bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of routes and funnels in protein folding by free energy functional methods
Authors: Plotkin Steven S. , Onuchic José N.
Published: July 2002

We use a free energy functional theory to elucidate general properties of heterogeneously ordering, fast folding proteins, and we test our conclusions with lattice simulations. We find that both structural and energetic heterogeneity can lower the free energy barrier to folding. Correlating stronger contact energies with entropically likely contacts of a given native structure lowers the barrier, and anticorrelating the energies has the reverse effect. Designing in relatively mild energetic heterogeneity can eliminate the barrier completely at the transition temperature. Sequences with native energies tuned to fold uniformly, as well as sequences tuned to fold reliably by a single or a few routes, are rare. Sequences with weak native energetic heterogeneity are more common; their folding kinetics is more strongly determined by properties of the native structure. Sequences with different distributions of stability throughout the protein may still be good folders to the same structure. A measure of folding route narrowness is introduced that correlates with rate and that can give information about the intrinsic biases in ordering arising from native topology. This theoretical framework allows us to investigate systematically the coupled effects of energy and topology in protein folding and to interpret recent experiments that investigate these effects.
Gastrulation Defective is a serine protease involved in activating the receptor Toll to polarize the
Drosophila
embryo
Authors: Han Jin-Hua , Lee Si Hyoung , Tan Yue-Qing , LeMosy Ellen K. , Hashimoto Carl
Published: July 2002

The dorsoventral axis of the
Drosophila
embryo is induced by a ventrally restricted ligand for the receptor Toll. The Toll ligand is generated by a proteolytic processing reaction, which occurs at the end of a proteolytic cascade and requires the
gastrulation defective
(
gd
),
nudel
,
pipe
, and
windbeutel
genes. Here we demonstrate that the GD protein is a serine protease and that the three other genes act to restrict GD activity to the ventral side of the embryo. Our data support a model in which the GD protease catalyzes the ventral activation of the proteolytic cascade that produces the Toll ligand.
Drug target validation: Lethal infection blocked by inducible peptide
Authors: Tao Jianshi , Wendler Philip , Connelly Gene , Lim Audrey , Zhang Jiansu , King Megan , Li Tongchuan , Silverman Jared A. , Schimmel Paul R. , Tally Francis P.
Published: July 2002

Genome projects are generating large numbers of potential new targets for drug discovery. One challenge is target validation, proving the usefulness of a specific target in an animal model. In this paper, we demonstrate a new approach to validation and assay development. We selected
in vitro
specific peptide binders to a potential pathogen target. By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued. Thus, by combining
in vitro
selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated. This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process.
Interaction of yeast kinetochore proteins with centromere–protein/transcription factor Cbf1
Authors: Hemmerich Peter , Stoyan Tanja , Wieland Gerhard , Koch Marianne , Lechner Johannes , Diekmann Stephan
Published: July 2002

The centromere–kinetochore complex ofSaccharomyces cerevisiaeis a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein–DNA and protein–protein interactions. We have usedin vitroassays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. GlutathioneS-transferase pull-down experiments showed the direct interaction ofin vitrotranslated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated thein vitroDNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNAin vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding atMET16promoters reveals the strong analogy between centromeres and theMET16promoter.
Leading and lagging strand DNA synthesis
in vitro
by a reconstituted herpes simplex virus type 1 replisome
Authors: Falkenberg Maria , Lehman I. R. , Elias Per
Published: July 2002

The synthesis of double-stranded DNA by a rolling circle mechanism was reconstituted
in vitro
with a replisome consisting of the DNA polymerase-UL42 complex and the heterotrimeric helicase-primase encoded by herpes simplex virus type 1. Okazaki fragments 3 kilobases in length and leading strands that may exceed 10 kilobases are produced. Lagging strand synthesis is stimulated by ribonucleoside triphosphates. DNA replication appears to be processive because it resists competition with an excess of (dT)
150
/(dA)
20
. The single-strand DNA binding protein ICP8 is not required, and high concentrations of ICP8 can, in fact, inhibit lagging strand synthesis. The inhibition can, however, be overcome by the addition of an excess of the UL8 component of the helicase-primase. Rolling circle replication by the herpesvirus and bacteriophage T7 replisomes appears to proceed by a similar mechanism.
Hyper‐Fractionated Radiotherapy for Soft Tissue Sarcoma: Results of theSecond Study of Hyper‐Fractionated Radiotherapy
Authors: Jacob R. , Gilligan D. , Robinson M. , Harmer C.
Published: July 2002

