bluuebunny/crossref_metadata_2025 huggingface dataset:
Potentiation of Fcε Receptor I–activated Ca2+ Current (ICRAC) by Cholera Toxin: Possible Mediation by ADP Ribosylation Factor
Authors: McCloskey Michael A. , Zhang Lei
Published: July 2002

Antigen-evoked influx of extracellular Ca2+ into mast cells may occur via store-operated Ca2+ channels called calcium release–activated calcium (CRAC) channels. In mast cells of the rat basophilic leukemia cell line (RBL-2H3), cholera toxin (CT) potentiates antigen-driven uptake of 45Ca2+ through cAMP-independent means. Here, we have used perforated patch clamp recording at physiological temperature to test whether cholera toxin or its substrate, Gs, directly modulates the activity of CRAC channels. Cholera toxin dramatically amplified (two- to fourfold) the Ca2+ release–activated Ca2+ current (ICRAC) elicited by suboptimal concentrations of antigen, without itself inducing ICRAC, and this enhancement was not mimicked by cAMP elevation. In contrast, cholera toxin did not affect the induction of ICRAC by thapsigargin, an inhibitor of organelle Ca2+ pumps, or by intracellular dialysis with low Ca2+ pipette solutions. Thus, the activity of CRAC channels is not directly controlled by cholera toxin or Gsα. Nor was the potentiation of ICRAC due to enhancement of phosphoinositide hydrolysis or calcium release. Because Gs and the A subunit of cholera toxin bind to ADP ribosylation factor (ARF) and could modulate its activity, we tested the sensitivity of antigen-evoked ICRAC to brefeldin A, an inhibitor of ARF-dependent functions, including vesicle transport. Brefeldin A blocked the enhancement of antigen-evoked ICRAC without inhibiting ADP ribosylation of Gsα, but it did not affect ICRAC induced by suboptimal antigen or by thapsigargin. These data provide new evidence that CRAC channels are a major route for Fcε receptor I–triggered Ca2+ influx, and they suggest that ARF may modulate the induction of ICRAC by antigen.
Mystery solved
Authors: Miner Jeffrey H.
Published: July 2002

Mutant mice lacking the integrin α8 subunit exhibit variable defects in kidney development with most mutants missing both kidneys. Several lines of evidence indicate that the known extracellular matrix ligands for integrin α8β1 are either dispensable for or not involved in α8β1 signaling during kidney development. This suggests the presence of an unknown ligand. A novel α8β1 ligand, nephronectin, has now been identified. Nephronectin is a new extracellular matrix protein associated with the Wolffian duct and the ureteric bud, epithelial structures with well-defined roles in kidney development.
Role of Interleukin 12 and Costimulators in T Cell Anergy In Vivo
Authors: Parijs Luk Van , Perez Victor L. , Biuckians Andre , Maki Robert G. , London Cheryl A. , Abbas Abul K.
Published: July 2002

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti–CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti–CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.
Differential Regulation of Constitutive Major Histocompatibility Complex Class I Expression in T and B Lymphocytes
Authors: Lee Chien-Kuo , Gimeno Ramon , Levy David E.
Published: July 2002

Major histocompatibility complex (MHC) class I antigens are constitutively expressed yet highly induced by interferon (IFN) during inflammation. We found that not only IFN-induced but also normal basal expression of MHC I required IFN receptors and signal transducer and activator of transcription (STAT)1, providing genetic evidence for continuous IFN signaling. Surprisingly, an IFN-independent requirement for STAT1 was also found, specifically in T lymphocytes, where MHC class I expression was not fully accounted for by IFN signaling. This IFN-independent pathway maintained tyrosine phosphorylation of STAT1 in T but not B lymphocytes even in the absence of IFN receptors. Interestingly, interleukin (IL)-7 selectively activated STAT1 and induced MHC class I in mature T but not B cells. These loss of function studies demonstrate an essential role of endogenous IFN and activated STAT1 for constitutive MHC class I expression in normal mice and define IL-7–dependent but IFN-independent regulation of STAT1 restricted to T lymphocytes.
The Src Family Tyrosine Kinase Fyn Regulates Natural Killer T Cell Development
Authors: Gadue Paul , Morton Neil , Stein Paul L.
Published: July 2002

