bluuebunny/crossref_metadata_2025 huggingface dataset:
Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium
Authors: O'Malley James P. , Moore Charlotte T. , Salpeter Miriam M.
Published: July 2002

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ∼1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ∼3–5 d) and a stabilized form (half-life ∼10 d), depending upon the state of innervation of the muscle.

Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca2+, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.
Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin
Authors: Tashiro Satoshi , Walter Joachim , Shinohara Akira , Kamada Nanao , Cremer Thomas
Published: July 2002

Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase (Tashiro, S., N. Kotomura, A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene. 12:2165–2170). However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.
Hsp70 and Antifibrillogenic Peptides Promote Degradation and Inhibit Intracellular Aggregation of Amyloidogenic Light Chains
Authors: Dul Jeanne L. , Davis David P. , Williamson Edward K. , Stevens Fred J. , Argon Yair
Published: July 2002

In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its β sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.
The Efficiency of CD4 Recruitment to Ligand-engaged TCR Controls the Agonist/Partial Agonist Properties of Peptide–MHC Molecule Ligands
Authors: Madrenas Joaquín , Chau Luan A. , Smith Judy , Bluestone Jeffrey A. , Germain Ronald N.
Published: July 2002

One hypothesis seeking to explain the signaling and biological properties of T cell receptor for antigen (TCR) partial agonists and antagonists is the coreceptor density/kinetic model, which proposes that the pharmacologic behavior of a TCR ligand is largely determined by the relative rates of (a) dissociation of ligand from an engaged TCR and (b) recruitment of lck-linked coreceptors to this ligand-engaged receptor. Using several approaches to prevent or reduce the association of CD4 with occupied TCR, we demonstrate that consistent with this hypothesis, the biological and biochemical consequence of limiting this interaction is to convert typical agonists into partial agonist stimuli. Thus, adding anti-CD4 antibody to T cells recognizing a wild-type peptide–MHC class II ligand leads to disproportionate inhibition of interleukin-2 (IL-2) relative to IL-3 production, the same pattern seen using a TCR partial agonist/antagonist. In addition, T cells exposed to wild-type ligand in the presence of anti-CD4 antibodies show a pattern of TCR signaling resembling that seen using partial agonists, with predominant accumulation of the p21 tyrosine-phosphorylated form of TCR-ζ, reduced tyrosine phosphorylation of CD3ε, and no detectable phosphorylation of ZAP-70. Similar results are obtained when the wild-type ligand is presented by mutant class II MHC molecules unable to bind CD4. Likewise, antibody coligation of CD3 and CD4 results in an agonist-like phosphorylation pattern, whereas bivalent engagement of CD3 alone gives a partial agonist-like pattern. Finally, in accord with data showing that partial agonists often induce T cell anergy, CD4 blockade during antigen exposure renders cloned T cells unable to produce IL-2 upon restimulation. These results demonstrate that the biochemical and functional responses to variant TCR ligands with partial agonist properties can be largely reproduced by inhibiting recruitment of CD4 to a TCR binding a wild-type ligand, consistent with the idea that the relative rates of TCR–ligand disengagement and of association of engaged TCR with CD4 may play a key role in determining the pharmacologic properties of peptide–MHC molecule ligands. Beyond this insight into signaling through the TCR, these results have implications for models of thymocyte selection and the use of anti-coreceptor antibodies in vivo for the establishment of immunological tolerance.
The Transcriptional Promoter Regulates Hypermutation of the Antibody Heavy Chain Locus
Authors: Tumas-Brundage Kathleen , Manser Tim
Published: July 2002

A somatic process introduces mutations into antibody variable (V) region genes at a high rate in many vertebrates, and is a major source of antibody diversity. The mechanism of this hypermutation process remains enigmatic, although retrospective studies and transgenic experiments have recently suggested a role for transcriptional regulatory elements. Here, we demonstrate that mouse heavy (H) chain loci in which the natural VH promoter has been replaced by a heterologous promoter undergo hypermutation. However, while the distribution of mutation in such loci appears normal, the frequency of mutation does not. Conversely, moving the VH promoter 750 bp upstream of its normal location results in a commensurate change in the site specificity of hypermutation in H chain loci, and the foreign DNA inserted into the VH leader intron to produce this promoter displacement is hypermutated in a manner indistinguishable from natural Ig DNA. These data establish a direct mechanistic link between the IgH transcription and hypermutation processes.
Commitment of Immature CD4+8+ Thymocytes to the CD4 Lineage Requires CD3 Signaling but Does Not Require Expression of Clonotypic T Cell Receptor (TCR) Chains
Authors: Suzuki Harumi , Shinkai Yoichi , Granger Lawrence G. , Alt Frederick W. , Love Paul E. , Singer Alfred
Published: July 2002