Purpose and Method. Hyper‐fractionated radiotherapy for treatment
of soft tissue sarcomas is designed to deliver a higher total dose of radiation without an
increase in late normal tissue damage. In a previous study at the Royal Marsden Hospital,
a total dose of 75 Gy using twice daily 1.25 Gy fractions resulted in a higher incidence of late
damage than conventional radiotherapy using 2 Gy daily fractions treating to a total of 60 Gy.
The current trial therefore used a lower dose per fraction of 1.2 Gy and lower total dose of 72
Gy, with 60 fractions given over a period of 6 weeks.

Subjects. A total of 37 patients (22 males and 15 females) with a median
age of 56 years (range 19–88 years) were treated.

Results. Of eight patients treated pre‐operatively, six showed a
partial response and in two the tumour was static. The maximum acute toxicities were grade
1 in eight, grade 2 in 14 and grade 3 in 15 patients. Late toxicities of the skin were
graded 1 in 10 and grade 2 in nine patients. Five patients complained of pain in the irradiated
bone and soft tissues, which was of moderate severity (grade 2). Stiffness was graded 2 in
three patients and severe (grade 3) in one.Three patients had moderate and one patient had
severe lymphoedema following treatment. The 5‐year recurrence‐free survival probability of
patients treated radically was 76%. Following excision of local recurrences the study group
had a disease‐free survival probability of 86% at 5 years.

Discussion. The regime is well tolerated with comparable local control
and late complication rates to standard daily fractionated therapy.The potential benefit of this
regime needs to be defined in a prospective randomized trial.
Digital Metastases of Giant Cell Rich Malignant Fibrous Histiocytoma
Authors: Brownlow Harry C. , Ioannidis George , Gibbons Max C. L. M. H. , Jones Adrian C. , Athanasou Nick
Published: July 2002

Background. Metastatic spread of soft tissue sarcomas to the digits is
extremely rare and metastasis of MFH to the fingers and toes has not been documented.

Purpose. We present two case reports of metastatic spread from a giant
cell rich malignant fibrous histiocytoma to the digits and discuss their management.
Lack of Activity of Docetaxel in Soft Tissue Sarcomas:Results of a Phase II Study of the Italian Group on Rare Tumors
Authors: Santoro Armando , Romanini Antonella , Rosso Alberto , Frustaci Sergio , Comandone Alessandro , Apice Gaetano , De Toma Domenico , Dogliotti Luigi , Lionetto Rita , Dani Carla , Bruzzi Paolo , Piolini Marco , Bergnolo Paola , Verusio Claudio
Published: July 2002

Purpose. The prognosis of advanced soft tissue sarcoma is poor, only
a few drugs showing some activity with response rates around 15– 25%. Consequently drug
development seems mandatory to improve treatment outcome. Following previous
favourable EORTC experience, the Italian Group on Rare Tumors started a phase II study
with docetaxel to confirm the activity of this drug in soft tissue sarcoma.

Patients and methods. Thirty‐seven patients with soft tissue sarcoma
resistant to at least one anthracyclinecontaining regimen were enrolled in a phase II multicenter
study evaluating docetaxel 100 mg/m2
in a 1‐h i.v. infusion q3
weeks.

Results.Thirty‐seven patients were enrolled onto this phase II study and
36 were evaluable for response. Only one partial remission was observed [2.8% with 95%
confidence interval (CI) 0.1– 16.2%]. Median progression‐free and overall survival
were 42 and 350 days, respectively. Neutropenia and leukopenia as well as cutaneous
manifestations were the most common toxicities.