T lymphocytes express two Src tyrosine kinases, Lck and Fyn. While thymocyte and T cell subsets are largely normal in fyn−/− mice, animals lacking Lck have impaired T cell development. Here, it is shown that Fyn is required for the rapid burst of interleukin (IL)-4 and IL-13 synthesis, which occurs promptly after T cell receptor activation. The lack of cytokine induction in fyn mutant mice is due to a block in natural killer (NK) T cell development. Studies using bone marrow chimeras indicate that the defect behaves in a cell-autonomous manner, and the lack of NK T cells is probably not caused by inappropriate microenvironmental cues. Both NK T cells and conventional T cells express similar levels of Lck, implying that Fyn and Lck have distinct roles in regulating NK T cell ontogeny. The fyn mutation defines the first signaling molecule that is selectively required for NK T cell, but not for T lymphocyte or NK cell development.
Antiidiotype Antibody against Platelet Anti-Gpiiia Contributes to the Regulation of Thrombocytopenia in HIV-1–Itp Patients
Authors: Nardi Michael , Karpatkin Simon
Published: July 2002

Patients with human immunodeficiency virus 1–associated immunological thrombocytopenia (HIV-1–ITP) have markedly elevated platelet-bound immunoglobulin (Ig)G, IgM, and C3C4, as well as serum circulating immune complexes (CICs) composed of the same. Affinity purification of IgGs from their CICs with fixed platelets reveals high-affinity antibody (Ab) against platelet glycoprotein (GP)IIIa 49–66, which correlates inversely with their platelet count. However, sera from these patients have little to no anti-GPIIIa activity. To investigate this, we assayed serum, purified serum IgG, and CIC-Ig from these patients. This revealed ∼150-fold greater Ab activity in purified serum IgG, and ∼4,000-fold greater reactivity in CIC-IgG. This was shown to be associated with the presence of antiidiotype Ab2 (both IgG and IgM) sequestered in the CIC-IgG. The IgM antiidiotype was predominantly blocking Ab, as demonstrated by specificity for F(ab′)2 fragments of anti–GPIIIa 49–66 of HIV-1–ITP patients and inhibition of reactivity with peptide GPIIIa 49–66, not with a control peptide. The IgM antiidiotype was not polyreactive. Similar measurements were made in nonthrombocytopenic HIV-1–infected patients. Their serum reactivity was not measurable, but serum Ig and CIC-IgG against platelet GPIIIa 49–66 was present, although considerably lower than that found in HIV-1–ITP patients (26- and 35-fold lower, respectively). In addition, their IgM antiidiotype reactivity was 12-fold greater than that found in HIV-1–ITP patients. The IgM antiidiotype Ab titer of both cohorts correlated with in vivo platelet count (r = 0.7, P = 0.0001, n = 32). To test the in vivo effectiveness of the IgM antiidiotype, thrombocytopenia was induced in mice with 25 μg of affinity-purified anti–GPIIIa 49–66 (mouse GPIIIa has 83% homology with human GPIIIa and Fc receptors for human IgG1). Maximum effect was obtained at 4–6 h after intraperitoneal injection into Balb/c mice with a platelet count of ∼30% baseline value. Preincubation of the anti-GPIIIa Ab with control IgM at molar ratios of IgM/IgG of 1:7 before intraperitoneal injection had no effect on the in vivo platelet count, whereas preincubation with patient IgM antiidiotype improved the platelet count to 50–80% of normal. Thrombocytopenia could be reversed after addition of IgM antiidiotype 4 h after induction of thrombocytopenia. Thus, CICs of HIV-1–infected patients contain IgM antiidiotype Ab against anti-GPIIIa, which appears to regulate their serum reactivity in vitro and their level of thrombocytopenia in vivo.
The First α Helix of Interleukin (Il)-2 Folds as a Homotetramer, Acts as an Agonist of the IL-2 Receptor β Chain, and Induces Lymphokine-Activated Killer Cells
Authors: Eckenberg Ralph , Rose Thierry , Moreau Jean-Louis , Weil Robert , Gesbert Franck , Dubois Sigrid , Tello Diana , Bossus Marc , Gras Hélène , Tartar André , Bertoglio Jacques , Chouaïb Salem , Goldberg Michel , Jacques Yannick , Alzari Pedro M. , Thèze Jacques
Published: July 2002