As a consequence of positive selection in the thymus, immature CD4+8+ double-positive, [DP] thymocytes selectively terminate synthesis of one coreceptor molecule and, as a result, differentiate into either CD4+ or CD8+ T cells. The decision by individual DP thymocytes to terminate synthesis of one or the other coreceptor molecule is referred to as lineage commitment. Previously, we reported that the intrathymic signals that induced commitment to the CD4 versus CD8 T cell lineages were markedly asymmetric. Notably, CD8 commitment appeared to require lineage-specific signals, whereas CD4 commitment appeared to occur in the absence of lineage-specific signals by default. Consequently, it was unclear whether CD4 commitment, as revealed by selective termination of CD8 coreceptor synthesis, occurred in all DP thymocytes, or whether CD4 commitment occurred only in T cell receptor (TCR)–CD3-signaled DP thymocytes. Here, we report that selective termination of CD8 coreceptor synthesis does not occur in DP thymocytes spontaneously. Rather, CD4 commitment in DP thymocytes requires signals transduced by either CD3 or ζ chains, which can signal CD4 commitment even in the absence of clonotypic TCR chains.
Dissociation of Intracellular Signaling Pathways in Response to Partial Agonist Ligands of the T Cell Receptor
Authors: Chau Luan A. , Bluestone Jeffrey A. , Madrenas Joaquín
Published: July 2002

The T cell receptor (TCR) is a versatile receptor able to generate different signals that result in distinct T cell responses. The pattern of early signals is determined by the TCR binding kinetics that control the ability of the ligand to coengage TCR and coreceptor. Coengagement of TCR and CD4 results in an agonist signaling pattern with complete tyrosine phosphorylation of TCR subunits, and recruitment and activation of ZAP-70. In contrast, TCR engagement without CD4 coengagement causes a partial agonist type of signaling, characterized by distinct phosphorylation of TCR subunits and recruitment but no activation of ZAP-70. The pathways triggered by partial agonist signaling are unknown. Here, we show that agonists cause association of active lck and active ZAP-70 with p120-GTPase–activating protein (p120-GAP). These associations follow engagement of CD4 or CD3, respectively. In contrast, partial agonists do not activate lck or ZAP-70, but induce association of p120-GAP with inactive ZAP-70. Despite these differences, both agonist and partial agonist signals activate the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation by partial agonists is transient, supporting a kinetic, CD4-dependent model for the mechanism of action of variant TCR ligands. Transient MAPK activation may explain some of the responses to TCR partial agonists and antagonists.
Murine Cytomegalovirus Inhibits Interferon γ–induced Antigen Presentation to CD4 T Cells by Macrophages Via Regulation of Expression of Major Histocompatibility Complex Class II–associated Genes
Authors: Heise Mark T. , Connick Megan , Virgin Herbert W.
Published: July 2002

CD4 T cells and interferon γ (IFN-γ) are required for clearance of murine cytomegalovirus (MCMV) infection from the salivary gland in a process taking weeks to months. To explain the inefficiency of salivary gland clearance we hypothesized that MCMV interferes with IFN-γ induced antigen presentation to CD4 T cells. MCMV infection inhibited IFN-γ–induced presentation of major histocompatibility complex (MHC) class II associated peptide antigen by differentiated bone marrow macrophages (BMMφs) to a T cell hybridoma via impairment of MHC class II cell surface expression. This effect was independent of IFN-α/β induction by MCMV infection, and required direct infection of the BMMφs with live virus. Inhibition of MHC class II cell surface expression was associated with a six- to eightfold reduction in IFN-γ induced IAb mRNA levels, and comparable decreases in IFN-γ induced expression of invariant chain (Ii), H-2Ma, and H-2Mb mRNAs. Steady state levels of several constitutive host mRNAs, including β-actin, cyclophilin, and CD45 were not significantly decreased by MCMV infection, ruling out a general effect of MCMV infection on mRNA levels. MCMV effects were specific to certain MHC genes since IFN-γ–induced transporter associated with antigen presentation (TAP)2 mRNA levels were minimally altered in infected cells. Analysis of early upstream events in the IFN-γ signaling pathway revealed that MCMV did not affect activation and nuclear translocation of STAT1α, and had minor effects on the early induction of IRF-1 mRNA and protein. We conclude that MCMV infection interferes with IFN-γ–mediated induction of specific MHC genes and the Ii at a stage subsequent to STAT1α activation and nuclear translocation. This impairs antigen presentation to CD4 T cells, and may contribute to the capacity of MCMV to spread and persist within the infected host.
A Hypermutable Insert in an Immunoglobulin Transgene Contains Hotspots of Somatic Mutation and Sequences Predicting Highly Stable Structures in the RNA Transcript
Authors: Storb Ursula , Klotz Emily L. , Hackett John , Kage Karen , Bozek Grazyna , Martin Terence E.
Published: July 2002

