bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling
Authors: Colgan Sean P. , Hershberg Robert M. , Furuta Glenn T. , Blumberg Richard S.
Published: July 2002

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a β2-microglobulin-associated 48-kDa glycoprotein and novel β2-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-γ signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.
Modulation of Ca
2+
entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca
2+
entry
Authors: Boulay Guylain , Brown Darren M. , Qin Ning , Jiang Meisheng , Dietrich Alexander , Zhu Michael Xi , Chen Zhangguo , Birnbaumer Mariel , Mikoshiba Katsuhiko , Birnbaumer Lutz
Published: July 2002

Homologues of
Drosophilia
transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca
2+
stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca
2+
-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione
S
-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca
2+
entry and to test for a role of their respective binding partners in Ca
2+
entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca
2+
entry. In contrast, F2g enhanced the two forms of Ca
2+
entry. We conclude that store depletion-activated Ca
2+
entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca
2+
from stores and activating Ca
2+
influx in response to either increasing IP3 or decreasing luminal Ca
2+
.
Disruption of the β subunit of the epithelial Na
+
channel in mice: Hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype
Authors: McDonald Fiona J. , Yang Baoli , Hrstka Ron F. , Drummond Heather A. , Tarr Deirdre E. , McCray Paul B. , Stokes John B. , Welsh Michael J. , Williamson Roger A.
Published: July 2002

The epithelial Na
+
channel (ENaC) is composed of three
homologous subunits: α, β and γ. We used gene targeting to
disrupt the β subunit gene of ENaC in mice. The βENaC-deficient
mice showed normal prenatal development but died within 2 days after
birth, most likely of hyperkalemia. In the −/− mice, we found an
increased urine Na
+
concentration despite hyponatremia and
a decreased urine K
+
concentration despite hyperkalemia.
Moreover, serum aldosterone levels were increased. In contrast to
αENaC-deficient mice, which die because of defective lung liquid
clearance, neonatal βENaC deficient mice did not die of respiratory
failure and showed only a small increase in wet lung weight that had
little, if any, adverse physiologic consequence. The results indicate
that,
in vivo
, the β subunit is required for ENaC
function in the renal collecting duct, but, in contrast to the α
subunit, the β subunit is not required for the transition from a
liquid-filled to an air-filled lung. The phenotype of the
βENaC-deficient mice is similar to that of humans with
pseudohypoaldosteronism type 1 and may provide a useful model to study
the pathogenesis and treatment of this disorder.
The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway
Authors: Ouyang Wenjun , Jacobson Nils G. , Bhattacharya Deepta , Gorham James D. , Fenoglio Domenic , Sha William C. , Murphy Theresa L. , Murphy Kenneth M.
Published: July 2002

Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon γ (IFN-γ) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-γ production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-γ production in Stat4-deficient T cells, but augmented IFN-γ expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-γ reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting.
Intermediate spectra and photocycle kinetics of the Asp96 → Asn mutant bacteriorhodopsin determined by singular value decomposition with self-modeling
Authors: Zimányi László , Kulcsár Ágnes , Lanyi Janos K. , Sears Donald F. , Saltiel Jack
Published: July 2002

Singular value decomposition with self-modeling is applied to resolve the intermediate spectra and kinetics of the Asp96 → Asn mutant bacteriorhodopsin. The search for the difference spectra of the intermediates is performed in eigenvector space on the stoichiometric plane. The analysis of data at pH values ranging from 4 to 8 and temperatures between 5 and 25°C reveals significant, early partial recovery of the initial state after photoexcitation. The derived spectra are not biased by assumed photocycles. The intermediate spectra derived in the initial step differ from spectra determined in prior analyses, which results in intermediate concentrations with improved stoichiometric properties. Increasingly more accurate photocycles follow with increasing assumed complexity, of which parallel models are favored, consistent with recent, independent experimental evidence.
Limiting dynamics of high-frequency electromechanical transduction of outer hair cells
Authors: Frank Gerhard , Hemmert Werner , Gummer Anthony W.
Published: July 2002