Interleukin (IL)-2 interacts with two types of functional receptors (IL-2Rαβγ and IL-2Rβγ) and acts on a broad range of target cells involved in inflammatory reactions and immune responses. For the first time, we show that a chemically synthesized fragment of the IL-2 sequence can fold into a molecule mimicking the quaternary structure of a hemopoietin. Indeed, peptide p1–30 (containing amino acids 1–30, covering the entire α helix A of IL-2) spontaneously folds into an α-helical homotetramer and stimulates the growth of T cell lines expressing human IL-2Rβ, whereas shorter versions of the peptide lack helical structure and are inactive. We also demonstrate that this neocytokine interacts with a previously undescribed dimeric form of IL-2Rβ. In agreement with its binding to IL-2Rβ, p1–30 activates Shc and p56lck but unlike IL-2, fails to activate Janus kinase (Jak)1, Jak3, and signal transducer and activator of transcription 5 (STAT5). Unexpectedly, we also show that p1–30 activates Tyk2, thus suggesting that IL-2Rβ may bind to different Jaks depending on its oligomerization. At the cellular level, p1–30 induces lymphokine-activated killer (LAK) cells and preferentially activates CD8low lymphocytes and natural killer cells, which constitutively express IL-2Rβ. A significant interferon γ production is also detected after p1–30 stimulation. A mutant form of p1–30 (Asp20→Lys), which is likely unable to induce vascular leak syndrome, remains capable of generating LAK cells, like the original p1–30 peptide. Altogether, our data suggest that p1–30 has therapeutic potential.
A Novel B Lymphocyte–Associated Adaptor Protein, Bam32, Regulates Antigen Receptor Signaling Downstream of Phosphatidylinositol 3-Kinase
Authors: Marshall Aaron J. , Niiro Hiroaki , Lerner Cara G. , Yun Theodore J. , Thomas Sushma , Disteche Christine M. , Clark Edward A.
Published: July 2002

We have identified and characterized a novel src homology 2 (SH2) and pleckstrin homology (PH) domain–containing adaptor protein, designated Bam32 (for B cell adaptor molecule of 32 kD). cDNAs encoding the human and mouse Bam32 coding sequences were isolated and the human bam32 gene was mapped to chromosome 4q25–q27. Bam32 is expressed by B lymphocytes, but not T lymphocytes or nonhematopoietic cells. Human germinal center B cells show increased Bam32 expression, and resting B cells rapidly upregulate expression of Bam32 after ligation of CD40, but not immunoglobulin M. Bam32 is tyrosine-phosphorylated upon B cell antigen receptor (BCR) ligation or pervanadate stimulation and associates with phospholipase Cγ2. After BCR ligation, Bam32 is recruited to the plasma membrane through its PH domain. Membrane recruitment requires phosphatidylinositol 3-kinase (PI3K) activity and an intact PI(3,4,5)P3-binding motif, suggesting that membrane association occurs through binding to 3-phosphoinositides. Expression of Bam32 in B cells leads to a dose-dependent inhibition of BCR-induced activation of nuclear factor of activated T cells (NF-AT), which is blocked by deletion of the PH domain or mutation of the PI(3,4,5)P3-binding motif. Thus, Bam32 represents a novel B cell–associated adaptor that regulates BCR signaling downstream of PI3K.
Regulation of Peripheral Lymph Node Genesis by the Tumor Necrosis Factor Family Member Trance
Authors: Kim Dongku , Mebius Reina E. , MacMicking John D. , Jung Steffen , Cupedo Tom , Castellanos Yaneth , Rho Jaerang , Wong Brian R. , Josien Regis , Kim Nacksung , Rennert Paul D. , Choi Yongwon
Published: July 2002

Proper lymph node (LN) development requires tumor necrosis factor–related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE−/− mice correlates with a significant reduction in lymphotoxin (LT)αβ+α4β7+CD45+CD4+CD3− cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE−/− mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTαβ expression on CD45+ CD4+CD3− cells, as LNs could not be induced in LTα−/− mice. LTα−/− mice also showed defects in the fate of CD45+CD4+CD3− cells similar to TRANCE−/− mice. Thus, we propose that both TRANCE and LTαβ regulate the colonization and cluster formation by CD45+ CD4+CD3− cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.
Differential Influence on Cytotoxic T Lymphocyte Epitope Presentation by Controlled Expression of Either Proteasome Immunosubunits or Pa28
Authors: van Hall Thorbald , Sijts Alice , Camps Marcel , Offringa Rienk , Melief Cornelis , Kloetzel Peter-M. , Ossendorp Ferry
Published: July 2002

The proteasome is the principal provider of major histocompatibility complex (MHC) class I–presented peptides. Interferon (IFN)-γ induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I–presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-γ–independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-γ–inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-γ–inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response.
Flice-Inhibitory Protein Is a Key Regulator of Germinal Center B Cell Apoptosis
Authors: Hennino Ana , Bérard Marion , Krammer Peter H. , Defrance Thierry
Published: July 2002

Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon “priming” of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain–containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1β–converting enzyme-inhibitory protein (c-FLIPL) but not the CD95L. In culture, c-FLIPL is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIPL plays an active role in positive selection of B cells in the GC.
Lyn Is Essential for Fcγ Receptor III–Mediated Systemic Anaphylaxis but Not for the Arthus Reaction
Authors: Yuasa Takae , Ono Masao , Watanabe Takeshi , Takai Toshiyuki
Published: July 2002

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcεRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcγRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcγRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcγRIIB, which otherwise downregulates FcγRIII-mediated cellular responses. FcγRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow–derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor α release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcγRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.
The Upstream Enhancer Is Necessary and Sufficient for the Expression of the Pre-T Cell Receptor α Gene in Immature T Lymphocytes
Authors: Reizis Boris , Leder Philip
Published: July 2002

The expression of the pre-T cell receptor α (pTa) gene occurs exclusively in immature T lymphocytes and is regulated by poorly defined mechanisms. We have analyzed the role of the upstream enhancer in pTa expression using conventional and bacterial artificial chromosome (BAC) reporter transgenes. The deletion of the enhancer completely abolished the expression of pTa BAC reporter in transgenic mice. Conversely, the combination of pTa enhancer and promoter targeted transgenes specifically to immature thymocytes, recapitulating the expression pattern of pTa. The core enhancer is conserved between mice and humans and contains a critical binding site for the transcription factor c-Myb. We also show that pTa promoter contains a conserved tandem E box site activated by E protein, HEB. These data establish the enhancer as a critical element regulating pTa gene expression and identify additional targets for c-Myb and E proteins in T cell development.
Ca2+-induced Ca2+ Release Phenomena in Mammalian Sympathetic Neurons Are Critically Dependent on the Rate of Rise of Trigger Ca2+
Authors: Hernández-Cruz Arturo , Escobar Ariel L. , Jiménez Nicolás
Published: July 2002

The role of ryanodine-sensitive intracellular Ca2+ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca2+ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca2+-mediated feedback loops, suggesting that it constitutes a Ca2+-induced Ca2+-release phenomenon. The dynamics of release is markedly affected when Sr2+ substitutes for Ca2+, indicating that Sr2+ release may operate with lower feedback gain than Ca2+ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca2+ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca2+ and Ca2+-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca2+ reservoir is full, the rapid initial Ca2+ rise determines a faster occupation of the ryanodine receptor Ca2+ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca2+ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store's Ca2+ content. This study suggests that transmembrane Ca2+ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca2+] near the Ca2+ release channels sufficiently fast to overcome their Ca2+-dependent inactivation. Conversely, caffeine-induced Ca2+ release can undergo considerable amplification especially when Ca2+ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca2+ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca2+ stores.
Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells
Authors: Sugihara Izumi
Published: July 2002

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).
Cooh-Terminal Truncated Alpha1S Subunits Conduct Current Better than Full-Length Dihydropyridine Receptors
Authors: Morrill James A. , Cannon Stephen C.
Published: July 2002

Skeletal muscle dihydropyridine (DHP) receptors function both as voltage-activated Ca2+ channels and as voltage sensors for coupling membrane depolarization to release of Ca2+ from the sarcoplasmic reticulum. In skeletal muscle, the principal or α1S subunit occurs in full-length (∼10% of total) and post-transcriptionally truncated (∼90%) forms, which has raised the possibility that the two functional roles are subserved by DHP receptors comprised of different sized α1S subunits. We tested the functional properties of each form by injecting oocytes with cRNAs coding for full-length (α1S) or truncated (α1SΔC) α subunits. Both translation products were expressed in the membrane, as evidenced by increases in the gating charge (Qmax 80–150 pC). Thus, oocytes provide a robust expression system for the study of gating charge movement in α1S, unencumbered by contributions from other voltage-gated channels or the complexities of the transverse tubules. As in recordings from skeletal muscle, for heterologously expressed channels the peak inward Ba2+ currents were small relative to Qmax. The truncated α1SΔC protein, however, supported much larger ionic currents than the full-length product. These data raise the possibility that DHP receptors containing the more abundant, truncated form of the α1S subunit conduct the majority of the L-type Ca2+ current in skeletal muscle. Our data also suggest that the carboxyl terminus of the α1S subunit modulates the coupling between charge movement and channel opening.
Algebras associated to elliptic curves
Authors: Stephenson Darin
Published: July 2002

