bluuebunny/crossref_metadata_2025 huggingface dataset:
Electroconvulsive shock and lidocaine reveal rapid consolidation of spatial working memory in the water maze.
Authors: Bohbot V , Otáhal P , Liu Z , Nadel L , Bures J
Published: July 2002

Head trauma leading to concussion and electroconvulsive shock (ECS) in humans causes amnesia for events that occurred shortly before the injury (retrograde amnesia). The present experiment investigated the amnesic effect of lidocaine and ECS in 25 rats trained on a working memory version of the Morris water task. Each day, the escape platform was moved to a new location; learning was evidenced by a decrease in the latency to find the platform from the first to the second trial. "Consolidation" of this newly encoded spatial engram was disrupted by bilateral inactivation of the dorsal hippocampus with 1 microliter of 4% lidocaine applied as soon as possible after the first trial. When trial 2 was given after recovery from the lidocaine (30 min after the injection), a normal decrease in latency indicated that the new engram was not disrupted. When trial 2 was given under the influence of lidocaine (5 min after injection), absence of latency decrease demonstrated both the success of the inactivation and the importance of hippocampus for the task. To examine the role of events immediately after learning, ECS (30 or 100 mA, 50 Hz, 1.2 sec) was applied 0 sec to 45 sec after a single escape to the new platform location. A 2-h delay between ECS and trial 2 allowed the effects of ECS to dissipate. ECS applied 45 sec or 30 sec after trial 1 caused no retrograde amnesia: escape latencies on trial 2 were the same as in control rats. However, ECS applied 0 sec or 15 sec after trial 1 induced clear retrograde amnesia: escape latencies on trial 2 were no shorter than on trial 1. It is concluded that the consolidation of a newly formed memory for spatial location can only be disrupted by ECS within 30 sec after learning.
Dominant-negative p53 mutations selected in yeast hit cancer hot spots.
Authors: Brachmann R K , Vidal M , Boeke J D
Published: July 2002

Clinically important mutant p53 proteins may be tumorigenic through a dominant-negative mechanism or due to a gain-of-function. Examples for both hypotheses have been described; however, it remains unclear to what extent they apply to TP53 mutations in general. Here it is shown that the mutational spectrum of dominant-negative p53 mutants selected in a novel yeast assay correlates tightly with p53 mutations in cancer. Two classes of dominant-negative mutations are described; the more dominant one affects codons that are essential for the stabilization of the DNA-binding surface of the p53 core domain and for the direct interaction of p53 with its DNA binding sites. These results predict that the vast majority of TP53 mutations leading to cancer do so in a dominant-negative fashion.
Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300
Authors: Horvai Andrew E. , Xu Lan , Korzus Edward , Brard Gyan , Kalafus Daniel , Mullen Tina-Marie , Rose David W. , Rosenfeld Michael G. , Glass Christopher K.
Published: July 2002

We report that interferon γ (IFN-γ) inhibits transcription of the macrophage scavenger receptor gene by antagonizing the Ras-dependent activities of AP-1 and cooperating ets domain transcription factors, apparently as a result of competition between AP-1/ets factors and activated STAT1 for limiting amounts of CBP and p300. Consistent with this model, STAT1α interacts directly with CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies blocks transcriptional responses to IFN-γ. Cells lacking STAT1 fail to inhibit AP-1/ets activity, and overexpression of CBP both potentiates IFN-γ-dependent transcription and relieves AP-1/ets repression. Thus, CBP and p300 integrate both positive and negative effects of IFN-γ on gene expression by serving as essential coactivators of STAT1α, modulating gene-specific responses to simultaneous activation of two or more signal transduction pathways.
Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase
Authors: Sun Xiaolin , Bognar Andrew L. , Baker Edward N. , Smith Clyde A.
Published: July 2002

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from
Lactobacillus casei
. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.
SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis
Authors: Ganiatsas Soula , Kwee Lia , Fujiwara Yuko , Perkins Andrew , Ikeda Tohru , Labow Mark A. , Zon Leonard I.
Published: July 2002

SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate
in vitro
in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1
in vivo
, we studied stress-induced signaling in SEK1
−/−
embryonic stem and fibroblast cells and evaluated the phenotype of SEK1
−/−
mouse embryos during development. Studies of SEK1
−/−
embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1
−/−
fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor α and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1
−/−
fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.
Growth stimulation of primary B cell precursors by the anti-phosphatase Sbf1
Authors: De Vivo Immaculata , Cui Xiangmin , Domen Jos , Cleary Michael L.
Published: July 2002