High-frequency resolution is one of the salient features of peripheral sound processing in the mammalian cochlea. The sensitivity originates in the active amplification of the travelling wave on the basilar membrane by the outer hair cells (OHCs), where electrically induced mechanical action of the OHC on a cycle-by-cycle basis is believed to be the crucial component. However, it is still unclear if this electromechanical action is sufficiently fast and can produce enough force to enhance mechanical tuning up to the highest frequencies perceived by mammals. Here we show that isolated OHCs in the microchamber configuration are able to overcome fluid forces with almost constant displacement amplitude and phase up to frequencies well above their place-frequency on the basilar membrane. The high-frequency limit of the electromotility, defined as the frequency at which the amplitude drops by 3 dB from its asymptotic low-frequency value, is inversely dependent on cell length. The frequency limit is at least 79 kHz. For frequencies up to 100 kHz, the electromotile response was specified by an overdamped (
Q
= 0.42) second-order resonant system. This finding suggests that the limiting factor for frequencies up to 100 kHz is not the speed of the motor but damping and inertia. The isometric force produced by the OHC was constant at least up to 50 kHz, with amplitudes as high as 53 pN/mV being observed. We conclude that the electromechanical transduction process of OHCs possesses the necessary high-frequency properties to enable amplification of the travelling wave over the entire hearing range.
An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice
Authors: Brandes Ralf P. , Schmitz-Winnenthal Friedrich-Hubertus , Félétou Michel , Gödecke Axel , Huang Paul L. , Vanhoutte Paul M. , Fleming Ingrid , Busse Rudi
Published: July 2002

In addition to nitric oxide (NO) and prostacyclin (PGI
2
), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS −/−) mice. Vasodilator responses to endothelium-dependent agonists were determined
in vivo
and
in vitro. In vivo
, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS −/− mice and was unaffected by treatment with
N
ω
-nitro-
l
-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS −/− mice, marked
N
ω
-nitro-
l
-arginine (
l
-NA, 300 μmol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of
l
-NA. This endothelium-dependent, NO/PGI
2
-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18α-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Δ
9
-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation
in vivo
and
in vitro
is not mediated by NO, PGI
2
, or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.
Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates
Authors: Schwesinger Falk , Ros Robert , Strunz Torsten , Anselmetti Dario , Güntherodt Hans-Joachim , Honegger Annemarie , Jermutus Lutz , Tiefenauer Louis , Plückthun Andreas
Published: July 2002

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.
Effects of saturation mutagenesis of the phage SP6 promoter on transcription activity, presented by activity logos
Authors: Shin Inkyung , Kim Jinsuk , Cantor Charles R. , Kang Changwon
Published: July 2002

A full set of SP6 promoter variants with all possible single substitutions at positions −17 to +5 was constructed. Transcription activities of these variants were individually measured
in vivo
and
in vitro
to determine the contribution of each base pair to the promoter activity. The
in vivo
activity was measured indirectly by transcriptional interference of the replication of promoter-bearing plasmids. This activity depends most highly on residues −11, −9, −8, −7, and +1 (initiation site). All substitutions at −11, −9, −8, and −7 abolished formation of closed complexes, except for A−8C. These residues are involved in base-specific interactions with the polymerase, and the substitutions exhibit the same strong inhibition
in vitro
. In contrast, the
in vitro
activities of some other variants, measured on linearized templates, were different from those
in vivo
. Some variants at −13, −4, and −2, among others, showed exceptionally higher activities
in vivo
than
in vitro
, supporting the possibility that these residues are involved in postbinding steps, including template melting and bending. The A−3T variant showed much lower activity
in vivo
than
in vitro
, but it bound to the polymerase 2-fold more than the consensus sequence and is possibly involved in polymerase binding. A quantitative hierarchy of all the base pairs is graphically displayed by activity logos, revealing the energetic contribution of each base pair to the activity.
Telecommuting and the Demand for Urban Living: A Preliminary Look at White-collar Workers
Authors: Ellen Ingrid Gould , Hempstead Katherine
Published: July 2002

With recent advances in communications technology, telecommuting appears to be an increasingly viable option for many workers. For urban researchers, the key question is whether this growing ability to telecommute is altering residential location decisions and leading households to live in smaller, lower-density and more remote locations. Using the Work Schedules supplement from the 1997 Current Population Study, this paper explores this question. Specifically, it examines the prevalence of telecommuting, explores the relationship between telecommuting and the residential choices of white-collar workers and, finally, speculates about future impacts on residential patterns and urban form.
Post-Communist Linguistic Problems in the North Adriatic Area*
Authors: Brezigar Bojan
Published: July 2002