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57–65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.
Interaction of Dendritic Cells with Skin Endothelium: A New Perspective on Immunosurveillance
Authors: Robert Caroline , Fuhlbrigge Robert C. , Kieffer J. David , Ayehunie Seyoum , Hynes Richard O. , Cheng Guiying , Grabbe Stephan , von Andrian Ulrich H. , Kupper Thomas S.
Published: July 2002

The goal of this study was to determine the mechanisms by which dendritic cells (DCs) in blood could interact with endothelium, a prerequisite to extravasation into tissues. Our results indicate that DCs express both HECA-452–reactive and nonreactive isoforms of P-selectin glycoprotein ligand 1 (PSGL-1) and can tether and roll efficiently on E- and P-selectin under flow conditions in vitro. Freshly isolated blood DCs were further observed to roll continuously along noninflamed murine dermal endothelium in vivo. This interaction is strictly dependent on endothelial selectins, as shown by experiments with blocking antibodies and with E- and P-selectin–deficient mice. We hypothesize that DCs in blood are constitutively poised at the interface of blood and skin, ready to extravasate upon induction of inflammation, and we showed that cutaneous inflammation results in a rapid recruitment of DCs from the blood to tissues. We propose that this is an important and previously unappreciated element of immunosurveillance.
Several Different Cell Surface Molecules Control Negative Selection of Medullary Thymocytes
Authors: Kishimoto Hidehiro , Sprent Jonathan
Published: July 2002

Repeated attempts to show that costimulation for negative selection is controlled by a single cell surface molecule have been unsuccessful. Thus, negative selection may involve multiple cell surface molecules acting in consort. In support of this idea, we show here that at least three cell surface molecules, namely CD28, CD5, and CD43, contribute to Fas-independent negative selection of the tolerance-susceptible population of heat-stable antigen (HSA)hiCD4+8− cells found in the medulla. The costimulatory function of these three molecules can be blocked by certain cytokines, IL-4 and IL-7, and coinjecting these cytokines with antigen in vivo abolishes negative selection; Fas-dependent negative selection, however, is maintained. The results suggest that efficient negative selection requires the combined functions of at least four cell surface molecules: CD28, CD5, CD43, and Fas.
Inhibitory Receptors Alter Natural Killer Cell Interactions with Target Cells Yet Allow Simultaneous Killing of Susceptible Targets
Authors: Eriksson Mikael , Leitz Guenther , Fällman Erik , Axner Ove , Ryan James C. , Nakamura Mary C. , Sentman Charles L.
Published: July 2002

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector–target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49–MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.
Impaired Nk1.1 T Cell Development in Mice Transgenic for a T Cell Receptor β Chain Lacking the Large, Solvent-Exposed Cβ Fg Loop
Authors: Degermann Sylvie , Sollami Giuseppina , Karjalainen Klaus
Published: July 2002

A striking feature of the T cell receptor (TCR) β chain structure is the large FG loop that protrudes freely into the solvent on the external face of the Cβ domain. We have already shown that a transgene-encoded Vβ8.2+ TCR β chain lacking the complete Cβ FG loop supports normal development and function of conventional α/β T cells. Thus, the FG loop is not absolutely necessary for TCR signaling. However, further analysis has revealed that a small population of α/β T cells coexpressing NK1.1 are severely depleted in these transgenic mice. The few remaining NK1.1 T cells have a normal phenotype but express very low levels of TCR. We find that the TCR Vβ8.2+ chain lacking the Cβ FG loop cannot pair efficiently with the invariant Vα14-Jα281 TCR α chain commonly expressed by this T cell family. Consequently, fewer NK1.1 T cells develop in these mice. Our results suggest that expression of the Vα14+ TCR α chain is particularly sensitive to TCR-β conformation. Development of NK1.1 T cells appears to need a TCR-β conformation dependent on the presence of the Cβ loop that is not necessarily required for assembly and function of TCRs on most α/β T cells.
Lck Domains Differentially Contribute to Pre–T Cell Receptor (Tcr)–And TCR-α/β–Regulated Developmental Transitions
Authors: Lin Kui , Longo Nancy S. , Wang Xin , Hewitt Judy A. , Abraham Kristin M.
Published: July 2002

