bluuebunny/crossref_metadata_2025 huggingface dataset:
Human and mouse homologs of
Escherichia coli
DinB (DNA polymerase IV), members of the UmuC/DinB superfamily
Authors: Gerlach Valerie L. , Aravind L. , Gotway Garrett , Schultz Roger A. , Koonin Eugene V. , Friedberg Errol C.
Published: July 2002

To understand the mechanisms underlying mutagenesis in eukaryotes better, we have cloned mouse and human homologs of the
Escherichia coli
dinB
gene.
E. coli
dinB
encodes DNA polymerase IV and greatly increases spontaneous mutations when overexpressed. The mouse and human DinB1 amino acid sequences share significant identity with
E. coli
DinB, including distinct motifs implicated in catalysis, suggesting conservation of the polymerase function. These proteins are members of a large superfamily of DNA damage-bypass replication proteins, including the
E. coli
proteins UmuC and DinB and the
Saccharomyces cerevisiae
proteins Rev1 and Rad30. In a phylogenetic tree, the mouse and human DinB1 proteins specifically group with
E. coli
DinB, suggesting a mitochondrial origin for these genes. The human
DINB1
gene is localized to chromosome 5q13 and is widely expressed.
A general strategy to enhance the potency of chimeric transcriptional activators
Authors: Natesan Sridaran , Molinari Elizabeth , Rivera Victor M. , Rickles Richard J. , Gilman Michael
Published: July 2002

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.
Electrophysiological evidence for a hyperpolarizing, galanin (1-15)-selective receptor on hippocampal CA3 pyramidal neurons
Authors: Xu Zhi-Qing David , Ma Xiaosong , Soomets Ursel , Langel Ulo , Hökfelt Tomas
Published: July 2002

The effects of the 29-amino acid neuropeptide galanin [GAL (1–29)], GAL(1–15), GAL(1–16), and the GAL subtype 2 receptor agonist
d
-tryptophan
2
-GAL(1–29) were studied in the dorsal hippocampus
in vitro
with intracellular recording techniques. GAL(1–15) induced, in the presence of tetrodotoxin, a dose-dependent hyperpolarization in hippocampal CA3 neurons. Most of the GAL(1–15)-sensitive neurons did not respond to GAL(1–29), GAL(1–16), or
d
-tryptophan
2
-GAL(1–29). These results indicate the presence of a distinct, yet-to-be cloned GAL(1–15)-selective receptor on CA3 neurons in the dorsal hippocampus.
Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization
Authors: Bleuel Kerstin , Popp Susanne , Fusenig Norbert E. , Stanbridge Eric J. , Boukamp Petra
Published: July 2002

The development of skin carcinomas presently is believed to be correlated with mutations in the p53 tumor suppressor and
ras
gene as well as with the loss of chromosome 9. We now demonstrate that, in addition, loss of chromosome 15 may be a relevant genetic defect. Reintroduction of an extra copy of chromosome 15, but not chromosome 4, into the human skin carcinoma SCL-I cells, lacking one copy of each chromosome, resulted in tumor suppression after s.c. injection in mice. Transfection with thrombospondin-1 (TSP-1), mapped to 15q15, induced the same tumor suppression without affecting cell proliferation
in vitro
or
in vivo
. Halted tumors remained as small cysts encapsulated by surrounding stroma and blood vessels. These cysts were characterized by increased TSP-1 matrix deposition at the tumor/stroma border and a complete lack of tumor vascularization. Coinjection of TSP-1 antisense oligonucleotides drastically reduced TSP-1 expression and almost completely abolished matrix deposition at the tumor/stroma border. As a consequence, the tumor phenotype reverted to a well vascularized, progressively expanding, solid carcinoma indistinguishable from that induced by the untransfected SCL-I cells. Thus, these data strongly suggest TSP-1 as a potential tumor suppressor on chromosome 15. The data further propose an unexpected mechanism of TSP-1-mediated tumor suppression. Instead of interfering with angiogenesis in general, in this system TSP-1 acts as a matrix barrier at the tumor/stroma border, which, by halting tumor vascularization, prevents tumor cell invasion and, thus, tumor expansion.
Modulation of nicotinic acetylcholine receptors by strychnine
Authors: García-Colunga Jesús , Miledi Ricardo
Published: July 2002

Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α
1
β
1
γδ, α
1
β
1
γ, and α
1
β
1
δ) and neuronal (α
2
β
2
and α
2
β
4
) nicotinic acetylcholine receptors (AcChoRs) expressed in
Xenopus
oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α
1
β
1
γδ > α
2
β
4
> α
2
β
2
. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex.
Structure-based design of submicromolar, biologically active inhibitors of trypanosomatid glyceraldehyde-3-phosphate dehydrogenase
Authors: Aronov Alex M. , Suresh Stephen , Buckner Frederick S. , Van Voorhis Wesley C. , Verlinde Christophe L. M. J. , Opperdoes Fred R. , Hol Wim G. J. , Gelb Michael H.
Published: July 2002

The bloodstream stage of
Trypanosoma brucei
and probably the intracellular (amastigote) stage of
Trypanosoma cruzi
derive all of their energy from glycolysis. Inhibiting glycolytic enzymes may be a novel approach for the development of antitrypanosomatid drugs provided that sufficient parasite versus host selectivity can be obtained. Guided by the crystal structures of human,
T. brucei
, and
Leishmania mexicana
glyceraldehyde-3-phosphate dehydrogenase, we designed adenosine analogs as tight binding inhibitors that occupy the pocket on the enzyme that accommodates the adenosyl moiety of the NAD
+
cosubstrate. Although adenosine is a very poor inhibitor, IC
50
≈ 50 mM, addition of substituents to the 2′ position of ribose and the
N
6
-position of adenosine led to disubstituted nucleosides with micromolar to submicromolar potency in glyceraldehyde-3-phosphate dehydrogenase assays, an improvement of 5 orders of magnitude over the lead. The designed compounds do not inhibit the human glycolytic enzyme when tested up to their solubility limit (≈40 μM). When tested against cultured bloodstream
T. brucei
and intracellular
T. cruzi
,
N
6
-(1-naphthalenemethyl)-2′-(3-chlorobenzamido)adenosine inhibited growth in the low micromolar range. Within minutes after adding this compound to bloodstream
T. brucei
, production of glucose-derived pyruvate ceased, parasite motility was lost, and a mixture of grossly deformed and lysed parasites was observed. These studies underscore the feasibility of using structure-based drug design to transform a mediocre lead compound into a potent enzyme inhibitor. They also suggest that energy production can be blocked in trypanosomatids with a tight binding competitive inhibitor of an enzyme in the glycolytic pathway.
Surgical Treatment of Chondrosarcoma of the Sternum
Authors: Ashford Robert U. , Stanton Jeremy , Khan Farid , Pringle Jean A. S. , Cannon Stephen R. , Briggs Timothy W. R.
Published: July 2002

Purpose: We reviewed all tumours of the sternum referred to The London Bone and Soft Tissue Tumour Service between
1956 and 1997 inclusive.

Patients and results: There were eight patients with this pathology, the male to female ratio was 3:1 and their mean age was
53 years. Of these patients, three are alive and disease free, one is alive with recurrence, and four have died, two of the consequences
of the disease and two of unrelated causes. Surgery is the principal treatment of these tumours both for excision and
subsequent reconstruction.