This paper completes the classification of Artin-Schelter regular algebras of global dimension three. For algebras generated by elements of degree one this has been achieved by Artin, Schelter, Tate and Van den Bergh. We are therefore concerned with algebras which are not generated in degree one. We show that there exist some exceptional algebras, each of which has geometric data consisting of an elliptic curve together with an automorphism, just as in the case where the algebras are assumed to be generated in degree one. In particular, we study the elliptic algebras

A
(
+
)

A(+)

,

A
(
−
)

A(-)

, and

A
(

a

)

A({\mathbf {a}})

, where

a

∈

P

2

{\mathbf {a}}\in \mathbb {P}^{2}

, which were first defined in an earlier paper. We omit a set

S
⊂

P

2

S\subset \mathbb {P}^2

consisting of 11 specified points where the algebras

A
(

a

)

A({\mathbf {a}})

become too degenerate to be regular.

Theorem. Let

A
A

represent

A
(
+
)

A(+)

,

A
(
−
)

A(-)

or

A
(

a

)

A({\mathbf {a}})

, where

a

∈

P

2

∖
S

{\mathbf {a}} \in \mathbb {P}^2\setminus S

. Then

A
A

is an Artin-Schelter regular algebra of global dimension three. Moreover,

A
A

is a Noetherian domain with the same Hilbert series as the (appropriately graded) commutative polynomial ring in three variables.

This, combined with our earlier results, completes the classification.
Boundary element monotone iteration scheme for semilinear elliptic partial differential equations
Authors: Deng Yuanhua , Chen Goong , Ni Wei-Ming , Zhou Jianxin
Published: July 2002

The monotone iteration scheme is a constructive method for solving a wide class of semilinear elliptic boundary value problems. With the availability of a supersolution and a subsolution, the iterates converge monotonically to one or two solutions of the nonlinear PDE. However, the rates of such monotone convergence cannot be determined in general. In addition, when the monotone iteration scheme is implemented numerically through the boundary element method, error estimates have not been analyzed in earlier studies. In this paper, we formulate a working assumption to obtain an exponentially fast rate of convergence. This allows a margin

δ
\delta

for the numerical implementation of boundary elements within the range of monotone convergence. We then interrelate several approximate solutions, and use the Aubin-Nitsche lemma and the triangle inequalities to derive error estimates for the Galerkin boundary-element iterates with respect to the

H

r

(
Ω
)

H^{r}(\Omega )

,

0
≤
r
≤
2

0\le r \le 2

, Sobolev space norms. Such estimates are of optimal order. Furthermore, as a peculiarity, we show that for the nonlinearities that are of separable type, “higher than optimal order” error estimates can be obtained with respect to the mesh parameter

h
h

. Several examples of semilinear elliptic partial differential equations featuring different situations of existence/nonexistence, uniqueness/multiplicity and stability are discussed, computed, and the graphics of their numerical solutions are illustrated.
Frame: detection of genomic sequencing errors.
Authors: Brown N P , Sander C , Bork P
Published: July 2002

MOTIVATION: The underlying error rate for genomic sequencing sometimes results in the introduction of artificial frameshifts and in-frame stop codons into putative protein encoding genes. Severe errors are then introduced into the inferred transcripts through mis-translation or premature termination. RESULTS: We describe a system for screening segments of DNA for frameshift and in-frame stop errors in coding regions. The method is based on homology matching using blastx to compare all six reading frames of the query nucleotide sequence against selected protein sequence databases. Fragments of protein matching neighbouring regions of the query DNA are united and extended laterally to define candidate open reading frames, within which, frameshifts and stops are identified. Suitable targets include prokaryotic or other intron-free genomic sequence and complementary DNAs. As an example of its use, we report here two frameshifted ORFs that deviate from the original TIGR sequence annotations for the recently released Helicobacter pylori genome. AVAILABILITY: The tool is accessible via the URL http://www.sander.ebi.ac.uk/frame/. CONTACT: brown@ebi.ac.uk.
GenomePixelizer—a visualization program for comparative
genomics within and between species
Authors: Kozik A. , Kochetkova E. , Michelmore R.
Published: July 2002

Summary: GenomePixelizer is a visualization tool that generates custom images of the physical or genetic positions of specified sets of genes in whole genomes or parts of genomes. Multiple sets of genes can be shown simultaneously with user-defined characteristics displayed. It allows the analysis of duplication events within and between species based on sequence similarities. The program is written in Tcl/Tk and works on any platform that supports the Tcl/Tk toolkit. GenomePixelizer generates HTML ImageMap tags for each gene in the image allowing links to databases. Images can be saved and presented on web pages.