Maturational changes at the CD4−CD8− double negative (DN) to CD4+CD8+ double positive (DP) transition are dependent on signals generated via the pre–T cell receptor (TCR) and the nonreceptor protein tyrosine kinase p56lck (Lck). How Lck activities are stimulated or relayed after pre-TCR formation remains obscure. Our structure–function mapping of Lck thymopoietic properties reveals that the noncatalytic domains of Lck are specialized to signal efficient cellular expansion at DN to DP transition. Moreover, although substitution of the Lck catalytic domain with FynT sequences minimally impacts DP development, single positive thymocytes are most efficiently produced in the presence of kinases containing both the NH2-terminal and catalytic regions of Lck. These findings demonstrate that the Lck structure is uniquely adapted to mediate signals at both major transitions in thymopoiesis.
Evidence for Class-Specific Factors in Immunoglobulin Isotype Switching
Authors: Shanmugam Ananth , Shi Meng-Jiao , Yauch Lauren , Stavnezer Janet , Kenter Amy L.
Published: July 2002

Immunoglobulin class switch recombination (SR) occurs by a B cell–specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate μ→α and μ→γ3 SR.
T Cell Receptor (Tcr)-Mediated Repertoire Selection and Loss of Tcr Vβ Diversity during the Initiation of a Cd4+ T Cell Response in Vivo
Authors: Fassò Marcella , Anandasabapathy Niroshana , Crawford Frances , Kappler John , Fathman C. Garrison , Ridgway William M.
Published: July 2002

We recently described a novel way to isolate populations of antigen-reactive CD4+ T cells with a wide range of reactivity to a specific antigen, using immunization with a fixed dose of nominal antigen and FACS® sorting by CD4high expression. Phenotypic, FACS®, functional, antibody inhibition, and major histocompatibility complex–peptide tetramer analyses, as well as T cell receptor Vβ sequence analyses, of the antigen-specific CD4high T cell populations demonstrated that a diverse sperm whale myoglobin 110–121–reactive CD4+ T cell repertoire was activated at the beginning (day 3 after immunization) of the immune response. Within 6 d of immunization, lower affinity clones were lost from the responding population, leaving an expanded population of oligoclonal, intermediate affinity (and residual high affinity) T cells. This T cell subset persisted for at least 4 wk after immunization and dominated the secondary immune response. These data provide evidence that CD4+ T cell repertoire selection occurs early in the immune response in vivo and suggest that persistence and expansion of a population of oligoclonal, intermediate affinity T cells is involved in CD4+ T cell memory.
Chromatin Remodeling by the T Cell Receptor (Tcr)-β Gene Enhancer during Early T Cell Development
Authors: Mathieu Noëlle , Hempel William M. , Spicuglia Salvatore , Verthuy Christophe , Ferrier Pierre
Published: July 2002

Gene targeting studies have shown that T cell receptor (TCR)-β gene expression and recombination are inhibited after deletion of an enhancer (Eβ) located at the 3′ end of the ∼500-kb TCR-β locus. Using knockout mouse models, we have measured, at different regions throughout the TCR-β locus, the effects of Eβ deletion on molecular parameters believed to reflect epigenetic changes associated with the control of gene activation, including restriction endonuclease access to chromosomal DNA, germline transcription, DNA methylation, and histone H3 acetylation. Our results demonstrate that, in early developing thymocytes, Eβ contributes to major chromatin remodeling directed to an ∼25-kb upstream domain comprised of the Dβ-Jβ locus regions. Accordingly, treatment of Eβ-deleted thymocytes with the histone deacetylase inhibitor trichostatin A relieved the block in TCR-β gene expression and promoted recombination within the Dβ-Jβ loci. Unexpectedly, however, epigenetic processes at distal Vβ genes on the 5′ side of the locus and at the 3′ proximal Vβ14 gene appear to be less dependent on Eβ, suggesting that Eβ activity is confined to a discrete region of the TCR-β locus. These findings have implications with respect to the developmental control of TCR-β gene recombination, and the process of allelic exclusion at this locus.
Dm Determines the Cryptic and Immunodominant Fate of T Cell Epitopes
Authors: Nanda Navreet K. , Sant Andrea J.
Published: July 2002