Discussion: Extended disease‐free survival is possible with correct diagnosis, complete excision at the first operation, appropriate
skeletal reconstruction, adequate skin cover and appropriate postoperative support and follow‐up.
The Clinical Value of Flow Cytometric DNA Content Analysis in Patientswith Soft Tissue Sarcomas
Authors: Samur Mustafa , Pamir Ali , Akbulut Hakan , Erekul Selim , Sağlik Yener , Yildiz Yusuf , Dinçol Dilek , Içli Fikri
Published: July 2002

Purpose. The purpose of this study was to evaluate: (1) the correlation
between grade and ploidy or S‐phase fraction (SPF), (2) the prognostic value of DNA flow
cytometric study in soft tissue sarcomas.

Patients /Methods. In all, 47 tissue samples from soft tissue sarcoma
patients, surgically treated in the same center, were included. Flow cytometric analyses
were performed according to a modified version of the original method of Hedley et al.

Results. DNA ploidy status could be determined in 44 samples out of
47 (success rate 94%). Of these 44, S‐phase fraction could be calculated in 34 samples (77%).
In the study group as a whole, aneuploidy was significantly correlated with high grade.
Survival analyses were carried out in 21 patients with soft tissue sarcoma, all surgically
treated in the same center, without chemotherapy or radiotherapy. In univariate analyses,
DNA ploidy was found to be a significant factor for overall survival (OAS) and metastasis‐free
survival MFS. Mean OAS for aneuploid tumors and diploid tumors were 35 and 65
months (p=0.034), and mean MFS 23 and 61 months, respectively (p=0.005) .

Discussion.There is a relation between histological grade and ploidy in
soft tissue sarcomas. It appears that low‐grade tumors are generally diploid, whereas
high‐grade tumors tend to be aneuploid. In a subgroup of patients treated only with
surgery, DNA ploidy was found to be an important factor for predicting OAS and MFS.
Primary Chondroblastic Osteosarcoma of the Lung
Authors: Wagner Monica S. , Reyes Cesar V.
Published: July 2002

Purpose. Primary extra‐osseous osteosarcomas are uncommon
lesions, and those originating within the lung are especially rare, with few case reports
existing in the literature.

Patient. We report the case of a 48‐year‐old male smoker with a primary
osteosarcoma of the right lower lung lobe.

Results. Diagnosis was based on histopathological findings of a poorly
differentiated sarcoma with malignant cellular components of osteoid and chondroid matrix,
along with immunohistochemical and electron microscopy confirmation.
Extensive clinical and radiographic evaluation failed to reveal any tumor at other anatomical
sites.
Cholera Toxin Is Exported from Microsomes by the Sec61p Complex
Authors: Schmitz Anton , Herrgen Helga , Winkeler Alexandra , Herzog Volker
Published: July 2002

After endocytosis cholera toxin is transported to the endoplasmic reticulum (ER), from where its A1 subunit (CTA1) is assumed to be transferred to the cytosol by an as-yet unknown mechanism. Here, export of CTA1 from the ER to the cytosol was investigated in a cell-free assay using either microsomes loaded with CTA1 by in vitro translation or reconstituted microsomes containing CTA1 purified from V. cholerae. Export of CTA1 from the microsomes was time- and adenosine triphosphate–dependent and required lumenal ER proteins. By coimmunoprecipitation CTA1 was shown to be associated during export with the Sec61p complex, which mediates import of proteins into the ER. Export of CTA1 was inhibited when the Sec61p complexes were blocked by nascent polypeptides arrested during import, demonstrating that the export of CTA1 depended on translocation-competent Sec61p complexes. Export of CTA1 from the reconstituted microsomes indicated the de novo insertion of the toxin into the Sec61p complex from the lumenal side. Our results suggest that Sec61p complex–mediated protein export from the ER is not restricted to ER-associated protein degradation but is also used by bacterial toxins, enabling their entry into the cytosol of the target cell.
Sustained Polymorphonuclear Leukocyte Transmigration Induces Apoptosis in T84 Intestinal Epithelial Cells
Authors: Le'Negrate Gaëlle , Selva Eric , Auberger Patrick , Rossi Bernard , Hofman Paul
Published: July 2002