SET binding factor 1 (Sbf1) was originally discovered by virtue of its interaction with a highly conserved motif (the SET domain) of unknown function in the protooncoprotein homolog ofDrosophila trithorax,Hrx. Sbf1 shares extensive sequence similarity with myotubularin, a dual specificity phosphatase (dsPTPase) that is mutated in a subset of patients with inherited myopathies. Both Sbf1 and myotubularin interact with the SET domains of Hrx and other epigenetic regulatory proteins, but Sbf1 lacks phosphatase activity due to several evolutionarily conserved amino acid changes in its structurally preserved catalytic pocket. Thus, Sbf1 has features of an anti-phosphatase that could competitively antagonize dsPTPases; however thein vivorole for such factors remains unknown. Given its ability to physically interact with Hrx, a developmental regulator subject to translocation-induced mutations in B cell precursor leukemias, the current studies were undertaken to assess the effects of Sbf1 on lymphopoiesis. After infection with recombinant Sbf1 retroviruses, bone marrow cells were plated under Whitlock-Witte conditions for long-term culture of B lineage cells. Sbf1-expressing cells rapidly dominated the cultures resulting in clonal outgrowths of B cell progenitors that retained a dependence on their primary bone marrow-derived stroma for continuous growthin vitro. Structure/function analyses demonstrated that the SET interaction domain of Sbf1 was necessary and sufficient for growth alterations of B cell progenitors. These observations support a model in which Sbf1 functions as a SET domain-dependent positive regulator of growth-inducing kinase signaling pathways that impinge on SET domain proteins. SET domain-dsPTPase interactions appear to be critically important for regulating the growth properties of B cell progenitors.
Association of
CYP
3
A
4
genotype with treatment-related leukemia
Authors: Felix Carolyn A. , Walker Amy H. , Lange Beverly J. , Williams Terence M. , Winick Naomi J. , Cheung Nai-Kong V. , Lovett Brian D. , Nowell Peter C. , Blair Ian A. , Rebbeck Timothy R.
Published: July 2002

Epipodophyllotoxins are associated with leukemias characterized by translocations of the
MLL
gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5′ promoter region of the
CYP3A4
gene (
CYP3A4-V
) that might alter the metabolism of anticancer drugs. We examined 99
de novo
and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the
CYP3A4-V
. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99
de novo
(19%) and 1 of 30 treatment-related (3%) leukemias carried the
CYP3A4-V
(
P
= 0.026; Fisher’s Exact Test, FET). Nine of 42
de novo
leukemias with
MLL
gene translocations (21%), and 0 of 22 treatment-related leukemias with
MLL
gene translocations carried the
CYP3A4-V
(
P
= 0.016, FET). This relationship remained significant when 19 treatment-related leukemias with
MLL
gene translocations that followed epipodophyllotoxin exposure were compared with the same 42
de novo
cases (
P
= 0.026, FET). These data suggest that individuals with
CYP3A4-W
genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The
CYP3A4-W
genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone.
Mutation of the Sry-related
Sox10
gene in
Dominant megacolon
, a mouse model for human Hirschsprung disease
Authors: Herbarth Beate , Pingault Veronique , Bondurand Nadege , Kuhlbrodt Kirsten , Hermans-Borgmeyer Irm , Puliti Aldamaria , Lemort Nicole , Goossens Michel , Wegner Michael
Published: July 2002

The spontaneous mouse mutant
Dominant megacolon
(
Dom
) is a valuable model for the study of human congenital megacolon (Hirschsprung disease). Here we report that the defect in the
Dom
mouse is caused by mutation of the gene encoding the Sry-related transcription factor Sox10. This assignment is based on (
i
) colocalization of the
Sox10
gene with the
Dom
mutation on chromosome 15; (
ii
) altered
Sox10
expression in the gut and in neural-crest derived structures of cranial ganglia of
Dom
mice; (
iii
) presence of a frameshift in the Sox10 coding region, and (
iv
) functional inactivation of the resulting truncated protein. These results identify the transcriptional regulator Sox10 as an essential factor in mouse neural crest development and as a further candidate gene for human Hirschsprung disease, especially in cases where it is associated with features of Waardenburg syndrome.
Titanospirillum velox
: A huge, speedy, sulfur-storing spirillum from Ebro Delta microbial mats
Authors: Guerrero Ricardo , Haselton Aaron , Solé Mónica , Wier Andrew , Margulis Lynn
Published: July 2002