The ability of the immune system to focus T cell responses against a select number of potential epitopes of a complex antigen is termed immunodominance. Epitopes that trigger potent T cell activation, after in vivo priming, are classified as immunodominant. By contrast, determinants that fail to elicit any response are called cryptic. DM, a major histocompatibility complex (MHC) heterodimer, plays a pivotal role in the presentation of MHC class II–restricted epitopes by catalyzing the exchange of class II–associated invariant chain peptide with the antigen-derived peptides within the MHC class II binding groove. Using L cells transfected with genes for MHC class II, invariant chain, and DM, we have studied the contribution of DM in the presentation of two cryptic (peptide 11–25 and peptide 20–35) and one dominant (peptide 106–116) epitope of hen egg white lysozyme (HEL). Cells lacking DM heterodimers efficiently display the determinants HEL 11–25 and HEL 20–35 to T cells. Strikingly, however, cells expressing DM are severely compromised in their ability to present the cryptic HEL 11–25/Ad and 20–35/Ad epitopes. DM-mediated antagonism of HEL 11–25/Ad and 20–35/Ad presentation could thus be central to 11–25/Ad and 20–35/Ad being cryptic epitopes in the HEL system. Interestingly, the display of the immunodominant epitope of HEL, 106–116/Ed, and of a dominant epitope of sperm whale myoglobin (SWM), 102–118/Ad, is entirely dependent on the expression of DM. Thus, cells lacking DM molecules are unable to efficiently express HEL 106–116/Ed and SWM 102–118/Ad determinants. We conclude that the DM heterodimers direct the immunodominant and cryptic fate of antigenic epitopes in vivo.
Histone Acetylation Determines the Developmentally Regulated Accessibility for T Cell Receptor γ Gene Recombination
Authors: Agata Yasutoshi , Katakai Tomoya , Ye Sang-Kyu , Sugai Manabu , Gonda Hiroyuki , Honjo Tasuku , Ikuta Koichi , Shimizu Akira
Published: July 2002

Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-γ gene, wherein predominant rearrangement is switched from Vγ3 to Vγ2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vγ3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow–derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vγ3 gene. These data demonstrate that histone acetyl- ation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.
Requirement for Transforming Growth Factor β1 in Controlling T Cell Apoptosis
Authors: Chen WanJun , Jin Wenwen , Tian Hongsheng , Sicurello Paula , Frank Mark , Orenstein Jan M. , Wahl Sharon M.
Published: July 2002

Transforming growth factor (TGF)-β1, a potent immunoregulatory molecule, was found to control the life and death decisions of T lymphocytes. Both thymic and peripheral T cell apoptosis was increased in mice lacking TGF-β1 (TGF-β1−/−) compared with wild-type littermates. Engagement of the T cell receptor enhanced this aberrant T cell apoptosis, as did signaling through either the death receptor Fas or the tumor necrosis factor α receptor in peripheral T cells. Strikingly, TGF-β was localized within the mitochondria of normal T cells, and the absence of TGF-β1 resulted in disruption of mitochondrial membrane potential (Δψm), which marks the point of no return in a cell condemned to die. This TGF-β–dependent regulation of viability appears dissociable from the TGF-β1 membrane receptor–Smad3 signaling pathway, but associated with a mitochondrial antiapoptotic protein Bcl–XL. Thus, TGF-β1 may protect T cells at multiple sites in the death pathway, particularly by maintaining the essential integrity of mitochondria. These findings may have broad implications not only for T cell selection and death in immune responses and in the generation of tolerance, but also for defining the mechanisms of programmed cell death in general.
Dendritic Cells Pulsed with Intact Streptococcus pneumoniae Elicit both Protein- and Polysaccharide-specific Immunoglobulin Isotype Responses In Vivo through Distinct Mechanisms
Authors: Colino Jesus , Shen Yi , Snapper Clifford M.
Published: July 2002

Immature bone marrow–derived myeloid dendritic cells (BMDCs) are induced to undergo phenotypic maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-10 when pulsed in vitro with intact Streptococcus pneumoniae. After transfer to naive mice, pulsed BMDCs induce immunoglobulin (Ig) isotype responses specific for both protein and polysaccharide pneumococcal antigens, having in common the requirement for viable BMDCs, T cells, and B7-dependent costimulation in the recipient mice. Whereas primary Ig isotype responses to bacterial proteins uniformly require BMDC expression of major histocompatibility complex class II, CD40, and B7, and the secretion of IL-6, but not IL-12, similar requirements for antipolysaccharide Ig responses were only observed for the IgG1 isotype.
Gat1 (Gaba:Na+:Cl−) Cotransport Function
Authors: Lu Chin-Chih , Hilgemann Donald W.
Published: July 2002