The population of the North Adriatic area has always been linguistically mixed. Slovene, Croatian and Italian populations have lived here for 14 centuries; German people moved here in the period of the Austro-Hungarian Empire. In the eighteenth and nineteenth centuries the city of Trieste became the official harbor of the Empire, and, as a consequence, the town grew, attracting others immigrants: Greeks, Jews, Serbs, and so on. Linguistically three main groups lived in Trieste: German, Italian and Slovene speakers. The Slovenes in Trieste owned many important activities, such as banks and trading companies. They were organized in the fields of education and culture with their own theater, private schools, library, daily newspaper and magazines. The Slovenes in Trieste also had their own political movement electing some representatives to the Parliament in Vienna. On the whole, they were considered a very well organized linguistic group. The Italians in Trieste were similarly well organized, and many supported a strong irredentist movement, economical and political organizations, as well as the theater. The German speakers were mostly immigrants who had moved to Trieste for business reasons or as civil servants.
The dynamics of cytokine d nitric oxide secretion in mice injected with Tityus serrulatus scorpion venom
Authors: Petricevich Vera L. , Peña Carlos F.
Published: July 2002

Aims: The effects of Tityus serrulatus venom (TSV) were analysed with respect to the susceptibility of four isogenic mouse, the symptoms following injection of venom and the inflammatory mediators in an experimental model of severe envenomation induced in mice.

Methods: The susceptibility was analysed by lethal dose (LD50) determination, including the symptoms observed during envenomating and glucose levels. The detection of cytokines in serum from mice were analysed using enzyme‐linked immunosorbent assay, and nitric oxide (NO) was analysed using nitrite determination.

Results: The estimated LD50 values were in micrograms per 100 microliters, and the susceptibility of mice to TSV varies with: (a) mouse strain and route of injection (A/J < BALB/c < C57Bl/6 = DBA); (b) mouse strain and sex (A/J female and male < BALB/c female and male); and (c) body weight (all groups of A/J < BALB/c groups).

Among the mouse strains studied, BALB/c mice presented moderate sensibility to TSV, with changes in specific signs and serum levels of glucose, several cytokines and NO, when injected intraperitoneally (i.p.) with 1 LD50 of venom. Sweating, salivation and tremor were the specific signs that preceded death. The maximum levels of glucose in sera from mice injected i.p. with 1 LD50 of TSV were observed 60‐90 min post‐injection. Significant differences were observed in the time‐course of cytokine levels, and the venom induced marked elevations of interleukin (IL)‐1α, IL‐1β, IL‐6, IL‐10 and interferon gamma (IFN‐γ). The maximum levels of IL‐1α and IL‐1β were observed 2 h post‐injection. The more pronounced levels of IL‐6 were observed 4 h post‐injection. There was an early increase in IFN‐γ followed by an even higher level after 4 h. IL‐10 levels peaked between 6 and 8 h, and this cytokine probably modulates the secretion of IFN‐γ. Tumor necrosis factor release was not detected in BALB/c mice injected with TSV. NO levels attained maximal release after 2 h, following venom injection, while a second peak for NO was at 6 h.

Conclusions: These findings indicate that the susceptibility to the systemic effects of the venom varies among mice of different haplotypes, and that the cytokines such as IL‐1, IL‐6, IFN‐γ and NO are strongly involved in the pathogenesis caused by this venom and are correlated with the severity of envenomation.
Disease Incidence and Results of Extremity Lesion Treatment: Mersey Region Soft Tissue Sarcomas (1975–1985)
Authors: Jane Michael J. , Hughes Peter J.
Published: July 2002

Purpose. The incidence and treatment results of extremity soft tissue sarcoma (STS) in the Mersey Region, in the absence of a Multi‐Disciplinary Unit, for the period 1975–1985, have been analysed.

Subjects and methods. Data from cases presenting with STS within the Mersey region, from 1 January 1975 until 31 December 1985, were reviewed. Only patients with sarcoma of head and neck, thoracic wall, abdominal wall, retroperitoneum, limb girdle or extremity were included. Extremity lesions were staged according to the MTS system. Pathological data also were assigned a grade according to tumour differentiation, mitosis count and tumour necrosis. Data from
patients with a minimum follow‐up of 5 years were collated, and patterns of treatment failure were investigated. Finally, time to first occurrence was analysed.