A long (20–30 μm), wide (3–5 μm) microbial-mat bacterium from the Ebro Delta (Tarragona, Spain) was grown in mixed culture and videographed live. Intracellular elemental sulfur globules and unique cell termini were observed in scanning-electron-microprobe and transmission-electron micrographs. A polar organelle underlies bundles of greater than 60 flagella at each indented terminus. These Gram-negative bacteria bend, flex, and swim in a spiral fashion; they translate at speeds greater than 10 body lengths per second. The large size of the spirillum permits direct observation of cell motility in single individual bacteria. After desiccation (i.e., absence of standing water for at least 24 h), large populations developed in mat samples remoistened with sea water. Ultrastructural observations reveal abundant large sulfur globules irregularly distributed in the cytoplasm. A multilayered cell wall, pliable and elastic yet rigid, distends around the sulfur globules. Details of the wall, multiflagellated termini, and large cytoplasmic sulfur globules indicate that these fast-moving spirilla are distinctive enough to warrant a genus and species designation:
Titanospirillum velox
genus nov., sp. nov. The same collection techniques at a similar habitat in the United States (Plum Island, northeast Essex County, Massachusetts) also yielded large populations of the bacterium among purple phototrophic and other inhabitants of sulfurous microbial-mat muds. The months-long survival of
T. velox
from Spain and from the United States in closed jars filled with mud taken from both localities leads us to infer that this large spirillum has a cosmopolitan distribution.
From snapshot to movie: φ analysis of protein folding transition states taken one step further
Authors: Ternström Tomas , Mayor Ugo , Akke Mikael , Oliveberg Mikael
Published: July 2002

Kinetic anomalies in protein folding can result from changes of the
kinetic ground states (D, I, and N), changes of the protein folding
transition state, or both. The 102-residue protein U1A has a
symmetrically curved chevron plot which seems to result mainly from
changes of the transition state. At low concentrations of denaturant
the transition state occurs early in the folding reaction, whereas at
high denaturant concentration it moves close to the native structure.
In this study we use this movement to follow continuously the formation
and growth of U1A's folding nucleus by φ analysis. Although U1A's
transition state structure is generally delocalized and displays a
typical nucleation–condensation pattern, we can still resolve a
sequence of folding events. However, these events are sufficiently
coupled to start almost simultaneously throughout the transition state
structure.
The N-terminal 209-aa domain of high molecular- weight 4.1R isoforms abrogates 4.1R targeting to the nucleus
Authors: Luque Carlos M. , Lallena María-José , Pérez-Ferreiro Carmen M. , de Isidro Yolanda , De Cárcer Guillermo , Alonso Miguel A. , Correas Isabel
Published: July 2002

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase–PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of ≈145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.
A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1
Authors: Ashery Uri , Koch Henriette , Scheuss Volker , Brose Nils , Rettig Jens
Published: July 2002

ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release
in vivo
is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the
Xenopus
neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.
Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus
Authors: Tomonaga Keizo , Kobayashi Takeshi , Lee Byeong- Jae , Watanabe Makiko , Kamitani Wataru , Ikuta Kazuyoshi
Published: July 2002

Borna disease virus (BDV) is a nonsegmented negative-strand RNA
virus that belongs to the
Mononegavirales
. Unlike other
animal viruses of this order, BDV replicates and transcribes in the
nucleus of infected cells. Previous studies have shown that BDV uses
RNA splicing machinery for its mRNA expression. In the present study,
we identified spliced RNAs that use an alternative 3′ splice site, SA3,
in BDV-infected cell lines as well as infected animal brain cells.
Transient transfection analysis of cDNA clones of BDV RNA revealed that
although SA3 is a favorable splice site in mammalian cells, utilization
of SA3 is negatively regulated in infected cells. This negative
splicing activity of the SA3 site is regulated by a putative cis-acting
region, the exon splicing suppressor (ESS), within the polymerase exon
of BDV. The BDV ESS contains similar motifs to other known ESSs present
in viral and cellular genes. Furthermore, our results indicated that a
functional polyadenylation signal just upstream of the BDV ESS is also
involved in the regulation of alternative splicing of BDV. These
observations represent the first documentation of complex RNA splicing
in animal RNA viruses and also provide new insight into the mechanism
of regulation of alternative splicing in animal viruses.
Evolutionary conservation of apoptosis mechanisms: Lepidopteran and baculoviral inhibitor of apoptosis proteins are inhibitors of mammalian caspase-9
Authors: Huang Qihong , Deveraux Quinn L. , Maeda Susumu , Salvesen Guy S. , Stennicke Henning R. , Hammock Bruce D. , Reed John C.
Published: July 2002