Availability: GenomePixelizer is freely available at http://niblrrs.ucdavis.edu/GenomePixelizer/GenomePixelizer_Welcome.html

Contact: akozik@atgc.org
EGF receptor/Rolled MAP kinase signalling protects cells against activated Armadillo in the Drosophila eye
Authors: Freeman Matthew , Bienz Mariann
Published: July 2002

β‐catenin/Armadillo are transcriptional co‐activators that mediate Wnt signalling in normal development. Activated forms of β‐catenin are oncogenic. We have constructed mutant forms of Drosophila Armadillo which correspond to common human oncogenic mutations, and find them to activate Armadillo constitutively. When expressed in the Drosophila eye, these eventually induce apoptosis in all cell types. Intriguingly, cells in the eye are resistant to the effects of activated Armadillo for a long period prior to the onset of cell death at the mid‐pupal stage. This latency is conferred by EGF receptor (EGFR)/MAP kinase signalling, which prevents activated Armadillo from inducing apoptosis; when EGFR signalling naturally ceases, the cells rapidly die. Nemo, the Drosophila homologue of NLK in mice and LIT‐1 in Caenorhabditis elegans, does not antagonize activated Armadillo, suggesting that the Nemo‐like MAP kinases may not generally interact with Armadillo/β‐catenin. Thus, our results show that activated Armadillo is subject to a specific negative control by EGFR/Rolled MAP kinase signalling.
Phosphorylation of histone H3 is functionally linked to retinoic acid receptor β promoter activation
Authors: Lefebvre Bruno , Ozato Keiko , Lefebvre Philippe
Published: July 2002

Ligand‐dependent transcriptional activation of retinoic acid receptors (RARs) is a multistep process culminating in the formation of a multimeric co‐activator complex on regulated promoters. Several co‐activator complexes harbor an acetyl transferase activity, which is required for retinoid‐induced transcription of reporter genes. Using murine P19 embryonal carcinoma cells, we examined the relationship between histone post‐translational modifications and activation of the endogenous RARβ2 promoter, which is under the control of a canonical retinoic acid response element and rapidly induced upon retinoid treatment. While histones H3 and H4 were constitutively acetylated at this promoter, retinoid agonists induced a rapid phosphorylation at Ser10 of histone H3. A retinoid antagonist, whose activity was independent of co‐repressor binding to RAR, could oppose this agonist‐induced H3 phosphorylation. Since such post‐translational modifications were not observed at several other promoters, we conclude that histone H3 phosphorylation may be a molecular signature of the activated, retinoid‐controlled mRARβ2 gene promoter.
Pharmacokinetics of the 8-methoxyquinolone, moxifloxacin: a comparison in humans and other mammalian species
Authors: Siefert H. M. , Domdey-Bette A. , Henninger K. , Hucke F. , Kohlsdorfer C. , Stass H. H.
Published: July 2002

The pharmacokinetics of moxifloxacin was investigated in NMRI mice, Wistar rats, rhesus monkeys, beagle dogs, Göttingen minipigs and healthy human volunteers after iv and oral administration of moxifloxacin-HCl (single doses of moxifloxacin 9.2 mg/kg bodyweight) in animals and 100 mg moxifloxacin (1.4 mg/kg bodyweight po and 1.2 mg/kg bodyweight iv) in humans. The plasma concentration vs time courses of the unchanged compound (determined by HPLC) and the derived pharmacokinetic parameters were used to evaluate the absorption process, to compare the pharmacokinetics in these species and to perform an interspecies scaling. The results of the pharmacokinetic investigations indicate a clear dependence on the species. Moxifloxacin is absorbed quickly (rats, dogs, humans > monkeys): the major portion of the dose reached the systemic circulation within the first 2 h. In the minipig absorption was slower. Bioavailability was high to moderate (91–52%) in all species. Protein binding ( fu ) was low (55–71%) in all species. The volume of distribution at steady state ( Vss ) was medium to large (2.0–4.9 L/kg) in all species. There were considerable differences in maximum concentrations ( Cmax,norm , 0.430–0.070 kg/L) and in AUC norm values (oral, 6.18–0.184 kg·h/L; iv, 7.51–0.237 kg·h/L). Total body clearance ( CL ) decreased with increasing bodyweight (4.21–0.132 L/(h·kg)). The mean residence time (MRT) decreased with decreasing bodyweight (15–0.88 h). The half-life ( t½ ) decreased with decreasing bodyweight (oral, 12–1.3 h, iv, 13–0.93 h). There was moderate to low renal excretion (iv, 20–6.2%), the renal clearance, ( CLR ) was in the range 0.615–0.0222 L/(h·kg). Regarding the pharmacokinetic parameters determined after oral administration, the dog was most similar to the human in terms of Cmax , AUC and t½ . There was good correlation between bodyweight and CL (coefficient of correlation ( r ) = 0.959), Vss ( r = 0.990) and MRT ( r = 0.943). On the basis of preclinical studies a terminal half-life appropriate for once-daily dosing in humans was predicted and confirmed by Phase I data.
Induction of scattering and cellular invasion by trefoil peptides in src‐ and RhoA‐transformed kidney and colonic epithelial cells
Authors: EMAMI SHAHIN , FLOCH NATHALIE LE , BRUYNEEL ERIK , THIM LARS , MAY FELICITY , WESTLEY BRUCE , RIO MARIE‐CHRISTINE , MAREEL MARC , GESPACH CHRISTIAN
Published: July 2002