Results and Discussion. The incidence of STS in this study was identical to that reported by the US Department of Health in 1976. Five year survival rate for Stage I tumours was only 51.7% which compares very unfavourably with contemporary series from Multi‐Disciplinary Units. Five year survival rate following wide local excision ± adjuvant therapy is 52.4%, while that following amputation ± adjuvant therapy is 45.5%. While not attaining the results reported by other centres, limb‐sparing surgery does not appear to appreciably prejudice long‐term survival.

Conclusions. STS are rare in the UK, leading to poor classification and suboptimal treatment of lesions. It is important to establish multidisciplinary teams of surgeons, radiologists, radiotherapists and oncologists to plan and organise multimodality therapy for STS.
Lipid Peroxidation and Activities of Antioxidant Enzymes in the Diabetic Kidney: Effect of Treatment with Angiotensin Convertase Inhibitors
Authors: Kedziora‐Kornatowska K. Z. , Luciak M. , Paszkowski J.
Published: July 2002

Effects of the angiotensin convertase inhibitors captopril (CAP) and enalapril (ENA) on the malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and catalase in the kidneys of rats with streptozotocin‐induced diabetes were studied. Induction of diabetes resulted in an increase of MDA concentration and progressive decreases of SOD and catalase activities after 6 and 12 weeks. CAP and ENA administration did not affect body weight changes or blood glucose and HbA contents in diabetic rats but decreased albuminuria and kidney weight increase, attenuated lipid peroxidation, and prevented the decreases in SOD and catalase activities. These results confirm the oxidative stress in streptozotocin‐induced experimental diabetes and point to the beneficial antioxidant effects of angiotensin convertase inhibitors.
Activated Phosphatidylinositol 3-Kinase and Akt Kinase Promote Survival of Superior Cervical Neurons
Authors: Philpott Karen L. , McCarthy Mary Jane , Klippel Anke , Rubin Lee L.
Published: July 2002

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.
Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells
Authors: Hirai Yohei , Lochter André , Galosy Sybille , Koshida Shogo , Niwa Shinichiro , Bissell Mina J.
Published: July 2002

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.
Pex18p and Pex21p, a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway
Authors: Edward Purdue P. , Yang Xudong , Lazarow Paul B.
Published: July 2002

We have identified ScPex18p and ScPex21p, two novel S. cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import receptor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting.
Activation of Myosin Phosphatase Targeting Subunit by Mitosis-specific Phosphorylation
Authors: Totsukawa Go , Yamakita Yoshihiko , Yamashiro Shigeko , Hosoya Hiroshi , Hartshorne David J. , Matsumura Fumio
Published: July 2002

It has been demonstrated previously that during mitosis the sites of myosin phosphorylation are switched between the inhibitory sites, Ser 1/2, and the activation sites, Ser 19/Thr 18 (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129– 137; Satterwhite, L.L., M.J. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595–605), suggesting a regulatory role of myosin phosphorylation in cell division. To explore the function of myosin phosphatase in cell division, the possibility that myosin phosphatase activity may be altered during cell division was examined. We have found that the myosin phosphatase targeting subunit (MYPT) undergoes mitosis-specific phosphorylation and that the phosphorylation is reversed during cytokinesis. MYPT phosphorylated either in vivo or in vitro in the mitosis-specific way showed higher binding to myosin II (two- to threefold) compared to MYPT from cells in interphase. Furthermore, the activity of myosin phosphatase was increased more than twice and it is suggested this reflected the increased affinity of myosin binding. These results indicate the presence of a unique positive regulatory mechanism for myosin phosphatase in cell division. The activation of myosin phosphatase during mitosis would enhance dephosphorylation of the myosin regulatory light chain, thereby leading to the disassembly of stress fibers during prophase. The mitosis-specific effect of phosphorylation is lost on exit from mitosis, and the resultant increase in myosin phosphorylation may act as a signal to activate cytokinesis.
Membrane Targeting and Stabilization of Sarcospan Is Mediated by the Sarcoglycan Subcomplex
Authors: Crosbie Rachelle H. , Lebakken Connie S. , Holt Kathleen H. , Venzke David P. , Straub Volker , Lee Jane C. , Grady R. Mark , Chamberlain Jeffery S. , Sanes Joshua R. , Campbell Kevin P.
Published: July 2002