We cloned a new inhibitor of apoptosis protein (IAP) homolog, SfIAP, from
Spodoptera frugiperda
Sf-21 cells, a host of insect baculoviruses. SfIAP contains two baculovirus IAP repeat domains followed by a RING domain. SfIAP has striking amino acid sequence similarity with baculoviral IAPs, CpIAP and OpIAP, suggesting that baculoviral IAPs may be host-derived genes. SfIAP and baculoviral CpIAP inhibit Bax but not Fas-induced apoptosis in human cells. Their apoptosis-suppressing activity in mammalian cells requires both baculovirus IAP repeat and RING domains. Further biochemical data suggest that SfIAP and CpIAP are specific inhibitors of mammalian caspase-9, the pinnacle caspase in the mitochondria/cytochrome
c
pathway for apoptosis, but are not inhibitors of downstream caspase-3 and caspase-7. Thus the mechanisms by which insect and baculoviral IAPs suppress apoptosis may involve inhibition of an insect caspase-9 homologue. Peptides representing the IAP-binding domain of the
Drosophila
cell death protein Grim abrogated human caspase suppression by SfIAP and CpIAP, implying evolutionary conservation of the functions of IAPs and their inhibitors.
Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes
Authors: Garagna Silvia , Marziliano Nicola , Zuccotti Maurizio , Searle Jeremy B. , Capanna Ernesto , Redi Carlo Alberto
Published: July 2002

In mammals, Robertsonian (Rb) translocation (the joining of two telo/acrocentric chromosomes at their centromere to form a metacentric) is the most effective process in chromosomal evolution leading to speciation; its occurrence also affects human health (through the induction of trisomies) and the fertility of farm animals. To understand the mechanism of Rb translocation, we used the house mouse as a model system and studied the organization of pericentromeric satellite DNAs (satDNA) of telocentrics and Rb chromosomes, both minor and major satDNA. The chromosome-orientation fluorescencein situhybridization (CO-FISH) technique was used to analyze the major satDNA. To detect the very small amount of minor satDNA, a procedure was developed that combines CO-FISH with primedin situlabeling and conventional FISH and is five times more sensitive than the CO-FISH procedure alone. It was found that both the major and the minor satDNA tandem repeats are oriented head-to-tail in telocentric and Rb chromosomes, and their polarity is always the same relative to the centromere. We suggest that all tandemly repetitive satDNAs in a species probably are locked into such a symmetry constraint as a universal consequence of chromosomal evolution. Rb translocation breakpoints were found localized within the minor satDNA of telocentrics, and these sequences contributed symmetrically to the formation of the centromeric region of the Rb chromosomes. These results are important for an understanding of the geometry of Rb translocations and suggest the study of DNA orientation as a new tool for investigating these rearrangements.
Language and Ethnicity Among Students in Teschen Silesia*
Authors: Hannan Kevin
Published: July 2002

This study examines attitudes towards language and ethnicity among nearly four hundred high-school students in the Teschen region of the Czech Republic and Poland. The borders of Poland, the Czech Republic, and Slovakia meet in this region of mountains, coal mines, and heavy industry which earlier in this century was bitterly contested by Poland and Czechoslovakia.
Drawing Together: Evaluation Of A Therapy Programme For Severe Aphasia
Authors: Sacchett Carol , Byng Sally , Marshall Jane , Pound Carole
Published: July 2002

