Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin
Authors: Govind Sheila , Kozma Robert , Monfries Clinton , Lim Louis , Ahmed Sohail
Published: July 2002

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.
Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer
Authors: Sato Ken , Sato Miyuki , Nakano Akihiko
Published: July 2002

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing α and γ subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.
Restriction of Secretory Granule Motion near the Plasma Membrane of Chromaffin Cells
Authors: Johns Laura M. , Levitan Edwin S. , Shelden Eric A. , Holz Ronald W. , Axelrod Daniel
Published: July 2002

We used total internal reflection fluorescence microscopy to study quantitatively the motion and distribution of secretory granules near the plasma membrane (PM) of living bovine chromaffin cells. Within the ∼300-nm region measurably illuminated by the evanescent field resulting from total internal reflection, granules are preferentially concentrated close to the PM. Granule motion normal to the substrate (the z direction) is much slower than would be expected from free Brownian motion, is strongly restricted over tens of nanometer distances, and tends to reverse directions within 0.5 s. The z-direction diffusion coefficients of granules decrease continuously by two orders of magnitude within less than a granule diameter of the PM as granules approach the PM. These analyses suggest that a system of tethers or a heterogeneous matrix severely limits granule motion in the immediate vicinity of the PM. Transient expression of the light chains of tetanus toxin and botulinum toxin A did not disrupt the restricted motion of granules near the PM, indicating that SNARE proteins SNAP-25 and VAMP are not necessary for the decreased mobility. However, the lack of functional SNAREs on the plasma or granule membranes in such cells reduces the time that some granules spend immediately adjacent to the PM.
The Small Muscle-Specific Protein Csl Modifies Cell Shape and Promotes Myocyte Fusion in an Insulin-like Growth Factor 1–Dependent Manner
Authors: Palmer Steve , Groves Nicola , Schindeler Aaron , Yeoh Thomas , Biben Christine , Wang Cheng-Chun , Sparrow Duncan B. , Barnett Louise , Jenkins Nancy A. , Copeland Neal G. , Koentgen Frank , Mohun Tim , Harvey Richard P.
Published: July 2002

We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic “myosacs.” The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer–binding factor (MEF)2, were also enhanced in an IGF-1 signaling–dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.
A role for nuclear lamins in nuclear envelope assembly
Authors: Lopez-Soler Reynold I. , Moir Robert D. , Spann Timothy P. , Stick Reimer , Goldman Robert D.
Published: July 2002

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina–pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These “nonfusogenic” vesicles lack lamin B3 (LB3) and do not bind LB3T; however, “fusogenic” vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.
The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis
Authors: Earles Cynthia A. , Bai Jihong , Wang Ping , Chapman Edwin R.
Published: July 2002

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.
MAST/Orbit has a role in microtubule–kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity
Authors: Maiato Helder , Sampaio Paula , Lemos Catarina L. , Findlay John , Carmena Mar , Earnshaw William C. , Sunkel Claudio E.
Published: July 2002

Multiple asters (MAST)/Orbit is a member of a new family of nonmotor microtubule-associated proteins that has been previously shown to be required for the organization of the mitotic spindle. Here we provide evidence that MAST/Orbit is required for functional kinetochore attachment, chromosome congression, and the maintenance of spindle bipolarity. In vivo analysis of Drosophila mast mutant embryos undergoing early mitotic divisions revealed that chromosomes are unable to reach a stable metaphase alignment and that bipolar spindles collapse as centrosomes move progressively closer toward the cell center and eventually organize into a monopolar configuration. Similarly, soon after depletion of MAST/Orbit in Drosophila S2 cells by double-stranded RNA interference, cells are unable to form a metaphase plate and instead assemble monopolar spindles with chromosomes localized close to the center of the aster. In these cells, kinetochores either fail to achieve end-on attachment or are associated with short microtubules. Remarkably, when microtubule dynamics is suppressed in MAST-depleted cells, chromosomes localize at the periphery of the monopolar aster associated with the plus ends of well-defined microtubule bundles. Furthermore, in these cells, dynein and ZW10 accumulate at kinetochores and fail to transfer to microtubules. However, loss of MAST/Orbit does not affect the kinetochore localization of D-CLIP-190. Together, these results strongly support the conclusion that MAST/Orbit is required for microtubules to form functional attachments to kinetochores and to maintain spindle bipolarity.
Absence of Respiratory Burst in X-linked Chronic Granulomatous Disease Mice Leads to Abnormalities in Both Host Defense and Inflammatory Response to Aspergillus fumigatus
Authors: Morgenstern David E. , Gifford Mary A.C. , Li Ling Lin , Doerschuk Claire M. , Dinauer Mary C.
Published: July 2002

