bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse
Authors: Moriyama Hiroaki , Abiru Norio , Paronen Johanna , Sikora Kamila , Liu Edwin , Miao Dongmei , Devendra Devasenan , Beilke Joshua , Gianani Roberto , Gill Ron G. , Eisenbarth George S.
Published: September 2003

It has been reported that an insulin 2 gene knockout, when bred onto
nonobese diabetic (NOD) mice, accelerates diabetes. We produced insulin 1 gene
knockout congenic NOD mice. In contrast to insulin 2, diabetes and insulitis
were markedly reduced in insulin 1 knockout mice, with decreased and delayed
diabetes in heterozygous females and no insulitis and diabetes in most
homozygous female mice. Lack of insulitis was found for insulin 1 female
homozygous knockout mice at 8, 12, and 37 weeks of age. Despite a lack of
insulitis, insulin 1 homozygous knockout mice spontaneously expressed insulin
autoantibodies. Administration of insulin peptide B:9-23 of both insulin 1 and
2 to NOD mice induced insulin autoantibodies. Insulin 1 is not the only
lymphocytic target of NOD mice. Insulin 1 homozygous knockout islets, when
transplanted into recently diabetic wild-type NOD mice, became infiltrated
with lymphocytes and only transiently reversed diabetes. These observations
indicate that loss of either insulin gene can influence progression to
diabetes of NOD mice and suggest that the preproinsulin 1 gene is crucial for
the spontaneous development of NOD insulitis and diabetes.
Drosophila spalt/spalt-related
mutants exhibit Townes-Brocks' syndrome phenotypes
Authors: Dong P. D. Si , Todi Sokol V. , Eberl Daniel F. , Boekhoff-Falk Grace
Published: September 2003

Mutations in
SALL1
, the human homolog of the
Drosophila
spalt
gene, result in Townes-Brocks' syndrome, which is characterized by
hand/foot, anogenital, renal, and ear anomalies, including sensorineural
deafness.
spalt
genes encode zinc finger transcription factors that
are found in animals as diverse as worms, insects, and vertebrates. Here, we
examine the effect of losing both of the
spalt
genes,
spalt
and
spalt-related
, in the fruit fly
Drosophila melanogaster
,
and report defects similar to those in humans with Townes-Brocks' syndrome.
Loss of both
spalt
and
spalt-related
function in flies
yields morphological defects in the testes, genitalia, and the antenna.
Furthermore,
spalt
/
spalt-related
mutant antennae show severe
reductions in Johnston's organ, the major auditory organ in
Drosophila
. Electrophysiological analyses confirm that
spalt
/
spalt-related
mutant flies are deaf. These
commonalities suggest that there is functional conservation for
spalt
genes between vertebrates and insects.
HILS1 is a spermatid-specific linker histone H1-like protein implicated in chromatin remodeling during mammalian spermiogenesis
Authors: Yan Wei , Ma Lang , Burns Kathleen H. , Matzuk Martin M.
Published: September 2003

Chromatin remodeling is a major event that occurs during mammalian
spermiogenesis, the process of spermatid maturation into spermatozoa. Nuclear
condensation during spermiogenesis is accomplished by replacing somatic
histones (linker histone H1 and core histones) and the testis-specific linker
histone, H1t, with transition proteins and protamines. It has long been
thought that H1t is the only testis-specific linker histone, and that all
linker histones are replaced by transition proteins, and subsequently by
protamines during spermiogenesis. Here, we report the identification and
characterization of a spermatid-specific linker histone H1-like protein
(termed HILS1) in the mouse and human. Both mouse and human
HILS1
genes are located in intron 8 of the α-sarcoglycan genes. HILS1 is
highly expressed in nuclei of elongating and elongated spermatids (steps
9-15). HILS1 displays several biochemical properties that are similar to those
of linker histones, including the abilities to bind reconstituted
mononucleosomes, produce a chromatosome stop during micrococcal nuclease
digestion, and aggregate chromatin. Because HILS1 is expressed in late
spermatids that do not contain core histones, HILS1 may participate in
spermatid nuclear condensation through a mechanism distinct from that of
linker histones. Because HILS1 also belongs to the large winged helix/forkhead
protein superfamily, HILS1 may also regulate gene transcription, DNA repair,
and/or other chromosome processes during mammalian spermiogenesis.
An ultrasensitive Ca
2+
/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling
Authors: Bradshaw J. Michael , Kubota Yoshi , Meyer Tobias , Schulman Howard
Published: September 2003