Trefoil factors (TFFs) are protease‐resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose‐response curves in src‐transformed MDCKts.src and PCmsrc cells (EC50 = 20–40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src‐pS2 cells and human colonic HCT8/S11‐pS2 cells established from a sporadic tumor. Furthermore, we found that TFF‐mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3′‐kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed byWestern blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.—Emami, S., Le Floch, N., Bruyneel, E., Thim, L., May, F., Westley, B., Rio, M.‐C., Mareel, M., Gespach, C. Induction of scattering and cellular invasion by trefoil peptides in src‐ and RhoA‐transformed kidney and colonic epithelial cells. FASEB J. 15, 351‐361 (2001)
Dielectric function for a material containing hyperspherical inclusions toO(c2): I. Multipole expansions
Authors: Choy T. C. , Alexopoulos Aris , Thorpe M. F.
Published: July 2002

By averaging over pairs ofhyperspheres, we have obtained the dielectric function for a binary mixture containinghypersphericalinclusions up to orderc2, wherecis the volume fraction of inclusions. The method used is based on multipole expansions for the potential of two spheres in a uniform field and is a generalization of the method of Jeffrey tod–dimensional space. Numerical results are presented for the second–order coefficient κ in the low–cexpansion of the dielectric constant for arbitraryd; these verify earlier known results, as well as showing the dependence of κ on dimensionality, which is particularly simple asd→ 1 and asd→ ∞.
Description of the glochidium of Margaritifera auricularia (Spengler 1793) (Bivalvia, Unionoidea)
Authors: Araujo R. , Ramos M. A.
Published: July 2002

The glochidium ofMargaritifera auriculariais described for the first time by using light microscopy, scanning electron microscopy and histological techniques. The larval mantle is formed by only two layers of cells; the inner one being much thicker, with microvilli. All cell masses of the glochidium are temporary aggregations that are the rudiments of organs of the subsequent juveniles which will be released after metamorphosis in the host tissues. In the glochidium there are three main masses of cells: (i) the muscle, which is in an anterior position; (ii) the oral plate in the centre of the larva; and (iii) the more ventrally and posteriorly situated ventral plate, or foot rudiment, flanged with lateral pits all bearing dense cilia. No rudimentary organs such as the pericardium, the kidney, the heart or nerve ganglia have developed. There are no visible hooks in the valve margins, but by using light microscopy we observed minute teeth covered by a rim of the periostracum. Near the margin of the shell there are two pairs of sensory hair tufts only observable by scanning electron microscopy. The glochidium ofM. auriculariais the largest of the family Margaritiferidae and intermediate between the glochidium of the known species of this family and those of Unionidae.
Immunohistochemical evidence for the Na+/Ca2+exchanger in squid olfactory neurons
Authors: Lucero Mary T. , Huang Wei , Dang Tu
Published: July 2002