This paper reports a therapy study that aims to promote communicative drawing in a group of seven people with severe and long‐standing aphasia. Therapy was conducted on individual and group bases over 12 weeks and entailed a range of techniques which are described in some detail. Treatment was evaluated using a novel generative drawing assessment in which subjects were required to draw absent items in response to photographic and conversational cues. Preand post‐therapy assessments of untreated skills, such as comprehension, naming and gesture, were also conducted. The results indicated that, as a group, the subjects' drawing improved. Unchanged performance on the other assessments indicated that the gains were specific to the content of therapy and could not be attributed to spontaneous recovery. Pre‐ and post‐therapy interviews with carers (i.e. relatives and close friends of the individuals) suggested that the effects of therapy were also being felt in the home. These results have important implications for therapy with people with severe aphasia.
Neisseria gonorrhoeae Epithelial Cell Interaction Leads to the Activation of the Transcription Factors Nuclear Factor κB and Activator Protein 1 and the Induction of Inflammatory Cytokines
Authors: Naumann Michael , Weßler Silja , Bartsch Cornelia , Wieland Björn , Meyer Thomas F.
Published: July 2002

We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-κB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-κB DNA–protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-κB–specific antibodies, we identified a NF-κB p50/p65 heterodimer. The NF-κB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor α and interluekin (IL)-1β occurred at later times and therefore did not account for NF-κB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-κB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-κB conferred by the protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-κB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.
Human Peripheral Blood Eosinophils Express a Functional c-kit Receptor for Stem Cell Factor that Stimulates Very Late Antigen 4 (VLA-4)–mediated Cell Adhesion to Fibronectin and Vascular Cell Adhesion Molecule 1 (VCAM-1)
Authors: Yuan Qian , Austen K. Frank , Friend Daniel S. , Heidtman Matthew , Boyce Joshua A.
Published: July 2002

We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine kinase, c-kit, the receptor for the stromal cell–derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0– 3.6-fold, average 2.8 ± 0.6-fold, greater than the negative control). The uniform and selective expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of 100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 μg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in 15 min by 7.7 ± 1.4-, 5.3 ± 3.3-, and 5.4 ± 0.2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 μg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 ± 9.2-, 3.8 ± 2.5-, and 1.7 ± 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml, 5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the α4 and β1 integrin subunits, revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer, very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4–mediated adhesion, which could contribute to the exit of these cells from the blood, their tissue localization, and their prominence in inflammatory lesions.
Viral and Bacterial Infections Interfere with Peripheral Tolerance Induction and Activate CD8+ T Cells to Cause Immunopathology
Authors: Ehl Stephan , Hombach Joachim , Aichele Peter , Rülicke Thomas , Odermatt Bernhard , Hengartner Hans , Zinkernagel Rolf , Pircher Hanspeter
Published: July 2002

We studied the impact of various infectious and proinflammatory agents on the induction of peripheral T cell tolerance. Adoptive transfer of CD8+ T cells from lymphocytic choriomeningitis virus (LCMV) T cell receptor transgenic mice into LCMV antigen transgenic mice expressing the LCMV glycoprotein epitope (gp) 33–41 under control of a major histocompatibility complex class I promoter led to efficient induction of peripheral tolerance after a period of transient activation. If, however, the recipient mice were challenged with viral or bacterial infections or proinflammatory agents (lipopolysaccharide or Poly:IC) early after cell transfer, tolerance induction was prevented and instead, CD8+ T cell activation leading to vigorous expansion and generation of cytolytic activity ensued. This became manifest in significant immunopathology mainly involving destruction of the splenic architecture and lysis of antigen-expressing lymphocyte and macrophage populations. Important parameters involved in the activation of host-reactive T cells by nonspecific infectious agents included the presence, localization, and quantity of the specific transgene-encoded self-antigen; in contrast, CD4+ T cells were not required. In mice surviving the acute phase, the transferred CD8+ T cells persisted at high levels in an anergic state; they were unable to generate cytolytic activity in vitro or to control LCMV infection in vivo. These results impinge on our understanding of the role of infectious agents in graft verus host reactions towards minor histocompatibility antigens.
Major Histocompatibility Complex Class I Viral Antigen Processing in the Secretory Pathway Defined by the trans-Golgi Network Protease Furin
Authors: Gil-Torregrosa Beatriz C. , Raúl Castaño A. , Del  Val Margarita
Published: July 2002

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.
Generation of Splenic Follicular Structure and B Cell Movement in Tumor Necrosis Factor–deficient Mice
Authors: Cook Matthew C. , Körner Heinrich , Sean Riminton D. , Lemckert Frances A. , Hasbold Jhagvaral , Amesbury Michelle , Hodgkin Philip D. , Cyster Jason G. , Sedgwick Jonathon D. , Basten Antony
Published: July 2002