The strength of hippocampal synapses can be persistently increased by
signals that activate Ca
2+
/calmodulin-dependent protein kinase II
(CaMKII). This CaMKII-dependent long-term potentiation is important for
hippocampal learning and memory. In this work we show that CaMKII exhibits an
intriguing switch-like activation that likely is important for changes in
synaptic strength. We found that autophosphorylation of CaMKII by itself
showed a steep dependence on Ca
2+
concentration [Hill coefficient
(n
H
) ≈ 5]. However, an even steeper Ca
2+
dependence
(n
H
≈ 8) was observed when autophosphorylation is balanced by
the dephosphorylation activity of protein phosphatase 1 (PP1). This
autophosphorylation-dephosphorylation switch was found to be reversible
because PP1 dephosphorylates CaMKII when Ca
2+
is lowered to a basal
level. The switch-like response of a CaMKII-PP1 system suggests that CaMKII
and PP1 may function together as a simple molecular device that specifically
translates only strong Ca
2+
signals into all-or-none potentiation
of individual hippocampal synapses.
Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte–specific expression in vivo
Authors: Zheng Qiping , Zhou Guang , Morello Roy , Chen Yuqing , Garcia-Rojas Xavier , Lee Brendan
Published: September 2003

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2+/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.
GPI-anchored uPAR requires Endo180 for rapid directional sensing during chemotaxis
Authors: Sturge Justin , Wienke Dirk , East Lucy , Jones Gareth E. , Isacke Clare M.
Published: September 2003

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in cell guidance and chemotaxis during normal and pathological events. uPAR is GPI-anchored and the mechanism by which it transmits intracellular polarity cues across the plasma membrane during directional sensing has not been elucidated. The constitutively recycling endocytic receptor Endo180 forms a trimolecular complex with uPAR in the presence of uPA, hence its alternate name uPAR-associated protein. Here, we demonstrate that Endo180 is a general promoter of random cell migration and has a more specific function in cell chemotaxis up a uPA gradient. Endo180 expression was demonstrated to enhance uPA-mediated filopodia production and promote rapid activation of Cdc42 and Rac. Expression of a noninternalizing Endo180 mutant revealed that promotion of random cell migration requires receptor endocytosis, whereas the chemotactic response to uPA does not. From these studies, we conclude that Endo180 is a crucial link between uPA–uPAR and setting of the internal cellular compass.
Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity
Authors: Wilson-Annan Julie , O'Reilly Lorraine A. , Crawford Simon A. , Hausmann George , Beaumont Jennifer G. , Parma Loes P. , Chen Lin , Lackmann Martin , Lithgow Trevor , Hinds Mark G. , Day Catherine L. , Adams Jerry M. , Huang David C.S.
Published: September 2003

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.
Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/β-catenin activity
Authors: Westfall Trudi A. , Brimeyer Ryan , Twedt Jen , Gladon Jean , Olberding Andrea , Furutani-Seiki Makoto , Slusarski Diane C.
Published: September 2003

We provide genetic evidence defining a role for noncanonical Wnt function in vertebrate axis formation. In zebrafish, misexpression of Wnt-4, -5, and -11 stimulates calcium (Ca2+) release, defining the Wnt/Ca2+ class. We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail. Embryos genetically depleted of both maternal and zygotic Wnt-5 product exhibit cell movement defects as well as hyperdorsalization and axis-duplication phenotypes. The dorsalized phenotypes result from increased β-catenin accumulation and activation of downstream genes. The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/β-catenin signaling.
Local signaling by the EGF receptor
Authors: Kempiak Stephan J. , Yip Shu-Chin , Backer Jonathan M. , Segall Jeffrey E.
Published: September 2003

Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.
Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3–independent β-catenin degradation
Authors: Topol Lilia , Jiang Xueyuan , Choi Hosoon , Garrett-Beal Lisa , Carolan Peter J. , Yang Yingzi
Published: September 2003

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing β-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of β-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and β-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote β-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.
Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and Gαq signaling
Authors: Jordan Theresa , Li Jinyuan , Jiang Hongbin , DiMario Joseph X.
Published: September 2003

Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and Gαq. Both mAchR and Gαq are abundant in medial adductor (MA) and PM fibers, and mAchR and Gαq interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased Gαq activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased Gαq activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, Gαq, and PKC.
Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding
Authors: McWhorter Michelle L. , Monani Umrao R. , Burghes Arthur H.M. , Beattie Christine E.
Published: September 2003

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by a loss of α motoneurons in the spinal cord. SMA is caused by low levels of the ubiquitously expressed survival motor neuron (Smn) protein. As it is unclear how low levels of Smn specifically affect motoneurons, we have modeled SMA in zebrafish, a vertebrate model organism with well-characterized motoneuron development. Using antisense morpholinos to reduce Smn levels throughout the entire embryo, we found motor axon–specific pathfinding defects. Reduction of Smn in individual motoneurons revealed that smn is acting cell autonomously. These results show for the first time, in vivo, that Smn functions in motor axon development and suggest that these early developmental defects may lead to subsequent motoneuron loss.
Differential requirements for AP-2 in clathrin-mediated endocytosis
Authors: Conner Sean D. , Schmid Sandra L.
Published: September 2003

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its μ2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in μ2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.
Inheritance of gene density–related higher order chromatin arrangements in normal and tumor cell nuclei
Authors: Cremer Marion , Küpper Katrin , Wagler Babett , Wizelman Leah , Hase Johann v. , Weiland Yanina , Kreja Ludwika , Diebold Joachim , Speicher Michael R. , Cremer Thomas
Published: September 2003

A gene density–related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119–1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei.
Differential αv integrin–mediated Ras-ERK signaling during two pathways of angiogenesis
Authors: Hood John D. , Frausto Ricardo , Kiosses William B. , Schwartz Martin A. , Cheresh David A.
Published: September 2003

Antagonists of αvβ3 and αvβ5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these αv integrins differentially contribute to sustained Ras-extracellular signal–related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or αvβ5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin αvβ3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-αvβ5 blocked upstream of Ras, whereas anti-αvβ3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/αvβ3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins αvβ3 and αvβ5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.
Telomere-independent homologue pairing and checkpoint escape of accessory ring chromosomes in male mouse meiosis
Authors: Voet Thierry , Liebe Bodo , Labaere Charlotte , Marynen Peter , Scherthan Harry
Published: September 2003

We analyzed transmission of a ring minichromosome (MC) through mouse spermatogenesis as a monosome and in the presence of a homologue. Mice, either monosomic or disomic for the MC, produced MC+ offspring. In the monosomic condition, most univalents underwent self-synapsis as indicated by STAG3, SCP3, and SCP1 deposition. Fluorescent in situ hybridization and three-dimensional fluorescence microscopy revealed that ring MCs did not participate in meiotic telomere clustering while MC homologues paired at the XY-body periphery. Self-synapsis of MC(s) and association with the XY-body likely allowed them to pass putative pachytene checkpoints. At metaphase I and II, MC kinetochores assembled MAD2 and BUBR1 spindle checkpoint proteins. Unaligned MCs triggered the spindle checkpoint leading to apoptosis of metaphase cells. Other MCs frequently associated with mouse pericentric heterochromatin, which may have allowed them to pass the spindle checkpoint. Our findings indicate a telomere-independent mechanism for pairing of mammalian MCs, illuminate escape routes to meiotic checkpoints, and give clues for genetic engineering of germ line–permissive chromosomal vectors.
Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis
Authors: Sekiya-Kawasaki Mariko , Groen Aaron Chris , Cope M. Jamie T.V. , Kaksonen Marko , Watson Hadiya A. , Zhang Chao , Shokat Kevan M. , Wendland Beverly , McDonald Kent L. , McCaffery J. Michael , Drubin David G.
Published: September 2003

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch–related process, and propose that Prk1p negatively regulates the actin assembly–stimulating activity of endocytic proteins.
Clathrin-mediated endocytosis in AP-2–depleted cells
Authors: Motley Alison , Bright Nicholas A. , Seaman Matthew N.J. , Robinson Margaret S.
Published: September 2003

We have used RNA interference to knock down the AP-2 μ2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2–depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2–depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2–depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.
When Wnts antagonize Wnts
Authors: Weidinger Gilbert , Moon Randall T.
Published: September 2003