Discussion. The results of this phase II study do not confirm a previous
EORTC repor t on the activity of docetaxel in soft tissue sarcoma, but are consistent with
other more recent phase II studies. The accumulated evidence does not justify the use of this
drug in the management of patients suffering from this disease, resistant to
anthracyclinecontaining regimens.
Quality of Life in Survivors of a Primary Bone Tumour:A Systematic Review
Authors: Eiser Christine , Grimer Robert J.
Published: July 2002

Purpose. We conducted a systematic search of published literature,
to assess (i) quality of life (QoL) for survivors of a bone tumour compared with the normal
population; (ii) QoL implications following amputation, successful or failed limb salvage;
(iii) adaptation of young children to amputation compared with older children or
adolescents.

Methods. Electronic databases were searched including Medline,
PsycLIT and Cinahl covering the years 1982– 1998.

Results. We identified 11 studies. Regardless of treatment, physical
functioning was poor compared with population norms or healthy siblings.There was less
consistent evidence regarding emotional functioning. Seven studies compared functioning
in amputees and limb salvage patients.Two reported advantages in physical function for the
limb salvage group, one for the amputees and the rest no differences. Evidence about social
functioning or marriage is inconclusive, but there are suggestions that amputees report more
job discrimination.

Discussion. The literature is inconclusive, largely because of
methodological problems. These include small and non‐representative samples, and lack of
sensitive and appropriate measures. Specific gaps in the literature include very little
work concerned with psychological outcomes for children, or for those experiencing failed
limb salvage. More attention needs to be given to gender differences in emotional response
to traumatic surgery.The implications of the results for helping families balance the merits of
different treatments are discussed.
Folding of Insulin Receptor Monomers Is Facilitated by the Molecular Chaperones Calnexin and Calreticulin and Impaired by Rapid Dimerization
Authors: Bass Joseph , Chiu Gavin , Argon Yair , Steiner Donald F.
Published: July 2002

Many complex membrane proteins undergo subunit folding and assembly in the ER before transport to the cell surface. Receptors for insulin and insulin-like growth factor I, both integral membrane proteins and members of the family of receptor tyrosine kinases (RTKs), are unusual in that they require homodimerization before export from the ER. To better understand chaperone mechanisms in endogenous membrane protein assembly in living cells, we have examined the folding, assembly, and transport of the human insulin receptor (HIR), a dimeric RTK. Using pulse-chase labeling and nonreducing SDS-PAGE analysis, we have explored the molecular basis of several sequential maturation steps during receptor biosynthesis. Under normal growth conditions, newly synthesized receptor monomers undergo disulfide bond formation while associated with the homologous chaperones calnexin (Cnx) and calreticulin (Crt). An inhibitor of glucose trimming, castanospermine (CST), abolished binding to Cnx/Crt but also unexpectedly accelerated receptor homodimerization resulting in misfolded oligomeric proreceptors whose processing was delayed and cell surface expression was also decreased by ∼30%. Prematurely-dimerized receptors were retained in the ER and more avidly associated with the heat shock protein of 70 kD homologue binding protein. In CST-treated cells, receptor misfolding followed disordered oligomerization. Together, these studies demonstrate a chaperone function for Cnx/Crt in HIR folding in vivo and also provide evidence that folding efficiency and homodimerization are counterbalanced.
Sec35p, a Novel Peripheral Membrane Protein, Is Required for ER to Golgi Vesicle Docking
Authors: VanRheenen Susan M. , Cao Xiaochun , Lupashin Vladimir V. , Barlowe Charles , Gerard Waters M.
Published: July 2002

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE–associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.
Structural Analysis of Drosophila Merlin Reveals Functional Domains Important for Growth Control and Subcellular Localization
Authors: LaJeunesse Dennis R. , McCartney Brooke M. , Fehon Richard G.
Published: July 2002