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin α (LTα). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF−/− and TNF/LTα−/− mice. By creating radiation bone marrow chimeras from wild-type and TNF−/− mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor–IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF−/− recipients, but not into TNF/LTα−/− recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTα−/− mice because TNF/LTα−/− B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.
Pre–T Cell Receptor (Tcr) and Tcr-Controlled Checkpoints in T Cell Differentiation Are Set by Ikaros
Authors: Winandy Susan , Wu Li , Wang Jin-Hong , Georgopoulos Katia
Published: July 2002

T cell differentiation relies on pre–T cell receptor (TCR) and TCR signaling events that take place at successive steps of the pathway. Here, we show that two of these T cell differentiation checkpoints are regulated by Ikaros. In the absence of Ikaros, double negative thymocytes can differentiate to the double positive stage without expression of a pre-TCR complex. Subsequent events in T cell development mediated by TCR involving transition from the double positive to the single positive stage are also regulated by Ikaros. Nonetheless, in Ikaros-deficient thymocytes, the requirement of pre-TCR expression for expansion of immature thymocytes as they progress to the double positive stage is still maintained, and the T cell malignancies that invariably arise in the thymus of Ikaros-deficient mice are dependent on either pre-TCR or TCR signaling. We conclude that Ikaros regulates T cell differentiation, selection, and homeostasis by providing signaling thresholds for pre-TCR and TCR.
Role of Antigen-Presenting Cells in Mediating Tolerance and Autoimmunity
Authors: Garza Kristine M. , Chan Steven M. , Suri Rakesh , Nguyen Linh T. , Odermatt Bernhard , Schoenberger Stephen P. , Ohashi Pamela S.
Published: July 2002

The mechanisms that determine whether receptor stimulation leads to lymphocyte tolerance versus activation remain poorly understood. We have used rat insulin promoter (RIP)-gp/P14 double-transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) on pancreatic β-islet cells together with T cells expressing an LCMV-gp–specific T cell receptor to assess the requirements for the induction of autoimmunity. Our studies have shown that administration of the gp peptide gp33 leads to the activation of P14-transgenic T cells, as measured by the upregulation of activation markers and the induction of effector cytotoxic activity. This treatment also leads to expansion and deletion of P14 T cells. Despite the induction of cytotoxic T lymphocyte activity, peptide administration is not sufficient to induce diabetes. However, the administration of gp peptide together with an activating anti-CD40 antibody rapidly induces diabetes. These findings suggest that the induction of tolerance versus autoimmunity is determined by resting versus activated antigen-presenting cells.
Expression of Natural Killer Receptor Alleles at Different Ly49 Loci Occurs Independently and Is Regulated by Major Histocompatibility Complex Class I Molecules
Authors: Tanamachi Dawn M. , Hanke Thomas , Takizawa Hisao , Jamieson Amanda M. , Raulet David H.
Published: July 2002

Ly49 receptor genes are expressed by subsets of natural killer (NK) cells in an overlapping fashion, accounting for the capacity of NK subsets to attack host cells that have selectively downregulated self–major histocompatibility complex (MHC) class I molecules. It was shown previously that most NK cells express only one or the other allele of a given Ly49 gene, while a smaller population expresses both alleles. However, the methods used to detect monoallelic and biallelic cells were nonquantitative. Here, new allele-specific antibodies were used to provide the first quantitative examination of biallelic and monoallelic expression of Ly49A and Ly49G2. The results demonstrate conclusively that most Ly49A+ and Ly49G2+ NK cells express the corresponding gene in a monoallelic fashion, with a smaller subset expressing both alleles. Unexpectedly, biallelic Ly49A+ NK cells were more numerous than predicted by completely independent allelic expression, suggesting some heterogeneity among NK progenitors in the potential to express a given Ly49 gene. The data also show that cells expressing one allele of Ly49G2 may express Ly49A from the same or opposite chromosome with equal likelihood, indicating that the expressed allele is chosen independently for different Ly49 genes. Finally, the data demonstrate that biallelic expression of Ly49A or Ly49G2 occurs least frequently in mice that express ligands for these receptors (H-2d mice), and most frequently in class I–deficient mice. Thus, biallelic expression of Ly49 genes is regulated by interactions of NK cell progenitors with MHC class I molecules.
Novel Cell Type–Specific Antiviral Mechanism of Interferon γ Action in Macrophages
Authors: Presti Rachel M. , Popkin Daniel L. , Connick Megan , Paetzold Susanne , Virgin Herbert W.
Published: July 2002