Secreted Wnt ligands appear to activate a variety of signaling pathways. Two papers in this issue now present genetic evidence that “noncanonical” Wnt signaling inhibits the “canonical” Wnt/β-catenin pathway. Westfall et al. (2003a) show that zebrafish embryos lacking maternal Wnt-5 function are dorsalized due to ectopic activation of β-catenin, whereas Topol et al. (2003) report that chondrogenesis in the distal mouse limb bud depends on inhibition of Wnt/β-catenin signaling by a paralogue of Wnt-5. These studies present the first genetic confirmation of the previous hypothesis that vertebrate Wnt signaling pathways can act in an antagonistic manner.
The Inducible CXCR3 Ligands Control Plasmacytoid Dendritic Cell Responsiveness to the Constitutive Chemokine Stromal Cell–derived Factor 1 (SDF-1)/CXCL12
Authors: Vanbervliet Béatrice , Bendriss-Vermare Nathalie , Massacrier Catherine , Homey Bernhard , de Bouteiller Odette , Brière Francine , Trinchieri Giorgio , Caux Christophe
Published: September 2003

The recruitment of selected dendritic cell (DC) subtypes conditions the class of the immune response. Here we show that the migration of human plasmacytoid DCs (pDCs), the blood natural interferon α–producing cells, is induced upon the collective action of inducible and constitutive chemokines. Despite expression of very high levels of CXCR3, pDCs do not respond efficiently to CXCR3 ligands. However, they migrate in response to the constitutive chemokine stromal cell–derived factor 1 (SDF-1)/CXCL12 and CXCR3 ligands synergize with SDF-1/CXCL12 to induce pDC migration. This synergy reflects a sensitizing effect of CXCR3 ligands, which, independently of a gradient and chemoattraction, decrease by 20–50-fold the threshold of sensitivity to SDF-1/CXCL12. Thus, the ability of the constitutive chemokine SDF-1/CXCL12 to induce pDC recruitment might be controlled by CXCR3 ligands released during inflammation such as in virus infection. SDF-1/CXCL12 and the CXCR3 ligands Mig/CXCL9 and ITAC/CXCL1 display adjacent expression both in secondary lymphoid organs and in inflamed epithelium from virus-induced pathologic lesions. Because pDCs express both the lymph node homing molecule l-selectin and the cutaneous homing molecule cutaneous lymphocyte antigen, the cooperation between inducible CXCR3 ligands and constitutive SDF-1/CXCL12 may regulate recruitment of pDCs either in lymph nodes or at peripheral sites of inflammation.
The Distinct Contributions of Murine T Cell Receptor (TCR)γδ+ and TCRαβ+ T Cells to Different Stages of Chemically Induced Skin Cancer
Authors: Girardi Michael , Glusac Earl , Filler Renata B. , Roberts Scott J. , Propperova Iva , Lewis Julia , Tigelaar Robert E. , Hayday Adrian C.
Published: September 2003

Epithelial tissues in which carcinomas develop often contain systemically derived T cell receptor (TCR)αβ+ cells and resident intraepithelial lymphocytes that are commonly enriched in TCRγδ+ cells. Recent studies have demonstrated that γδ cells protect the host against chemically induced cutaneous malignancy, but the role of αβ T cells has been enigmatic, with both protective and tumor-enhancing contributions being reported in different systems. This study aims to clarify the contributions of each T cell type to the regulation of squamous cell carcinoma induced in FVB mice by a two-stage regimen of 7,12-dimethylbenz[a]anthracene initiation followed by repetitive application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. This protocol permits one to monitor the induction of papillomas and the progression of those papillomas to carcinomas. The results show that whereas γδ cells are strongly protective, the nonredundant contributions of αβ T cells to the host's protection against papillomas are more modest. Furthermore, at both high and low doses of carcinogens, αβ T cells can contribute to rather than inhibit the progression of papillomas to carcinomas. As is likely to be the case in humans, this study also shows that the contribution of T cells to tumor immunosurveillance is regulated by modifier genes.
Impaired T Cell Death and Lupus-like Autoimmunity in T Cell–specific Adapter Protein–deficient Mice
Authors: Drappa Jorn , Kamen Lynn A. , Chan Elena , Georgiev Maria , Ashany Dalit , Marti Francesc , King Philip D.
Published: September 2003