Acute colitis is characterized by a large number of polymorphonuclear leukocytes (PMNLs) migrating across the columnar epithelium in response to inflammatory stimuli. Several of these inflammatory factors have been characterized as proapoptotic inducers for intestinal epithelial cells. Our aim was to elucidate the role of PMNL transmigration in the onset of intestinal epithelial cell apoptosis. We found that PMNL migration, in response to N-formyl-methionyl-leucyl-phenylalanine across monolayers of intestinal epithelial cells (T84), was associated with activation of caspase-2, -3, and -9 and poly(ADP-ribose) polymerase cleavage within epithelial cells. Moreover, dihydrocytochalasin B treatment of T84 cells induced apoptosis with similar characteristics. Although Fas and Fas ligand were expressed on T84 cells and PMNLs, treatment of epithelial cells with an antagonistic anti-Fas antibody failed to prevent apoptosis induced by migrating PMNLs. Owing to the F-actin reorganization accompanying PMNL transmigration, these findings indicate a direct relationship between PMNL migration and induction of apoptosis in epithelial cells. This apoptotic process appears to involve remodeling of the actin cytoskeleton of enterocytes independent of the Fas/Fas ligand pathway.
C-Jun Activation-Dependent Tumorigenic Transformation Induced Paradoxically by Overexpression or Block of S-Adenosylmethionine Decarboxylase
Authors: Paasinen-Sohns Aino , Kielosto Mari , Kääriäinen Essi , Eloranta Terho , Laine Aire , Jänne Olli A. , Birrer Michael J. , Hölttä Erkki
Published: July 2002

All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH2-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.
Simple centromere, complex kinetochore
Authors: Cheeseman Iain M. , Drubin David G. , Barnes Georjana
Published: July 2002

Although the budding yeast centromere is extremely short (125 bp) compared to those of other eukaryotes, the kinetochore that assembles on this DNA displays a rich molecular complexity. Here, we describe recent advances in our understanding of kinetochore function in budding yeast and present a model describing the attachment that is formed between spindle microtubules and centromeric DNA. This analysis may provide general principles for kinetochore function and regulation.
Translocation of PKCθ in T cells is mediated by a nonconventional, PI3-K– and Vav-dependent pathway, but does not absolutely require phospholipase C
Authors: Villalba Martin , Bi Kun , Hu Junru , Altman Yoav , Bushway Paul , Reits Eric , Neefjes Jacques , Baier Gottfried , Abraham Robert T. , Altman Amnon
Published: July 2002

PKCθ plays an essential role in activation of mature T cells via stimulation of AP-1 and NF-κB, and is known to selectively translocate to the immunological synapse in antigen-stimulated T cells. Recently, we reported that a Vav/Rac pathway which depends on actin cytoskeleton reorganization mediates selective recruitment of PKCθ to the membrane or cytoskeleton and its catalytic activation by anti-CD3/CD28 costimulation. Because this pathway acted selectively on PKCθ, we addressed here the question of whether the translocation and activation of PKCθ in T cells is regulated by a unique pathway distinct from the conventional mechanism for PKC activation, i.e., PLC-mediated production of DAG. Using three independent approaches, i.e., a selective PLC inhibitor, a PLCγ1-deficient T cell line, or a dominant negative PLCγ1 mutant, we demonstrate that CD3/CD28-induced membrane recruitment and COOH-terminal phosphorylation of PKCθ are largely independent of PLC. In contrast, the same inhibitory strategies blocked the membrane translocation of PKCα. Membrane or lipid raft recruitment of PKCθ (but not PKCα) was absent in T cells treated with phosphatidylinositol 3-kinase (PI3-K) inhibitors or in Vav-deficient T cells, and was enhanced by constitutively active PI3-K. 3-phosphoinositide-dependent kinase-1 (PDK1) also upregulated the membrane translocation of PKCθ, but did not associate with it. These results provide evidence that a nonconventional PI3-K– and Vav-dependent pathway mediates the selective membrane recruitment and, possibly, activation of PKCθ in T cells.
The Vav Binding Site (Y315) in ZAP-70 Is Critical for Antigen Receptor–mediated Signal Transduction
Authors: Wu Jun , Zhao Qihong , Kurosaki Tomohiro , Weiss Arthur
Published: July 2002