Merlin, the product of the Neurofibromatosis type 2 (NF2) tumor-suppressor gene, is a member of the protein 4.1 superfamily that is most closely related to ezrin, radixin, and moesin (ERM). NF2 is a dominantly inherited disease characterized by the formation of bilateral acoustic schwannomas and other benign tumors associated with the central nervous system. To understand its cellular functions, we are studying a Merlin homologue in Drosophila. As is the case for NF2 tumors, Drosophila cells lacking Merlin function overproliferate relative to their neighbors. Using in vitro mutagenesis, we define functional domains within Merlin required for proper subcellular localization and for genetic rescue of lethal Merlin alleles. Remarkably, the results of these experiments demonstrate that all essential genetic functions reside in the plasma membrane– associated NH2-terminal 350 amino acids of Merlin. Removal of a seven–amino acid conserved sequence within this domain results in a dominant-negative form of Merlin that is stably associated with the plasma membrane and causes overproliferation when expressed ectopically in the wing. In addition, we provide evidence that the COOH-terminal region of Merlin has a negative regulatory role, as has been shown for ERM proteins. These results provide insights into the functions and functional organization of a novel tumor suppressor gene.
Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei
Authors: Haaf Thomas , Raderschall Elke , Reddy Gurucharan , Ward David C. , Radding Charles M. , Golub Efim I.
Published: July 2002

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.
p24 Proteins and Quality Control of LIN-12 and GLP-1 Trafficking in Caenorhabditis elegans
Authors: Wen Chenhui , Greenwald Iva
Published: July 2002

Mutations in the Caenorhabditis elegans sel-9 gene elevate the activity of lin-12 and glp-1, which encode members of the LIN-12/NOTCH family of receptors. Sequence analysis indicates SEL-9 is one of several C. elegans p24 proteins. Allele-specific genetic interactions suggest that reducing sel-9 activity increases the activity of mutations altering the extracellular domains of LIN-12 or GLP-1. Reducing sel-9 activity restores the trafficking to the plasma membrane of a mutant GLP-1 protein that would otherwise accumulate within the cell. Our results suggest a role for SEL-9 and other p24 proteins in the negative regulation of transport of LIN-12 and GLP-1 to the cell surface, and favor a role for p24 proteins in a quality control mechanism for endoplasmic reticulum–Golgi transport.
The Receptor Tyrosine Phosphatase Crypα Promotes Intraretinal Axon Growth
Authors: Ledig Matthias M. , Haj Fawaz , Bixby John L. , Stoker Andrew W. , Mueller Bernhard K.
Published: July 2002

Retinal ganglion cell axons grow towards the optic fissure in close contact with the basal membrane, an excellent growth substratum. One of the ligands of receptor tyrosine phosphatase CRYPα is located on the retinal and tectal basal membranes. To analyze the role of this RPTP and its ligand in intraretinal growth and guidance of ganglion cell axons, we disrupted ligand- receptor interactions on the retinal basal membrane in culture. Antibodies against CRYPα strongly reduced retinal axon growth on the basal membrane, and induced a dramatic change in morphology of retinal growth cones, reducing the size of growth cone lamellipodia. A similar effect was observed by blocking the ligand with a CRYPα ectodomain fusion protein. These effects did not occur, or were much reduced, when axons were grown either on laminin-1, on matrigel or on basal membranes with glial endfeet removed. This indicates that a ligand for CRYPα is located on glial endfeet. These results show for the first time in vertebrates that the interaction of a receptor tyrosine phosphatase with its ligand is crucial not only for promotion of retinal axon growth but also for maintenance of retinal growth cone lamellipodia on basal membranes.
Caspase Recruitment Domain (Card-Dependent) Cytoplasmic Filaments Mediate Bcl10-Induced Nf-κb Activation
Authors: Guiet Chantal , Vito Pasquale
Published: July 2002

Mucosa-associated lymphoid tissue (MALT) lymphomas are associated with overexpression and constitutive activity of bcl10, a caspase recruitment domain (CARD)-containing protein that activates NF-κB. Here, we show that arrangement of overexpressed bcl10 protein in cytoplasmic filaments is essential for recruitment of signal transducer molecules-involved NF-κB activation. We also show that cytoskeleton elements regulate bcl10 signaling.