T cell–specific adaptor protein (TSAd) is a T lineage–restricted signaling adaptor molecule that is thought to participate in the assembly of intracellular signaling complexes in T cells. Previous studies of TSAd-deficient mice have revealed a role for TSAd in the induction of T cell interleukin 2 secretion and proliferation. We now show that TSAd-deficient mice are susceptible to lupus-like autoimmune disease. On the nonautoimmune-prone C57BL/6 genetic background, TSAd deficiency results in hypergammaglobulinemia that affects all immunoglobulin (Ig)G subclasses. Older C57BL/6 TSAd-deficient mice (1 yr of age) accumulate large numbers of activated T and B cells in spleen, produce autoantibodies against a variety of self-targets including single stranded (ss) and double stranded (ds) DNA, and, in addition, develop glomerulonephritis. We further show that immunization of younger C57BL/6 TSAd-deficient mice (at age 2 mo) with pristane, a recognized nonspecific inflammatory trigger of lupus, results in more severe glomerulonephritis compared with C57BL/6 controls and the production of high titer ss and ds DNA antibodies of the IgG subclass that are not normally produced by C57BL/6 mice in this model. The development of autoimmunity in TSAd-deficient mice is associated with defective T cell death in vivo. These findings illustrate the role of TSAd as a critical regulator of T cell death whose absence promotes systemic autoimmunity.
The Strategy of T Cell Antigen-presenting Cell Encounter in Antigen-draining Lymph Nodes Revealed by Imaging of Initial T Cell Activation
Authors: Bajénoff Marc , Granjeaud Samuel , Guerder Sylvie
Published: September 2003

The development of an immune response critically relies on the encounter of rare antigen (Ag)-specific T cells with dendritic cells (DCs) presenting the relevant Ag. How two rare cells find each other in the midst of irrelevant other cells in lymph nodes (LNs) is unknown. Here we show that initial T cell activation clusters are generated near high endothelial venules (HEVs) in the outer paracortex of draining LNs by retention of Ag-specific T cells as they exit from HEVs. We further show that tissue-derived DCs preferentially home in the vicinity of HEVs, thus defining the site of cluster generation. At this location DCs efficiently scan all incoming T cells and selectively retain those specific for the major histocompatibility complex–peptide complexes the DCs present. Such strategic positioning of DCs on the entry route of T cells into the paracortex may foster T cell–DC encounter and thus optimize initial T cell activation in vivo.
Inhibition of Respiration by Nitric Oxide Induces aMycobacterium tuberculosisDormancy Program
Authors: Voskuil Martin I. , Schnappinger Dirk , Visconti Kevin C. , Harrell Maria I. , Dolganov Gregory M. , Sherman David R. , Schoolnik Gary K.
Published: September 2003

An estimated two billion persons are latently infected with Mycobacterium tuberculosis. The host factors that initiate and maintain this latent state and the mechanisms by which M. tuberculosis survives within latent lesions are compelling but unanswered questions. One such host factor may be nitric oxide (NO), a product of activated macrophages that exhibits antimycobacterial properties. Evidence for the possible significance of NO comes from murine models of tuberculosis showing progressive infection in animals unable to produce the inducible isoform of NO synthase and in animals treated with a NO synthase inhibitor. Here, we show that O2 and low, nontoxic concentrations of NO competitively modulate the expression of a 48-gene regulon, which is expressed in vivo and prepares bacilli for survival during long periods of in vitro dormancy. NO was found to reversibly inhibit aerobic respiration and growth. A heme-containing enzyme, possibly the terminal oxidase in the respiratory pathway, likely senses and integrates NO and O2 levels and signals the regulon. These data lead to a model postulating that, within granulomas, inhibition of respiration by NO production and O2 limitation constrains M. tuberculosis replication rates in persons with latent tuberculosis.
The Four Distal Tyrosines Are Required for LAT-dependent Signaling in FcεRI-mediated Mast Cell Activation
Authors: Saitoh Shin-ichiroh , Odom Sandra , Gomez Gregorio , Sommers Connie L. , Young Howard A. , Rivera Juan , Samelson Lawrence E.
Published: September 2003

The linker for activation of T cells (LAT) is an adaptor protein critical for FcεRI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and FcεRI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C–γ1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in FcεRI-mediated mast cell activation, bone marrow–derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow–derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C–γ-binding tyrosine had a significant effect on antigen-induced histamine release.
Thymic Medullary Epithelial Cell Differentiation, Thymocyte Emigration, and the Control of Autoimmunity Require Lympho–Epithelial Cross Talk via LTβR
Authors: Boehm Thomas , Scheu Stefanie , Pfeffer Klaus , Bleul Conrad C.
Published: September 2003