Neurotransmitter transporters are reported to mediate transmembrane ion movements that are poorly coupled to neurotransmitter transport and to exhibit complex “channel-like” behaviors that challenge the classical “alternating access” transport model. To test alternative models, and to develop an improved model for the Na+- and Cl−-dependent γ-aminobutyric acid (GABA) transporter, GAT1, we expressed GAT1 in Xenopus oocytes and analyzed its function in detail in giant membrane patches. We detected no Na+- or Cl−- dependent currents in the absence of GABA, nor did we detect activating effects of substrates added to the trans side. Outward GAT1 current (“reverse” transport mode) requires the presence of all three substrates on the cytoplasmic side. Inward GAT1 current (“forward” transport mode) can be partially activated by GABA and Na+ on the extracellular (pipette) side in the nominal absence of Cl−. With all three substrates on both membrane sides, reversal potentials defined with specific GAT1 inhibitors are consistent with the proposed stoichiometry of 1GABA:2Na+:1Cl−. As predicted for the “alternating access” model, addition of a substrate to the trans side (120 mM extracellular Na+) decreases the half-maximal concentration for activation of current by a substrate on the cis side (cytoplasmic GABA). In the presence of extracellular Na+, the half-maximal cytoplasmic GABA concentration is increased by decreasing cytoplasmic Cl−. In the absence of extracellular Na+, half-maximal cytoplasmic substrate concentrations (8 mM Cl−, 2 mM GABA, 60 mM Na+) do not change when cosubstrate concentrations are reduced, with the exception that reducing cytoplasmic Cl− increases the half-maximal cytoplasmic Na+ concentration. The forward GAT1 current (i.e., inward current with all extracellular substrates present) is inhibited monotonically by cytoplasmic Cl− (Ki, 8 mM); cytoplasmic Na+ and cytoplasmic GABA are without effect in the absence of cytoplasmic Cl−. In the absence of extracellular Na+, current–voltage relations for reverse transport current (i.e., outward current with all cytoplasmic substrates present) can be approximated by shallow exponential functions whose slopes are consistent with rate-limiting steps moving 0.15–0.3 equivalent charges. The slopes of current–voltage relations change only little when current is reduced four- to eightfold by lowering each cosubstrate concentration; they increase twofold upon addition of 100 mM Na+ to the extracellular (pipette) side.
Tensor products of Minimal Holomorphic Representations
Authors: Zhang Genkai
Published: July 2002

LetD=G/KD=G/Kbe an irreducible bounded symmetric domain with genusppandHν(D)H^{\nu }(D)the weighted Bergman spaces of holomorphic functions forν>p−1\nu >p-1. The spacesHν(D)H^\nu (D)form unitary (projective) representations of the groupGGand have analytic continuation inν\nu; they give also unitary representations whenν\nuin the Wallach set, which consists of a continuous part and a discrete part ofrrpoints. The first non-trivial discrete pointν=a2\nu =\frac a2gives the minimal highest weight representation ofGG. We give the irreducible decomposition of tensor productHa2⊗Ha2¯H^{\frac a2}\otimes \overline {H^{\frac a2}}. As a consequence we discover some new spherical unitary representations ofGGand find the expansion of the corresponding spherical functions in terms of theKK-invariant (Jack symmetric) polynomials, the coefficients being continuous dual Hahn polynomials.
The Eukaryotic Two-Component Histidine Kinase Sln1p RegulatesOCH1via the Transcription Factor, Skn7p
Authors: Li Sheng , Dean Susan , Li Zhijian , Horecka Joe , Deschenes Robert J. , Fassler Jan S.
Published: July 2002

The yeast “two-component” osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with acis-acting element identified upstream ofOCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.
Golgi Vesicle Proteins Are Linked to the Assembly of an Actin Complex Defined by mAbp1
Authors: Fucini Raymond V. , Chen Ji-Long , Sharma Catherine , Kessels Michael M. , Stamnes Mark
Published: July 2002