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor–mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav– ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor–mediated signal transduction.
Role of Tyrosine Phosphorylation of HS1 in B Cell Antigen Receptor-mediated Apoptosis
Authors: Yamanashi Yuji , Fukuda Takahiro , Nishizumi Hirofumi , Inazu Tetsuya , Higashi Ken-ichi , Kitamura Daisuke , Ishida Takaomi , Yamamura Hirohei , Watanabe Takeshi , Yamamoto Tadashi
Published: July 2002

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.
Enhanced Intracellular Dissociation of Major Histocompatibility Complex Class I–associated Peptides: A Mechanism for Optimizing the Spectrum of Cell Surface–Presented Cytotoxic T Lymphocyte Epitopes
Authors: Sijts Alice J.A.M. , Pamer Eric G.
Published: July 2002

Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses. Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves. We find that two H2-Kd–restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics. Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates. p60 217-225 forms complexes with H2-Kd with a half-life >6 h, while p60 449-457 dissociates from H2-Kd with a half-life of ∼1 h. We find that p60 449-457–H2-Kd complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes. While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-Kd molecules maintain a remarkable capacity to bind new T cell epitopes. We find that intracellular H2-Kd molecules can bind new CTL epitopes for up to 3 h after their synthesis. Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells. We propose that the increased intracellular lability of peptide–MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.
Role of Different T Cell Receptors in the Development of Pre–T Cells
Authors: Buer Jan , Aifantis Iannis , DiSanto James P. , Fehling Hans Joerg , Boehmer Harald von
Published: July 2002

The development of pre–T cells with productive TCR-β rearrangements can be mediated by each the pre–T cell receptor (pre-TCR), the TCR-αβ as well as the TCR-γδ, albeit by distinct mechanisms. Although the TCR-γδ affects CD4−8− precursor cells irrespective of their rearrangement status by TCR-β mechanisms not involving TCR-β selection, both the preTCR and the TCR-αβ select only cells with productive TCR-β genes for expansion and maturation. The TCR-αβ appears to be much less effective than the pre-TCR because of the paucity of TCR-α proteins in TCR-β–positive precursors since an early expressed transgenic TCR-αβ can largely substitute for the pre-TCR. Thus, the TCR-αβ can assume a role not only in the rescue from programmed cell death of CD4+8+ but also of CD4−8− thymocytes. In evolution this double function of the TCR-αβ may have been responsible for the maturation of αβ T cells before the advent of the pre–TCR-α chain.
Major Histocompatibility Complex Class I Molecules Modulate Activation Threshold and Early Signaling of T Cell Antigen Receptor–γ/δ Stimulated by Nonpeptidic Ligands
Authors: Carena Ilaria , Shamshiev Abdijapar , Donda Alena , Colonna Marco , Libero Gennaro De
Published: July 2002