Interferon (IFN)-γ and macrophages (Mϕ) play key roles in acute, persistent, and latent murine cytomegalovirus (MCMV) infection. IFN-γ mechanisms were compared in embryonic fibroblasts (MEFs) and bone marrow Mϕ (BMMϕ). IFN-γ inhibited MCMV replication in a signal transducer and activator of transcription (STAT)-1α–dependent manner much more effectively in BMMϕ (∼100-fold) than MEF (5–10-fold). Although initial STAT-1α activation by IFN-γ was equivalent in MEF and BMMϕ, microarray analysis demonstrated that IFN-γ regulates different sets of genes in BMMϕ compared with MEFs. IFN-γ inhibition of MCMV growth was independent of known mechanisms involving IFN-α/β, tumor necrosis factor α, inducible nitric oxide synthase, protein kinase RNA activated (PKR), RNaseL, and Mx1, and did not involve IFN-γ–induced soluble mediators. To characterize this novel mechanism, we identified the viral targets of IFN-γ action, which differed in MEF and BMMϕ. In BMMϕ, IFN-γ reduced immediate early 1 (IE1) mRNA during the first 3 h of infection, and significantly reduced IE1 protein expression for 96 h. Effects of IFN-γ on IE1 protein expression were independent of RNaseL and PKR. In contrast, IFN-γ had no significant effects on IE1 protein or mRNA expression in MEFs, but did decrease late gene mRNA expression. These studies in primary cells define a novel mechanism of IFN-γ action restricted to Mϕ, a cell type key for MCMV pathogenesis and latency.
Discrete Cleavage Motifs of Constitutive and Immunoproteasomes Revealed by Quantitative Analysis of Cleavage Products
Authors: Toes R.E.M. , Nussbaum A.K. , Degermann S. , Schirle M. , Emmerich N.P.N. , Kraft M. , Laplace C. , Zwinderman A. , Dick T.P. , Müller J. , Schönfisch B. , Schmid C. , Fehling H.-J. , Stevanovic S. , Rammensee H.G. , Schild H.
Published: July 2002

Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon γ–inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex–like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously.

We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell–specificity as indicated by the observation that LMP-7−/− mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.
Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels
Authors: Kellenberger Stephan , West James W. , Scheuer Todd , Catterall William A.
Published: July 2002

Fast Na+ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in LIII-IV of the rat brain type IIA Na+ channel. We have analyzed macroscopic and single channel currents from Na+ channels with mutations within and adjacent to hydrophobic clusters in LIII-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in LIII-IV also affected the latency to first opening, indicating coupling between LIII-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na+ channels.
Modulation of Voltage-dependent Properties of a Swelling-activated Cl− Current
Authors: Voets Thomas , Droogmans Guy , Nilius Bernd
Published: July 2002

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl− current (ICl,swell) in BC3H1 myoblasts. This Cl− current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl− currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (∼60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10–80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl− concentration of the extracellular solution. Replacing extracellular Cl− by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN− > I− > NO3− > Cl− >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH , external divalent cations, and by the nature of the permeant anion.
Principal Eigenvalues with Indefinite Weight Functions
Authors: Jin Zhiren
Published: July 2002

Both existence and non-existence results for principal eigenvalues of an elliptic operator with indefinite weight function have been proved. The existence of a continuous family of principal eigenvalues is demonstrated.
THE ARMY OFFICER CORPS IN THE ALL‐VOLUNTEER FORCE
Authors: Meese Michael J.
Published: July 2002

The remarkable success of the all‐volunteer force (AVF) in the past 30 years belies the controversial decision to implement such a force, which was the result of effective economic arguments and propitious political factors. In spite of significant work by the Gates Commission, the success of the AVF was not preordained, but was significantly influenced by ways in which the army adapted since 1973 to make the most effective use of volunteer soldiers. This adaptation included careful evaluation of standards, pay, education, training, non‐monetary compensation, quality of life, and promotion of diversity. Each of these factors had to be carefully managed to enhance the effectiveness of the military labor force. Military personnel policies have a long‐term impact not only on the military but also on society as a whole.