Recent studies indicate that regulation of the actin cytoskeleton is important for protein trafficking, but its precise role is unclear. We have characterized the ARF1-dependent assembly of actin on the Golgi apparatus. Actin recruitment involves Cdc42/Rac and requires the activation of the Arp2/3 complex. Although the actin-binding proteins mAbp1 (SH3p7) and drebrin share sequence homology, they are differentially segregated into two distinct ARF-dependent actin complexes. The binding of Cdc42 and mAbp1, which localize to the Golgi apparatus, but not drebrin, is blocked by occupation of the p23 cargo-protein-binding site on coatomer. Exogenously expressed mAbp1 is mislocalized and inhibits Golgi transport in whole cells. The ability of ARF, vesicle-coat proteins, and cargo to direct the assembly of cytoskeletal structures helps explain how only a handful of vesicle types can mediate the numerous trafficking steps in the cell.
A new fourier transform approach for protein coding measure based on the format of the Z curve.
Authors: Yan M , Lin Z S , Zhang C T
Published: July 2002

MOTIVATION: At the core of most protein gene-finding algorithms are the coding measures used to make a decision on coding/non-coding. Of the protein coding measures, the Fourier measure is one of the most important. However, due to the limited length of the windows usually used, the accuracy of the measure is not satisfactory. This paper is devoted to improving the accuracy by lengthening the sequence to amplify the periodicity of 3 in the coding regions. RESULTS: A new algorithm is presented called the lengthen-shuffle Fourier transform algorithm. For the same window length, the percentage accuracy of the new algorithm is 6-7% higher than that of the ordinary Fourier transform algorithm. The resulting percentage accuracy (average of specificity and sensitivity) of the new measure is 84.9% for the window length 162 bp. AVAILABILITY: The program is available on request fromC.-T. Zhang. Contact: ctzhang@tju.edu.cn
Female genital structures in several families of Centropagoidea (Copepoda: Calanoida)
Authors: Barthélémy Roxane-M. , Cuoc Corinne , Defaye Danielle , Brunet Michel , Mazza Jacques
Published: July 2002

The female genital structures of 21 calanoid species belonging to the families Candaciidae, Centropagidae, Pontellidae, Sulcanidae, Temoridae and Tortanidae were studied using light and electron microscopy (scanning electron microscopy, transmission electron microscopy). Except the monotypic Sulcanidae, their organization conforms to a common pattern characterized by egg–laying ducts opening through paired adjacent gonopores into a small cavity, the genital atrium, covered by a genital operculum and opening to the outside through a distal atrial slit. No seminal receptacle is present and the seminal products must be stored for a brief period in the genital atrium.The genital structures occupy a well delimited genital area, the main changes in which concern their position on the ventral face of the genital double–somite, and operculum morphology. Their organization and possible mode of operation are compared with those of other calanoid families. A possible correlation between absence of seminal receptacles and mating behaviour is discussed.
Depth ionization curves for an unmodulated proton beam measured with different ionization chambers
Authors: Mobit Paul N. , Sandison George A. , Bloch Charles
Published: July 2002

Differences in depth dose curves for a 78 MeV unmodulated proton beam were measured with four commercially available ionization chambers. Measurements were performed both in water and in a commercially available solid water phantom. A depth scaling factor was determined from the ratio of depths distal to the Bragg peak where the dose is reduced to 80% of the maximum dose in water and in the solid water phantom. This scaling factor provides good agreement between the ionization curves at all depths in water and in this solid water phantom. There is no significant difference in the value of the depth scaling factor between the ratios and or for 78 MeV unmodulated proton beams. The depth scaling factor for this commercially available solid water phantom is 1.023. An effective point of measurement for a cylindrical ionization chamber was found to be slightly greater than the 50% of the cavity radius proposed by the AAPM‐TG25 dosimetry protocol for electron beams and amounts to 62.5% of the cavity radius of cylindrical ionization chambers. The ion collection efficiency, and the polarity correction factor, for all the ionization chambers studied are within 1% and 0.4% of unity, respectively. Absolute doses measured with a parallel plate ionization chamber in water and in the solid water phantom show that the doses measured in the solid water phantom are 1.4% ± 0.5% lower than in water. The dose rate dependent response of the beam line monitor chamber was also investigated. Agreement between all the chambers was within 1.5% at the dose rates studied but the results showed that all four ionization chambers are less dose rate dependent than the monitor chamber.
A new algorithm for computer tomographic reconstruction from partial view projections
Authors: Brunetti A. , Golosio B.
Published: July 2002