Thus, organized assemblage of proteins in ordered structures linked to the cytoskeleton network may represent a general mechanism for intracellular signaling.
Skeletor, a Novel Chromosomal Protein That Redistributes during Mitosis Provides Evidence for the Formation of a Spindle Matrix
Authors: Walker Diana L. , Wang Dong , Jin Ye , Rath Uttama , Wang Yanming , Johansen Jørgen , Johansen Kristen M.
Published: July 2002

A spindle matrix has been proposed to help organize and stabilize the microtubule spindle during mitosis, though molecular evidence corroborating its existence has been elusive. In Drosophila, we have cloned and characterized a novel nuclear protein, skeletor, that we propose is part of a macromolecular complex forming such a spindle matrix. Skeletor antibody staining shows that skeletor is associated with the chromosomes at interphase, but redistributes into a true fusiform spindle structure at prophase, which precedes microtubule spindle formation. During metaphase, the spindle, defined by skeletor antibody labeling, and the microtubule spindles are coaligned. We find that the skeletor-defined spindle maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate. Consequently, the properties of the skeletor-defined spindle make it an ideal substrate for providing structural support stabilizing microtubules and counterbalancing force production. Furthermore, skeletor metaphase spindles persist in the absence of microtubule spindles, strongly implying that the existence of the skeletor-defined spindle does not require polymerized microtubules. Thus, the identification and characterization of skeletor represents the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus before microtubule spindle formation.
The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin
Authors: Dyson Jennifer M. , O'Malley Cindy J. , Becanovic Jelena , Munday Adam D. , Berndt Michael C. , Coghill Imogen D. , Nandurkar Harshal H. , Ooms Lisa M. , Mitchell Christina A.
Published: July 2002

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.
Resistance to Fas-mediated Apoptosis of Peripheral T Cells in Human T Lymphocyte Virus Type I (HTLV-I) Transgenic Mice with Autoimmune Arthropathy
Authors: Kishi Shuji , Saijyo Shinobu , Arai Masaaki , Karasawa Shigeru , Ueda Susumu , Kannagi Mari , Iwakura Yoichiro , Fujii Masahiro , Yonehara Shin
Published: July 2002

Transgenic mice carrying the env-pX region of human T lymphocyte virus type I (HTLV-I) develop autoimmune arthropathy in high incidence. Adopting the approach that Fas-mediated apoptosis has a critical function in the elimination of self-reactive T cells, we examined the involvement of this apoptosis in the induction of autoimmunity in HTLV-I transgenic mice. Splenic T cells derived from the transgenic mice were more resistant to apoptosis induced by anti-Fas mAb than those of the nontransgenic mice, whereas no appreciable difference in apoptosis was detected for thymocytes from either mouse's type. The resistance of transgenic T cells may be due to Tax coded in the pX region, since Tax mediates the inhibition of anti-Fas– induced apoptosis in mature T cell line, Jurkat. Among the transgenic mice, the extent of the resistance to Fas-mediated apoptosis was further enhanced in transgenic T cells with disease. These results suggest that the escape of self-reactive T cells from Fas-mediated apoptosis in the periphery, is critical for the development of autoimmune arthropathy in HTLV-I transgenic mice.
Polymerase Chain Reaction Selects a Novel Disintegrin Proteinase from CD40-Activated Germinal Center Dendritic Cells
Authors: Mueller Chris G.F. , Rissoan Marie-Clotilde , Salinas Barbara , Ait-Yahia Smina , Ravel Odile , Bridon Jean-Michel , Briere Francine , Lebecque Serge , Liu Yong-Jun
Published: July 2002

To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction–based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor α and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.
Selection of Antigen-specific T Cells by a Single IEk Peptide Combination
Authors: Liu Chih-Pin , Parker David , Kappler John , Marrack Philippa
Published: July 2002