Killer cell inhibitory receptors and CD94-NKG2-A/B heterodimers are major histocompatibility complex class I–specific inhibitory receptors expressed by natural killer cells, T cell antigen receptor (TCR)-γ/δ cells, and a subset of TCR-α/β cells. We studied the functional interaction between TCR-γ/δ and CD94, this inhibitory receptor being expressed on the majority of γ/δ T cells. When engaged by human histocompatibility leukocyte antigen class I molecules, CD94 downmodulates activation of human TCR-γ/δ by phosphorylated ligands. CD94-mediated inhibition is more effective at low than at high doses of TCR ligand, which may focus T cell responses towards antigen-presenting cells presenting high amounts of antigen. CD94 engagement has major effects on TCR signaling cascade. It facilitates recruitment of SHP-1 phosphatase to TCR–CD3 complex and affects phosphorylation of Lck and ZAP-70 kinase, but not of CD3 ζ chain upon TCR triggering. These events may cause abortion of proximal TCR-mediated signaling and set a higher TCR activation threshold.
Outer Periarteriolar Lymphoid Sheath Arrest and Subsequent Differentiation of Both Naive and Tolerant Immunoglobulin Transgenic B Cells Is Determined by B Cell Receptor Occupancy
Authors: Cook Matthew C. , Basten Antony , Groth Barbara Fazekas de St.
Published: July 2002

T-dependent B cell responses in the spleen are initiated in the outer periarteriolar lymphoid sheath (PALS) and culminate in the generation of proliferative foci and germinal center reactions. By pulsing anti–hen egg lysozyme (HEL) immunoglobulin transgenic (IgTg) B cells with various concentrations of HEL in vitro before adoptive transfer into normal recipients, it was shown that a critical number of B cell receptors (BCRs) must be ligated for B cells to undergo arrest in the outer PALS. T cell help was manipulated independently of the BCR stimulus by incubating B cells expressing the appropriate major histocompatibility complex class II antigen with a peptide recognized by CD4+ TCR Tg T cells. B cells which either failed to arrest in the outer PALS due to a subthreshold BCR stimulus, or arrested only transiently due to the brevity of the BCR stimulus, underwent an abortive response within the follicles when provided with T cell help. In contrast, naive B cells stimulated by a sustained, suprathreshold concentration of either foreign or self-antigen and given T cell help, proliferated in the outer PALS and then differentiated. Outer PALS arrest was not influenced by the nature of the B cells occupying the follicle, but appeared to be determined solely by the magnitude of BCR stimulation. Thus antigen-pulsed B cells arrested in the outer PALS in an identical manner irrespective of whether the follicles comprised a population of normal B cells with multiple specificities, a monoclonal naive population, or a monoclonal population of tolerant B cells. In addition, tolerant B cells were found to relocate from the follicles to the outer PALS of HEL/anti-HEL double Tg mice in which the concentration of soluble self-antigen had been increased by zinc feeding. Similarly, when anti-HEL Tg mice were crossed with a second HEL Tg strain expressing a higher concentration of soluble HEL, the tolerant anti-HEL Tg B cells were located constitutively in the outer PALS. Thus, subtle variations in antigen concentration resulted in dramatic changes in positioning of B cells within the spleen. A series of mixed bone marrow chimeras in which the effective antigen concentration was inversely related to the number of self-reactive B cells due to absorption of antigen by transgene-encoded membrane and secreted Ig, was used to confirm that alteration in B cell position previously attributed to changes in follicular composition could be explained on the basis of available antigen concentration, rather than the diversity of the repertoire.
Generation of Lytic Natural Killer 1.1+, Ly-49− Cells from Multipotential Murine Bone Marrow Progenitors in a Stroma-free Culture: Definition of Cytokine Requirements and Developmental Intermediates
Authors: Williams Noelle Sevilir , Moore Thomas A. , Schatzle John D. , Puzanov Igor J. , Sivakumar P.V. , Zlotnik Albert , Bennett Michael , Kumar Vinay
Published: July 2002