Conventional algorithms for tomographic reconstruction require the acquisition of a complete set of projections at uniform angular displacements. In many cases, however, the geometry of the sample or a loss of data can significantly reduce the range of the available projections. Several algorithms have been proposed in literature to handle such situations, but their performances are low or they require strong constraints and hypothesis about the nature of the sample or the data. Here a new method is proposed. It is based on a novel morphing technique, which affords in general terms the problem of curve matching and is here specialized to the case of tomographic reconstruction. The proposed algorithm is very fast in comparison to other approaches having similar effectiveness; furthermore, it allows one to obtain good quality images even when a significant fraction of the views is absent, without any hypothesis about the nature of the sample or the kind of measurement. The results obtained by applying this technique to the Shepp–Logan phantom and to a clinical scan are reported here and discussed.
Computer‐aided bootstrap generation of characteristic curves for radiographic imaging systems
Authors: Bednarek Daniel R. , Rudin Stephen
Published: July 2002

Determination of the characteristic curve is essential for quantitative evaluation of digital as well as screen‐film radiographic imaging systems. When it is not practical to generate the entire curve through variation of a single exposure parameter, bootstrap methods can be used. For the bootstrap method used here, curve segments are generated by varying one exposure parameter and multiple segments are produced at different exposure levels by varying a second parameter. The segments then are joined to form a single composite characteristic curve. If the second parameter is one for which the sensitivity of the receptor is not constant (such as x‐ray tube potential or beam filtration), the shift of each segment along the log relative‐exposure axis needed to join the overlapping curve sections is not known and some form of segment matching must be employed. A spline interpolation method and a polynomial fit integral method were developed for automatic segment matching and compared. For both methods, the shifts between successive segment pairs which result in optimal overlap are determined and the cumulative shifts to obtain the complete composite curve are calculated for all segments. The two methods are evaluated for several image receptors and with a known curve generated from an analytic function. Both methods closely agreed in the shifts determined for the image receptors and provided a good visual matching of the curve segments. The spline interpolation method more accurately determined the appropriate shifts for the mathematically generated curve segments. The bootstrap methods can provide complete, accurate characteristic curves, and the segment joining program makes the process fast and precise.
High resolution polymer gel dosimetry by parameter selective MR‐microimaging on a whole body scanner at 3 T
Authors: Berg A. , Ertl A. , Moser E.
Published: July 2002

High dose variations across small spatial distances, as present in brachytherapeutic applications or radiosurgery and especially γ‐knife therapy, are difficult to quantify by standard dosimetry. We demonstrate the possibility to obtain planar spatial resolutions for dose imaging at pixel sizes below 200 μm within multislice parameter selective MR imaging on polymer gels. The sensitivity of the transversal and longitudinal relaxation time as well as diffusivity on dose is shown. High spatial resolution is achieved by parameter selective microimaging of polymer gels on a high‐field (3 T) whole‐body MR system equipped with a dedicated strong gradient system and a small probe head matched to the sample size. In addition to the spin–spin relaxation rate we investigate the sensitivity of the longitudinal relaxation rate and the diffusivity in acrylic polymer gels on irradiation up to dose levels of about 20 Gy. Dose images are obtained after calibration of the corresponding MR parameters by known dose levels of γ irradiation. Also the MR‐parameter may be used for dose imaging. The impact of all of the three parameters and diffusivity on obtained signal intensities in irradiated regions has to be taken into account in nonoptimized pulse sequences. Further, very high spatial resolution imposes several restrictions on the evaluation of which have to be considered for quantitative dosimetry. These restrictions are discussed in detail. We also demonstrate the importance of such a high spatial resolution in case of a set of differently sized γ‐knife stereotactic irradiation schemes. Gel dosimetry based on MR parameter selective microimaging represents a potent alternative for the detection of dose distributions characterized by steep dose gradients, typical in brachytherapeutic and radiosurgical applications.
A controlled phantom study of a noise equalization algorithm for detecting microcalcifications in digital mammograms
Authors: Gürün Özgür Ozan , Fatouros Panos P. , Kuhn Gary M. , Shaw de Paredes Ellen
Published: July 2002

We report on some extensions and further developments of a well‐known microcalcification detection algorithm based on adaptive noise equalization. Tissue equivalent phantom images with and without labeled microcalcifications were subjected to this algorithm, and analyses of results revealed some shortcomings in the approach. Particularly, it was observed that the method of estimating the width of distributions in the feature space was based on assumptions which resulted in the loss of similarity preservation characteristics. A modification involving a change of estimator statistic was made, and the modified approach was tested on the same phantom images. Other modifications for improving detectability such as downsampling and use of alternate local contrast filters were also tested. The results indicate that these modifications yield improvements in detectability, while extending the generality of the approach. Extensions to real mammograms and further directions of research are discussed.