In normal mice, major histocompatibility complex (MHC) proteins are bound to many different peptides, derived from the proteins of their host. In the thymus, the diversity of this collection of MHC + peptide ligands allows thymocytes bearing many different T cell receptors (TCRs) to mature by low avidity reactions between the MHC + peptide ligands and the thymocyte TCRs. To investigate this problem, the selection of T cells specific for a well-studied combination of MHC + peptide, IEk + moth cytochrome c 88–103 (MCC), was investigated. Mice were created that expressed IEk bound to a single peptide, either a variant of MCC in which a critical TCR contact residue, 99K, was changed to A, or a variant of a mouse hemoglobin 64–76 (Hb) peptide, 72A. IEk bound to the MCC variant caused the clonal deletion of some T cells specific for the IEk + MCC ligand; nevertheless, it also positively selected many T cells that could react with this ligand. Some of the TCRs on the selected T cells were related to those on cells from normal mice and some were not. IEk bound to the Hb variant, on the other hand, did not select any T cells which could react with IEk + MCC. These results demonstrate that although positive selection is a partially degenerate event, the sequence of the peptide involved in positive selection controls the selected repertoire.
The HIV-1 vpr Protein Acts as a Negative Regulator of Apoptosis in a Human Lymphoblastoid T Cell Line: Possible Implications for the Pathogenesis of AIDS
Authors: Conti L. , Rainaldi G. , Matarrese P. , Varano B. , Rivabene R. , Columba S. , Sato A. , Belardelli F. , Malorni W. , Gessani S.
Published: July 2002

Although apoptosis is considered one of the major mechanisms of CD4+ T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor α (TNF-α), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-α as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.
Hapten-induced Colitis Is Associated with Colonic Patch Hypertrophy and T Helper Cell 2–Type Responses
Authors: Dohi Taeko , Fujihashi Kohtaro , Rennert Paul D. , Iwatani Koichi , Kiyono Hiroshi , McGhee Jerry R.
Published: July 2002

To investigate the potential involvement of T helper (Th)2-type responses in murine models of intestinal inflammation, we used trinitrobenzene sulfonic acid (TNBS)–hapten to induce inflammatory bowel disease in situations where Th1-type responses with interferon (IFN)-γ synthesis are either diminished or do not occur. Intracolonic administration of TNBS to either normal (IFN-γ+/+) or Th1-deficient IFN-γ knockout (IFN-γ−/−) BALB/c mice resulted in significant colitis. In IFN-γ−/− mice, crypt inflammation was more severe than in IFN-γ+/+ mice and was accompanied by hypertrophy of colonic patches with a lymphoepithelium containing M cells and distinct B and T cell zones resembling Peyer's patches. Hapten-specific, colonic patch T cells from both mouse groups exhibited a Th2 phenotype with interleukin (IL)-4 and IL-5 production. TNBS colitis in normal mice treated with anti–IL-4 antibodies or in IL-4−/− mice was less severe than in either IFN-γ+/+ or IFN-γ−/− mice. Our findings now show that the Th2-type responses in TNBS colitis are associated with colonic patch enlargement and inflammation of the mucosal layer and may represent a model for ulcerative colitis.
High Pathogenic Potential of Low-Affinity Autoantibodies in Experimental Autoimmune Hemolytic Anemia
Authors: Fossati-Jimack Liliane , Reininger Luc , Chicheportiche Yves , Clynes Raphael , Ravetch Jeffrey V. , Honjo Tasuku , Izui Shozo
Published: July 2002

To assess the potency of low-affinity anti–red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti–mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a–injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.
Inhibition of T Helper Cell Type 2 Cell Differentiation and Immunoglobulin E Response by Ligand-Activated Vα14 Natural Killer T Cells
Authors: Cui Junqing , Watanabe Naohiro , Kawano Tetsu , Yamashita Masakatsu , Kamata Tohru , Shimizu Chiori , Kimura Motoko , Shimizu Eiko , Koike Jyunzo , Koseki Haruhiko , Tanaka Yujiro , Taniguchi Masaru , Nakayama Toshinori
Published: July 2002