We have developed a stroma-free culture system in which mouse marrow or thymus cells, known to be enriched for lymphoid progenitors, can be driven to generate natural killer (NK) cells. Culture of lineage marker (Lin)−, c-kit+, Sca2+, interleukin (IL)-2/15Rβ (CD122)− marrow cells in IL-6, IL-7, stem cell factor (SCF), and flt3 ligand (flt3-L) for 5–6 d followed by IL-15 alone for an additional 4–5 d expanded the starting population 30–40-fold and gave rise to a virtually pure population of NK1.1+, CD3− cells. Preculture in IL-6, IL-7, SCF, and flt3-L was necessary for inducing IL-15 responsiveness in the progenitors because the cells failed to significantly expand when cultured in IL-15 alone from the outset. Although culture of the sorted progenitors in IL-6, IL-7, SCF, and flt3-L for the entire 9–11-d culture period caused significant expansion, no lytic NK1.1+ cells were generated if IL-15 was not added, demonstrating a critical role for IL-15 in NK differentiation. Thus, two distinct populations of NK progenitors, IL-15 unresponsive and IL-15 responsive, have been defined. Similar results were obtained with Lin−, CD44+, CD25−, c-kit+ lymphoid progenitors obtained from adult thymus. The NK cells generated by this protocol lysed the NK-sensitive target YAC-1 and expressed markers of mature NK cells with the notable absence of Ly-49 major histocompatibility complex (MHC) receptors. However, despite the apparent lack of these inhibitory MHC receptors, the NK cells generated could distinguish MHC class I+ from class I− syngeneic targets, suggesting the existence of novel class I receptors.
Analysis of the Role of  Variation of Major Histocompatibility Complex Class II Expression on Nonobese Diabetic (NOD) Peripheral T Cell Response
Authors: Ridgway William M. , Ito Hiroaki , Fassò Marcella , Yu Chen , Garrison Fathman C.
Published: July 2002

The current paradigm of major histocompatibility complex (MHC) and disease association suggests that efficient binding of autoantigens by disease-associated MHC molecules leads to a T cell–mediated immune response and resultant autoimmune sequelae. The data presented below offer a different model for this association of MHC with autoimmune diabetes. We used several mouse lines expressing different levels of I-Ag7 and I-Ak on the nonobese diabetic (NOD) background to evaluate the role of MHC class II in the previously described NOD T cell autoproliferation. The ratio of I-Ag7 to I-Ak expression correlated with the peripheral T cell autoproliferative phenotype in the mice studied. T cells from the NOD, [NOD × NOD.I-Anull]F1, and NOD I-Ak transgenic mice demonstrated autoproliferative responses (after priming with self-peptides), whereas the NOD.H2h4 (containing I-Ak) congenic and [NOD × NOD.H2h4 congenic]F1 mice did not. Analysis of CD4+ NOD I-Ak transgenic primed lymph node cells showed that autoreactive CD4+ T cells in the NOD I-Ak transgenic mice were restricted exclusively by I-Ag7. Considered in the context of the avidity theory of T cell activation and selection, the reported poor peptide binding capacity of NOD I-Ag7 suggested a new hypothesis to explain the effects of MHC class II expression on the peripheral autoimmune repertoire in NOD mice. This new explanation suggests that the association of MHC with diabetes results from “altered” thymic selection in which high affinity self-reactive (potentially autoreactive) T cells escape negative selection. This model offers an explanation for the requirement of homozygous MHC class II expression in NOD mice (and in humans) in susceptibility to insulin-dependent diabetes mellitus.
An Opposite Pattern of Selection of a Single T Cell Antigen Receptor in the Thymus and among Intraepithelial Lymphocytes
Authors: Cruz Daniel , Sydora Beate C. , Hetzel Kristine , Yakoub Gian , Kronenberg Mitchell , Cheroutre Hilde
Published: July 2002