Mice with X-linked chronic granulomatous disease (CGD) generated by targeted disruption of the gp91phox subunit of the NADPH–oxidase complex (X-CGD mice) were examined for their response to respiratory challenge with Aspergillus fumigatus. This opportunistic fungal pathogen causes infection in CGD patients due to the deficient generation of neutrophil respiratory burst oxidants important for damaging A. fumigatus hyphae. Alveolar macrophages from X-CGD mice were found to kill A. fumigatus conidia in vitro as effectively as alveolar macrophages from wild-type mice. Pulmonary disease in X-CGD mice was observed after administration of doses ranging from 105 to 48 spores, none of which produced disease in wild-type mice. Higher doses produced a rapidly fatal bronchopneumonia in X-CGD mice, whereas progression of disease was slower at lower doses, with development of chronic inflammatory lesions. Marked differences were also observed in the response of X-CGD mice to the administration of sterilized Aspergillus hyphae into the lung. Within 24 hours of administration, X-CGD mice had significantly higher numbers of alveolar neutrophils and increased expression of the proinflammatory cytokines IL-1β and TNF-α relative to the responses seen in wild-type mice. By one week after administration, pulmonary inflammation was resolving in wild-type mice, whereas X-CGD mice developed chronic granulomatous lesions that persisted for at least six weeks. This is the first experimental evidence that chronic inflammation in CGD does not always result from persistent infection, and suggests that the clinical manifestations of this disorder reflect both impaired microbial killing as well as other abnormalities in the inflammatory response in the absence of a respiratory burst.
Defects in Macrophage Recruitment and Host Defense in Mice Lacking the CCR2 Chemokine Receptor
Authors: Kurihara Takao , Warr Glenn , Loy James , Bravo Rodrigo
Published: July 2002

Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene. Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2−/− mice failed to recruit macrophages in an experimental peritoneal inflammation model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.
Kinetics of Eotaxin Generation and Its Relationship to Eosinophil Accumulation in Allergic Airways Disease: Analysis in a Guinea Pig Model In Vivo
Authors: Humbles Alison A. , Conroy Dolores M. , Marleau Sylvie , Rankin Sara M. , Palframan Roger T. , Proudfoot Amanda E.I. , Wells Timothy N.C. , Li Dechun , Jeffery Peter K. , Griffiths-Johnson David A. , Williams Timothy J. , Jose Peter J.
Published: July 2002

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12–24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.
Accelerated Neutrophil Apoptosis in Mice Lacking A1-a, a Subtype of the bcl-2–related A1 Gene
Authors: Hamasaki Azumi , Sendo Fujiro , Nakayama Keiko , Ishida Noriko , Negishi Izumi , Nakayama Kei-ichi , Hatakeyama Shigetsugu
Published: July 2002

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a−/− mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a−/− mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/− mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a−/− and A1-a+/− animals. On the other hand, the extent of tumor necrosis factor α–induced acceleration of neutrophil apoptosis did not differ among A1-a−/−, A1-a+/−, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/−, and A1-a−/−. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.
Interleukin 7 Receptor Control of  T Cell Receptor γ Gene Rearrangement: Role of Receptor-associated Chains and Locus Accessibility
Authors: Durum Scott K. , Candèias Serge , Nakajima Hiroshi , Leonard Warren J. , Baird Allison M. , Berg Leslie J. , Muegge Kathrin
Published: July 2002

VDJ recombination of T cell receptor and immunoglobulin loci occurs in immature lymphoid cells. Although the molecular mechanisms of DNA cleavage and ligation have become more clear, it is not understood what controls which target loci undergo rearrangement. In interleukin 7 receptor (IL-7R)α−/− murine thymocytes, it has been shown that rearrangement of the T cell receptor (TCR)-γ locus is virtually abrogated, whereas other rearranging loci are less severely affected. By examining different strains of mice with targeted mutations, we now observe that the signaling pathway leading from IL-7Rα to rearrangement of the TCR-γ locus requires the γc receptor chain and the γc-associated Janus kinase Jak3. Production of sterile transcripts from the TCR-γ locus, a process that generally precedes rearrangement of a locus, was greatly repressed in IL-7Rα−/− thymocytes. The repressed transcription was not due to a lack in transcription factors since the three transcription factors known to regulate this locus were readily detected in IL-7Rα−/− thymocytes. Instead, the TCR-γ locus was shown to be methylated in IL-7Rα−/− thymocytes. Treatment of IL-7Rα−/− precursor T cells with the specific histone deacetylase inhibitor trichostatin A released the block of TCR-γ gene rearrangement. This data supports the model that IL-7R promotes TCR-γ gene rearrangement by regulating accessibility of the locus via demethylation and histone acetylation of the locus.
Coordinate Activation of Activator Protein 1 and Inflammatory Cytokines in Response to Neisseria gonorrhoeae Epithelial Cell Contact Involves Stress Response Kinases
Authors: Naumann Michael , Rudel Thomas , Wieland Björn , Bartsch Cornelia , Meyer Thomas F.
Published: July 2002

Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1–dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.