Murine Vα14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Vα14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-γ upon in vivo stimulation with a specific ligand, α-galactosylceramide (α-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Vα14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Vα14 NKT-deficient mice that Vα14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with α-GalCer, Vα14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Vα14 NKT– or IFN-γ–deficient mice. Consistent with these results, we also found that ligand-activated Vα14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Vα14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-γ. In marked contrast, our studies have revealed that IL-4 produced by Vα14 NKT cells has only a minor effect on Th2 cell differentiation.
A20 Inhibits Cytokine-Induced Apoptosis and Nuclear Factor κB–Dependent Gene Activation in Islets
Authors: Grey Shane T. , Arvelo Maria B. , Hasenkamp Wendy , Bach Fritz H. , Ferran Christiane
Published: July 2002

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease resulting from apoptotic destruction of β cells in the islets of Langerhans. Low expression of antioxidants and a predilection to produce nitric oxide (NO) have been shown to underscore β cell apoptosis. With this perspective in mind, we questioned whether β cells could mount an induced protective response to inflammation. Here we show that human and rat islets can be induced to rapidly express the antiapoptotic gene A20 after interleukin (IL)-1β activation. Overexpression of A20 by means of adenovirus-mediated gene transfer protects islets from IL-1β and interferon γ–induced apoptosis. The cytoprotective effect of A20 against apoptosis correlates with and is dependent on the abrogation of cytokine-induced NO production. The inhibitory effect of A20 on cytokine-stimulated NO production is due to transcriptional blockade of inducible NO synthase (iNOS) induction; A20 inhibits the activation of the transcription factor nuclear factor κB at a level upstream of IκBα degradation. These data demonstrate a dual antiapoptotic and antiinflammatory function for A20 in β cells. This qualifies A20 as part of the physiological cytoprotective response of islets. We propose that A20 may have therapeutic potential as a gene therapy candidate to achieve successful islet transplantation and the cure of IDDM.
Deamidation of Asparagine in a Major Histocompatibility Complex–Bound Peptide Affects T Cell Recognition but Does Not Explain Type B Reactivity
Authors: Cirrito Thomas P. , Pu Zheng , Deck M. Brian , Unanue Emil R.
Published: July 2002

We have analyzed a panel of T cell hybridomas specific for the chemically dominant epitope of hen egg-white lysozyme 48–61 which has asparagine 59 as an important T cell receptor contact residue. A number of T cells recognize 48–61 with asparagine at position 59, but not the aspartic acid or isoaspartic acid derivatives. Conversely, we find T cells that specifically recognize 48–61 bearing an isoaspartic acid at residue 59, but not asparagine. For other T cells, asparagine, aspartic acid, or isoaspartic acid at residue 59 is irrelevant. We present evidence that our previous distinction between type A and type B T cells is not explained by asparagine deamidation at residue 59.
The B Lymphocyte Adaptor Molecule of 32 kD (Bam32) Regulates B Cell Antigen Receptor Signaling and Cell Survival
Authors: Niiro Hiroaki , Maeda Akito , Kurosaki Tomohiro , Clark Edward A.
Published: July 2002

The B lymphocyte–associated adaptor protein 32 kD in size (Bam32) is expressed at high levels in germinal center (GC) B cells. It has an NH2-terminal src homology 2 (SH2) domain which binds phospholipase C (PLC)γ2, and a COOH-terminal pleckstrin homology (PH) domain. Thus, Bam32 may function to integrate protein tyrosine kinase (PTK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways in B cells. To further define the role Bam32 plays in B cells, we generated Bam32-deficient DT40 cells. These Bam32−/− cells exhibited lower levels of B cell antigen receptor (BCR)-induced calcium mobilization with modest decreases in tyrosine phosphorylation of phospholipase C (PLC)γ2. Moreover, BCR-induced activation of extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) pathways was impaired in Bam32−/− cells but not the activation of Akt-related pathways. Activation of downstream transcription factors such as nuclear factor of activated T cells (NF-AT) and nuclear factor of κ binding (NF-κB) was also impaired in Bam32−/− cells. Furthermore, Bam32−/− cells were more susceptible to BCR-induced death. Taken together, these findings suggest that Bam32 functions to regulate BCR-induced signaling and cell survival most likely in germinal centers.
Maximal HIV-1 Replication in Alveolar Macrophages during Tuberculosis Requires both Lymphocyte Contact and Cytokines
Authors: Hoshino Yoshihiko , Nakata Koh , Hoshino Satomi , Honda Yoshihiro , Tse Doris B. , Shioda Tatsuo , Rom William N. , Weiden Michael
Published: July 2002