Thymocytes depend on the interaction with thymic epithelial cells for the generation of a diverse, nonautoreactive T cell repertoire. In turn, thymic epithelial cells acquire their three-dimensional cellular organization via instructive signals from developing thymocytes. The nature of these signals has been elusive so far. We show that thymocytes and medullary epithelial cells (MECs) communicate via the lymphotoxin β receptor (LTβR) signaling axis. Normal differentiation of thymic MECs requires LTβR ligand on thymocytes and LTβR together with nuclear factor–κB-inducing kinase (Nik) in thymic epithelial cells. Impaired lympho–epithelial cross talk in the absence of the LTβR causes aberrant differentiation and reduced numbers of thymic MECs, leads to the retention of mature T lymphocytes, and is associated with autoimmune phenomena, suggesting an unexpected role for LTβR signaling in central tolerance induction.
Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages
Authors: Schnappinger Dirk , Ehrt Sabine , Voskuil Martin I. , Liu Yang , Mangan Joseph A. , Monahan Irene M. , Dolganov Gregory , Efron Brad , Butcher Philip D. , Nathan Carl , Schoolnik Gary K.
Published: September 2003

Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2–deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon γ– and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and β-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of σE-dependent, sodium dodecyl sulfate–regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.
A haplotype-based molecular analysis of CFTR mutations associated with respiratory and pancreatic diseases
Authors: Lee Ji Hyun , Choi Ji Ha , Namkung Wan , Hanrahan John W. , Chang Joon , Song Si Young , Park Seung Woo , Kim Dong Soo , Yoon Joo-Heon , Suh Yousin , Jang In-Jin , Nam Joo Hyun , Kim Sung Joon , Cho Mi-Ook , Lee Jong-Eun , Kim Kyung Hwan , Lee Min Goo
Published: September 2003

Aberrant membrane transport caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with a wide spectrum of respiratory and digestive diseases as well as cystic fibrosis. Using a gene scanning method, we found 11 polymorphisms and mutations of the CFTR gene in the Korean population. Individual variants at these sites were analyzed by conventional DNA screening in 117 control and 75 patients having bronchiectasis or chronic pancreatitis. In a haplotype determination based on a Bayesian algorithm, 15 haplotypes were assembled in the 192 individuals tested. Several haplotypes, especially with Q1352H, IVS8 T5, and E217G, were found to have disease associations in a case–control study. Notably, a common polymorphism of M470V appears to affect the intensity of the disease association. Among the two haplotypes having IVS8 T5, the T5-V470 haplotype showed higher disease association than the T5-M470 haplotype. In addition, a Q1352H mutation found in a V470 background showed the strongest disease association. The physiological significances of the identified mutations were rigorously analyzed. Non-synonymous E217G and Q1352H mutations in the M470 background caused a 60–80% reduction in CFTR-dependent Cl− currents and HCO3−-transport activities. Surprisingly, the additional M470V polymorphic variant with the Q1352H mutation completely abolished CFTR-dependent anion transport activities. These findings provide the first evidence on the importance of CFTR mutations in the Asian population. Importantly, the results also reveal that interactions between multiple genetic variants in cis affect the final function of the gene products.
Human microvascular endothelial cells resist Shiga toxins by IFN-γ treatment in vitro
Authors: Yoshida Tomoaki , Sugiyama Tsuyoshi , Koide Naoki , Mori Isamu , Yokochi Takashi
Published: August 2003

Shiga toxins (Stxs) produced by enterohaemorrhagicEscherichia coliorShigella dysenteriaedamage human endothelial cells predominantly in cooperation with pro-inflammatory cytokines, such as TNF-α. However, in this study,in vitroIFN-γpre-treatment resulted in human lung microvascular endothelial cells becoming over 10 000-fold less sensitive to Stxs. In contrast, in their basal condition, they were extremely sensitive to Stxs. Interestingly, TNF-αaddition to IFN-γreverted the Stx-resistant phenotype, which corresponded with its well-established enhancing effect on Stx toxicity. Toxin binding to the cell was barely affected by IFN-γ. Also, the toxin uptake in the Stx-resistant phenotype was more than 100-fold greater than that of normal cells, when compared at Stx concentrations resulting in equivalent degrees of cell damage. Protein synthesis was inhibited by nearly 90 % in the Stx-resistant phenotype after 24 h toxin exposure. This indicated that the intracellular toxin was active as anN-glycosidase, while cells were still over 60 % viable, suggesting a possible unknown cytotoxic function of Stx. In conclusion, this study shows a unique effect of IFN-γin the suppression of the toxicity of Stxs in a human microvascular endothelial cell model and the involvement of a novel mechanism in this suppression.
The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium
Authors: Tanaka Kousei , Kobayashi Kazuo , Ogasawara Naotake
Published: August 2003