The dystrophin–glycoprotein complex (DGC) is a multisubunit complex that spans the muscle plasma membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual components are manifested as muscular dystrophy. We recently identified a unique tetraspan-like dystrophin-associated protein, which we have named sarcospan (SPN) for its multiple sarcolemma spanning domains (Crosbie, R.H., J. Heighway, D.P. Venzke, J.C. Lee, and K.P. Campbell. 1997. J. Biol. Chem. 272:31221–31224). To probe molecular associations of SPN within the DGC, we investigated SPN expression in normal muscle as a baseline for comparison to SPN's expression in animal models of muscular dystrophy. We show that, in addition to its sarcolemma localization, SPN is enriched at the myotendinous junction (MTJ) and neuromuscular junction (NMJ), where it is a component of both the dystrophin– and utrophin–glycoprotein complexes. We demonstrate that SPN is preferentially associated with the sarcoglycan (SG) subcomplex, and this interaction is critical for stable localization of SPN to the sarcolemma, NMJ, and MTJ. Our experiments indicate that assembly of the SG subcomplex is a prerequisite for targeting SPN to the sarcolemma. In addition, the SG– SPN subcomplex functions to stabilize α-dystroglycan to the muscle plasma membrane. Taken together, our data provide important information about assembly and function of the SG–SPN subcomplex.
Nuclear Eukaryotic Initiation Factor 4e (Eif4e) Colocalizes with Splicing Factors in Speckles
Authors: Dostie Josée , Lejbkowicz Flavio , Sonenberg Nahum
Published: July 2002

The eukaryotic initiation factor 4E (eIF4E) plays a pivotal role in the control of protein synthesis. eIF4E binds to the mRNA 5′ cap structure, m7GpppN (where N is any nucleotide) and promotes ribosome binding to the mRNA. It was previously shown that a fraction of eIF4E localizes to the nucleus (Lejbkowicz, F., C. Goyer, A. Darveau, S. Neron, R. Lemieux, and N. Sonenberg. 1992. Proc. Natl. Acad. Sci. USA. 89:9612–9616). Here, we show that the nuclear eIF4E is present throughout the nucleoplasm, but is concentrated in speckled regions. Double label immunofluorescence confocal microscopy shows that eIF4E colocalizes with Sm and U1snRNP. We also demonstrate that eIF4E is specifically released from the speckles by the cap analogue m7GpppG in a cell permeabilization assay. However, eIF4E is not released from the speckles by RNase A treatment, suggesting that retention of eIF4E in the speckles is not RNA-mediated. 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) treatment of cells causes the condensation of eIF4E nuclear speckles. In addition, overexpression of the dual specificity kinase, Clk/Sty, but not of the catalytically inactive form, results in the dispersion of eIF4E nuclear speckles.
Rabies Virus-Induced Membrane Fusion Pathway
Authors: Gaudin Yves
Published: July 2002

Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fusion at lower membrane concentrations and this inhibition was compensated by the presence of oleic acid. However, under suboptimal fusion conditions, short chain LPCs, which were translocated in the inner leaflet of the membranes, considerably reduced the lag time preceding membrane merging, resulting in faster kinetics of fusion. This indicated that the rate limiting step for fusion is the formation of a fusion pore in a diaphragm of restricted hemifusion. The previously described cold-stabilized prefusion complex was also characterized. This intermediate is at a well-advanced stage of the fusion process when the hemifusion diaphragm is destabilized, but lipid mixing is still restricted, probably by a ring-like complex of glycoproteins. I provide evidence that this state has a dynamic character and that its lipid organization can reverse back to two lipid bilayers.
Fishing out proteins that bind to titin
Authors: Sanger Joseph W. , Sanger Jean M.
Published: July 2002