HIV-1 replication is markedly upregulated in alveolar macrophages (AM) during pulmonary tuberculosis (TB). This is associated with loss of an inhibitory CCAAT enhancer binding protein β (C/EBPβ) transcription factor and activation of nuclear factor (NF)-κB. Since the cellular immune response in pulmonary TB requires lymphocyte–macrophage interaction, a model system was developed in which lymphocytes were added to AM. Contact between lymphocytes and AM reduced inhibitory C/EBPβ, activated NF-κB, and enhanced HIV-1 replication. If contact between lymphocytes and macrophages was prevented, inhibitory C/EBPβ expression was maintained and the HIV-1 long terminal repeat (LTR) was not maximally stimulated although NF-κB was activated. Antibodies that cross-linked macrophage expressed B-7, and vascular cell adhesion molecule and CD40 were used to mimic lymphocyte contact. All three cross-linking antibodies were required to abolish inhibitory C/EBPβ expression. However, the HIV-1 LTR was not maximally stimulated and NF-κB was not activated. Maximal HIV-1–LTR stimulation required both lymphocyte-derived soluble factors, and cross-linking of macrophage expressed costimulatory molecules. High level HIV-1–LTR stimulation was also achieved when IL-1β, IL-6, and TNF-β were added to macrophages with cross-linked costimulatory molecules. Contact between activated lymphocytes and macrophages is necessary to down-regulate inhibitory C/EBPβ, thereby derepressing the HIV-1 LTR. Lymphocyte-derived cytokines activate NF-κB, further enhancing the HIV-1 LTR.
Probing the Structure of the Diphtheria Toxin Channel
Authors: Huynh Paul D. , Cui Can , Zhan Hangjun , Oh Kyoung Joon , Collier R. John , Finkelstein Alan
Published: July 2002

Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328–376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α-helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.
Topography of Diphtheria Toxin's T Domain in the Open Channel State
Authors: Senzel Lisa , Gordon Michael , Blaustein Robert O. , Oh K. Joon , Collier R. John , Finkelstein Alan
Published: July 2002

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a “double dagger” model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed “daggers,” TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ∼70 residues, is translocated across the membrane to the trans side.
Functional Properties of Human Nicotinic Achrs Expressed by Imr-32 Neuroblastoma Cells Resemble Those of α3β4 Achrs Expressed in Permanently Transfected Hek Cells
Authors: Nelson Mark E. , Wang Fan , Kuryatov Alexander , Choi Catherine H. , Gerzanich Volodymyr , Lindstrom Jon
Published: July 2002

We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human α3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express α3, α5, α7, β2, and β4 AChR subunits. From these subunits, several types of α3 AChRs as well as homomeric α7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect α3β4 AChRs. α7 AChR function was not detected, yet we estimate that there are 70% as many surface α7 AChRs in IMR-32 when compared with α3 AChRs. Agonist potencies (EC50 values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16±1 μM) > nicotine (Nic; 48 ± 7 μM) ≥ cytisine (Cyt; 57 ± 3 μM) = acetylcholine (ACh; 59 ± 6 μM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s−1 (300 μM ACh; −60 mV). Assays that used mAbs confirmed the predominance of α3- and β4-containing AChRs in IMR-32 cells. Although 18% of total α3 AChRs contained β2 subunits, no β2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface α3β2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in α3β2 transfected HEK cells. Characterization of various α3 AChRs expressed in HEK cell lines revealed that the functional properties of the α3β4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC50 values) for this line was DMPP (14 ± 1 μM) = Cyt (18 ± 1 μM) > Nic (56 ± 15 μM > ACh (79 ± 8 μM). The efficacies of both Cyt and DMPP were ∼80% when compared with ACh and the desensitization rate was 2 s−1. These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to α3β4 AChRs.