The olfactory organs from the squidLolliguncula brevisare composed of a pseudostratified epithelium containing five morphological subtypes of chemosensory neurons and ciliated support cells. Physiological recordings have been made from two of the subtypes and only the type 4 neuron has been studied in detail. Odour–stimulated increases in intracellular calcium and rapid activation of an electrogenic Na+/Ca2+exchanger current in type 4 neurons suggest that the exchanger proteins are localized very close to the transduction machinery. Electrophysiological studies have shown that olfactory signal transduction takes place in the apical ciliary regions of olfactory neurons. Using polyclonal antiserum against squid Na+/Ca2+proteins, we observed specific staining in the ciliary region of cells that resemble type 2, 3, 4 and 5 neurons. Staining was also observed in axon bundles, and in muscle tissue. Collectively, these data support the model that Na+/Ca2+exchanger proteins are localized to transduction machinery in cilia of type 4 neurons and suggest that the other olfactory subtypes also use Ca2+during chemosensory responses.
Envelope-Class Retrovirus-Like Elements Are Widespread, Transcribed and Spliced, and Insertionally Polymorphic in Plants
Authors: Vicient Carlos M. , Kalendar Ruslan , Schulman Alan H.
Published: July 2002

Retrotransposons and retroviruses share similar intracellular life cycles and major encoded proteins, but retrotransposons lack the envelope (env) critical for infectivity. Retrotransposons are ubiquitous and abundant in plants and active retroviruses are known in animals. Although a few env-containing retroelements,gypsy-like Athila, Cyclops, andCalypso and copia-like SIRE-1, have been identified in plants, the general presence and functionality of the domain remains unclear. We show here that env-class elements are present throughout the flowering plants and are widely transcribed. Within the grasses, we show the transcription of the envdomain itself for Bagy-2 and related retrotransposons, all members of the Athila group. Furthermore, Bagy-2transcripts undergo splicing to generate a subgenomic envproduct as do those of retroviruses. Transcription and the polymorphism of their insertion sites in closely related barley cultivars suggests that at least some are propagationally active. The putative ENV polypeptides of Bagy-2 and rice Rigy-2 contain predicted leucine zipper and transmembrane domains typical of retroviral ENVs. These findings raise the prospect of active retroviral agents among the plants.

[The sequence data described in this paper have been deposited as follows: Bagy-2 elements, EMBL accession nos.AF254799 and AJ279072; an alignment of 328 gypsy-likert sequences, accession no. DS44537; barley envsequences, accession nos. AJ298028–AJ298032; cDNA sequences for the spliced env subgenomic RNAs, accession nos.AJ311200–AJ311202; genomic sequences for env-classrt, accession nos. AJ295085–AJ295111; cDNA sequences forenv-class rt, accession nos. AJ295112–AJ295139; representatives of polymorphic Bagy-2 bands from IRAP gels, accession nos. AF363958, AF 363959, and AY029538.]
Methylation-Specific Oligonucleotide Microarray: A New Potential for High-Throughput Methylation Analysis
Authors: Gitan Raad S. , Shi Huidong , Chen Chuan-Mu , Yan Pearlly S. , Huang Tim Hui-Ming
Published: July 2002

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19–23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the humanestrogen receptor α (ERα) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.

[The sequence data described in this paper have been submitted to the data library under accession no. X03635.1 G1:31233.]
Fluoroscopic study of tumor motion due to breathing: Facilitating precise radiation therapy for lung cancer patients
Authors: Chen Qin‐Sheng , Weinhous Martin S. , Deibel F. Christopher , Ciezki Jay P. , Macklis Roger M.
Published: July 2002

Target motion due to breathing is one of the major obstacles in dose escalation of radiation therapy to some tumors in the thoracoabdominal region. The development of beam gating or target motion tracking techniques provides a possibility to reduce normal tissue volume in a treatment field. Tumor motion monitoring in those techniques plays a crucial role, but has not yet been adequately explored. This paper reports our preliminary investigation on breath introduced tumor motion. Tumor locations and motion properties were determined from digitized fluoroscopic videos acquired during patient simulation. Image distortion due to irregularities in the imaging chain, such as the pincushion distortion, was corrected with a polynomial unwarping method. Temporal Fourier transformation of the fluoroscopic video was introduced to convert the motion information over time to a static view of a motion field, in which regions with different motion ranges can be directly measured. Patient breathing patterns vary from patient to patient and so does the kinematic behavior of individual tumors. In order to evaluate the feasibility for tracking internal target motion with nonionizing‐radiation techniques, motion patterns between internal targets and external radio opaque markers placed on patient's chest during fluoroscopic video acquisition were compared. For some patients, significant motion phase discrepancies between an internal target and an external marker have been observed. Quantitative measurements are reported. These results will be useful in the design of a motion tracking or gated radiotherapy system.