Another giant protein has been detected in cross-striated muscle cells. Given the name obscurin, it was discovered in a yeast two-hybrid screen in which the bait was a small region of titin that is localized near the Z-band. Obscurin is about 720 kD, similar in molecular weight to nebulin, but present at about one tenth the level (Young et al., 2001). Like titin, obscurin contains multiple immunoglobulin-like domains linked in tandem, but in contrast to titin it contains just two fibronectin-like domains. It also contains sequences that suggest obscurin may have roles in signal transduction. During embryonic development, its localization changes from the Z-band to the M-band. With these intriguing properties, obscurin may not remain obscure for long.
Quantitative Impact of Thymic Clonal Deletion on the T Cell Repertoire
Authors: van Meerwijk Joost P.M. , Marguerat Samuel , Lees Rosemary K. , Germain Ronald N. , Fowlkes B.J. , MacDonald H. Robson
Published: July 2002

Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras. This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.
P-Selectin Glycoprotein Ligand-1 (PSGL-1) on T Helper 1 but Not on T Helper 2 Cells Binds to P-Selectin and Supports Migration into Inflamed Skin
Authors: Borges Eric , Tietz Wolfgang , Steegmaier Martin , Moll Thomas , Hallmann Rupert , Hamann Alf , Vestweber Dietmar
Published: July 2002

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin–Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin–binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.
Impaired Bone Marrow Microenvironment and Immune Function in T Cell Protein Tyrosine Phosphatase–deficient Mice
Authors: You-Ten Kong E. , Muise Eric S. , Itié Annick , Michaliszyn Eva , Wagner John , Jothy Serge , Lapp Wayne S. , Tremblay Michel L.
Published: July 2002

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP–deficient mutant mice. The three genotypes (+/+, +/−, −/−) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3–5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP −/− animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.
Memory B Cells Are Biased Towards Terminal Differentiation: A Strategy That May Prevent Repertoire Freezing
Authors: Arpin Christophe , Banchereau Jacques , Liu Yong-Jun
Published: July 2002

Isolation of large numbers of surface IgD+CD38− naive and surface IgD−CD38− memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40–activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.
A Role for Fas in Negative Selection of Thymocytes In Vivo
Authors: Kishimoto Hidehiro , Surh Charles D. , Sprent Jonathan
Published: July 2002

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1–100 μg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vβ8+ cells at the level of the semi-mature population of HSAhi CD4+ 8− cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vβ8+ HSAhi CD4+ 8− thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (≤1 μg) but not by high doses (100 μg). Similar findings applied when T cell receptor transgenic mice were injected with specific peptide. These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.
T Cell Affinity Maturation by Selective Expansion during Infection
Authors: Busch Dirk H. , Pamer Eric G.
Published: July 2002

T lymphocyte recognition of infected cells is mediated by T cell receptors (TCRs) interacting with their ligands, self–major histocompatibility complex (MHC) molecules complexed with pathogen-derived peptides. Serial TCR interactions with potentially small numbers of MHC/ peptide complexes on infected cells transmit signals that result in T lymphocyte expansion and activation of effector functions. The impact of TCR affinity for MHC/peptide complexes on the rate or extent of in vivo T cell expansion is not known. Here we show that in vivo expansion of complex T cell populations after bacterial infection is accompanied by an increase in their overall affinity for antigen. T cell populations that have undergone additional rounds of in vivo expansion express a narrower range of TCRs, have increased sensitivity for antigen in cytotoxic T lymphocyte assays, and bind MHC/peptide complexes with greater affinity. The selective expansion of higher affinity T cells provides an in vivo mechanism for optimizing the early detection of infected cells.
SHP2-interacting Transmembrane Adaptor Protein (SIT), A Novel Disulfide-linked Dimer Regulating Human T Cell Activation
Authors: Marie-Cardine Anne , Kirchgessner Henning , Bruyns Eddy , Shevchenko Andrej , Mann Matthias , Autschbach Frank , Ratnofsky Sheldon , Meuer Stefan , Schraven Burkhart
Published: July 2002