The Gram-positive bacterium Bacillus subtilis has a complete set of enzymes for the tricarboxylic acid (TCA) cycle and can grow aerobically using most of the TCA cycle intermediates (malate, fumarate, succinate and citrate) as a sole carbon source. The B. subtilis genome sequence contains three paralogous two-component regulatory systems, CitST, DctSR and YufLM. CitST and DctSR activate the expression of a transporter of the Mg2+–citrate complex (CitM) and a fumarate and succinate transporter (DctP), respectively. These findings prompted an investigation of whether the YufL sensor and its cognate regulator, YufM, play a role in malate uptake. This paper reports that the YufM regulator shows in vitro binding to the promoter region of two malate transporter genes, maeN and yflS, and is responsible for inducing their expression in vivo. It was also found that inactivation of the yufM or maeN genes resulted in bacteria that could not grow in a minimal salts medium containing malate as a sole carbon source, indicating that the induction of the MaeN transporter by the YufM regulator is essential for the utilization of malate as a carbon source. Inactivation of the yufL gene resulted in the constitutive expression of MaeN. This expression was suppressed by reintroduction of the kinase domain of YufL, indicating that the YufL sensor is required for proper signal detection and signalling specificity. The authors propose that a phosphatase activity of YufL plays an important role in the YufLM two-component regulatory system. The studies reported here have revealed that members of a set of paralogous two-component regulatory systems in B. subtilis, CitST, DctSR and YufLM, are involved in a related function – uptake (and metabolism) of the TCA cycle intermediates – but with distinct substrate specificities.
Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1
Authors: Kim Eun-Jin , Sabra Wael , Zeng An-Ping
Published: August 2003

Pseudomonas aeruginosaPAO1 was recently found to exhibit two remarkable physiological responses to oxidative stress: (1) a strong reduction in the efficiency of oxygen transfer from the gas phase into the liquid phase, thus causing oxygen limitation in the culture and (2) formation of a clear polysaccharide capsule on the cell surface. In this work, it has been shown that the iron concentration in the culture plays a crucial role in evoking these phenomena. The physiological responses of twoP. aeruginosaPAO1 isolates (NCCB 2452 and ATCC 15692) were examined in growth media with varied iron concentrations. In a computer-controlled bioreactor cultivation system for controlled dissolved oxygen tension (pO2), a strong correlation between the exhaustion of iron and the onset of oxygen limitation was observed. The oxygen transfer rate of the culture, characterized by the volumetric oxygen transfer coefficient,kLa, significantly decreased under iron-limited conditions. The formation of alginate and capsule was more strongly affected by iron concentration than by oxygen concentration. The reduction of the oxygen transfer rate and the subsequent oxygen limitation triggered by iron deficiency may represent a new and efficient way forP. aeruginosaPAO1 to adapt to growth conditions of iron limitation. Furthermore, the secretion of proteins into the culture medium was strongly enhanced by iron limitation. The formation of the virulence factor elastase and the iron chelators pyoverdine and pyochelin also significantly increased under iron-limited conditions. These results have implications for lung infection of cystic fibrosis patients byP. aeruginosain view of the prevalence of iron limitation at the site of infection and the respiratory failure leading to death.
Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis
Authors: Inácio José Manuel , Costa Carla , de Sá-Nogueira Isabel
Published: August 2003

The Bacillus subtilis proteins involved in the utilization of l-arabinose are encoded by the araABDLMNPQ–abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by l-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription ‘roadblocking’ of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.
The variation of dTDP-l-rhamnose pathway genes in Vibrio cholerae
Authors: Li Qun , Hobbs Matthew , Reeves Peter R.
Published: August 2003

The genetic variation in the dTDP-l-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5′ end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5′ end rmlB gene, but very divergent and strain-specific sequences at the 3′ end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5′ and 3′ parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.