T lymphocytes express several low molecular weight transmembrane adaptor proteins that recruit src homology (SH)2 domain–containing intracellular molecules to the cell membrane via tyrosine-based signaling motifs. We describe here a novel molecule of this group termed SIT (SHP2 interacting transmembrane adaptor protein). SIT is a disulfide-linked homodimeric glycoprotein that is expressed in lymphocytes. After tyrosine phosphorylation by src and possibly syk protein tyrosine kinases SIT recruits the SH2 domain–containing tyrosine phosphatase SHP2 via an immunoreceptor tyrosine-based inhibition motif. Overexpression of SIT in Jurkat cells downmodulates T cell receptor– and phytohemagglutinin-mediated activation of the nuclear factor of activated T cells (NF-AT) by interfering with signaling processes that are probably located upstream of activation of phospholipase C. However, binding of SHP2 to SIT is not required for inhibition of NF-AT induction, suggesting that SIT not only regulates NF-AT activity but also controls NF-AT unrelated pathways of T cell activation involving SHP2.
Microbial Epitopes Act as Altered Peptide Ligands to Prevent Experimental Autoimmune Encephalomyelitis
Authors: Ruiz Pedro J. , Garren Hideki , Hirschberg David L. , Langer-Gould Annette M. , Levite Mia , Karpuj Marcela V. , Southwood Scott , Sette Alessandro , Conlon Paul , Steinman Lawrence
Published: July 2002

Molecular mimicry refers to structural homologies between a self-protein and a microbial protein. A major epitope of myelin basic protein (MBP), p87–99 (VHFFKNIVTPRTP), induces experimental autoimmune encephalomyelitis (EAE). VHFFK contains the major residues for binding of this self-molecule to T cell receptor (TCR) and to the major histocompatibility complex. Peptides from papilloma virus strains containing the motif VHFFK induce EAE. A peptide from human papilloma virus type 40 (HPV 40) containing VHFFR, and one from HPV 32 containing VHFFH, prevented EAE. A sequence from Bacillus subtilis (RKVVTDFFKNIPQRI) also prevented EAE. T cell lines, producing IL-4 and specific for these microbial peptides, suppressed EAE. Thus, microbial peptides, differing from the core motif of the self-antigen, MBPp87–99, function as altered peptide ligands, and behave as TCR antagonists, in the modulation of autoimmune disease.
Impairment of Natural Killer Cytotoxic Activity and Interferon γ Production in Ccaat/Enhancer Binding Protein γ–Deficient Mice
Authors: Kaisho Tsuneyasu , Tsutsui Hiroko , Tanaka Takashi , Tsujimura Tohru , Takeda Kiyoshi , Kawai Taro , Yoshida Nobuaki , Nakanishi Kenji , Akira Shizuo
Published: July 2002

We have investigated in vivo roles of CCAAT/enhancer binding protein γ (C/EBPγ) by gene targeting. C/EBPγ-deficient (C/EBPγ2/−) mice showed a high mortality rate within 48 h after birth. To analyze the roles of C/EBPγ in lymphoid lineage cells, bone marrow chimeras were established. C/EBPγ2/− chimeras showed normal T and B cell development. However, cytolytic functions of their splenic natural killer (NK) cells after stimulation with cytokines such as interleukin (IL)-12, IL-18, and IL-2 were significantly reduced as compared with those of control chimera NK cells. In addition, the ability of C/EBPγ−/− chimera splenocytes to produce interferon (IFN)-γ in response to IL-12 and/or IL-18 was markedly impaired. NK cells could be generated in vitro with normal surface marker expression in the presence of IL-15 from C/EBPγ2/− newborn spleen cells. However, they also showed lower cytotoxic activity and IFN-γ production when stimulated with IL-12 plus IL-18 than control NK cells, as observed in C/EBPγ2/− chimera splenocytes. In conclusion, our study reveals that C/EBPγ is a critical transcription factor involved in the functional maturation of NK cells.
Distinct Signal Thresholds for the Unique Antigen Receptor–Linked Gene Expression Programs in Mature and Immature B Cells
Authors: Benschop Robert J. , Melamed Doron , Nemazee David , Cambier John C.
Published: July 2002

Although it is well established that immature B lymphocytes are exquisitely sensitive to tolerance induction compared with their mature counterparts, the molecular basis for this difference is unknown. We demonstrate that signaling by B cell antigen receptors leads to distinct and mutually exclusive biologic responses in mature and immature B cells: upregulation of CD86, CD69, and MHC class II in mature cells and receptor editing in immature cells. These responses can be induced simply by elevation of intracellular free calcium levels, as occurs after receptor aggregation. Importantly, induction of immature B cell responses requires much smaller increases in intracellular free calcium than does induction of mature B cell responses. These differences in biologic response and sensitivity to intracellular free calcium likely contributes to selective elimination at the immature stage of even those B cells that express low affinity for self-antigens.