The differentiation of intestinal intraepithelial lymphocytes (IEL) remains controversial, which may be due in part to the phenotypic complexity of these T cells. We have investigated here the development of IEL in mice on the recombination activating gene (RAG)-2−/− background which express a T cell antigen receptor (TCR) transgene specific for an H-Y peptide presented by Db (H-Y/Db × RAG-2− mice). In contrast to the thymus, the small intestine in female H-Y/Db × RAG-2− mice is severely deficient in the number of IEL; TCR transgene+ CD8αα and CD8αβ are virtually absent. This is similar to the number and phenotype of IEL in transgenic mice that do not express the Db class I molecule, and which therefore fail positive selection. Paradoxically, in male mice, the small intestine contains large numbers of TCR+ IEL that express high levels of CD8αα homodimers. The IEL isolated from male mice are functional, as they respond upon TCR cross-linking, although they are not autoreactive to stimulator cells from male mice. We hypothesize that the H-Y/Db TCR fails to undergo selection in IEL of female mice due to the reduced avidity of the TCR for major histocompatibility complex peptide in conjunction with the CD8αα homodimers expressed by many cells in this lineage. By contrast, this reduced TCR/CD8αα avidity may permit positive rather than negative selection of this TCR in male mice. Therefore, the data presented provide conclusive evidence that a TCR which is positively selected in the thymus will not necessarily be selected in IEL, and furthermore, that the expression of a distinct CD8 isoform by IEL may be a critical determinant of the differential pattern of selection of these T cells.
Independent and Opposing Roles For Btk and Lyn in B and Myeloid Signaling Pathways
Authors: Satterthwaite Anne B. , Lowell Clifford A. , Khan Wasif N. , Sideras Paschalis , Alt Frederick W. , Witte Owen N.
Published: July 2002

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk−/− and lyn−/− mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk−/−lyn−/− mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk−/−lyn−/− mice. However, Btk−/−lyn−/− animals had a more severe immunodeficiency than Btk−/− mice. B cell numbers and response to T cell–dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn−/− mice. A transgene expressing Btk at ∼25% of endogenous levels (Btklo) was crossed onto Btk−/− and Btk−/−lyn−/− backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.
Class Switching in B Cells Lacking 3′ Immunoglobulin Heavy Chain Enhancers
Authors: Manis John P. , van der Stoep Nienke , Tian Ming , Ferrini Roger , Davidson Laurie , Bottaro Andrea , Alt Frederick W.
Published: July 2002

The 40-kb region downstream of the most 3′ immunoglobulin (Ig) heavy chain constant region gene (Cα) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5′ enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3aΔ and HS1,2Δ, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3aΔ or HS1,2Δ mutations. In addition, induced expression of the specifically targeted pgk-neor genes was regulated similarly to that of germline CH genes. Our findings implicate a 3′ CSR regulatory locus that appears remarkably similar in organization and function to the β-globin gene 5′ LCR and which we propose may regulate differential CSR via a promoter competition mechanism.
Transfer of Small Resting B Cells into Immunodeficient Hosts Results in the Selection of a Self-renewing Activated B Cell Population
Authors: Agenès Fabien , Freitas António A.
Published: July 2002

We studied the role of bone marrow B cell production in the renewal of peripheral B cells and the feedback mechanisms that control the entry of newly formed B cells into the peripheral B cell pools. When resting lymph node B cells are injected into B cell–deficient hosts, a fraction of the transferred cells expands and constitutes a highly selected population that survives for prolonged periods of time by continuous cell renewal at the periphery. Although the number of donor B cells recovered is low, a significant fraction shows an activated phenotype, and the serum immunoglobulin (Ig)M levels are as in normal mice. This population of activated B cells is resistant to replacement by a new cohort of B cells and is able to feedback regulate both the entry of newly formed B cells into the peripheral pool and terminal differentiation. These findings suggest that peripheral B cell selection follows the first come, first served rule and that IgM-secreting cells are generated from a pool of stable activated B cells with an independent homeostasis.
In Vivo Cytotoxic T Lymphocyte Elicitation by Mycobacterial Heat Shock Protein 70 Fusion Proteins Maps to a Discrete Domain and Is Cd4+ T Cell Independent
Authors: Huang Qian , Richmond Joan F.L. , Suzue Kimiko , Eisen Herman N. , Young Richard A.
Published: July 2002

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8+ cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4+ T lymphocytes. We found that the ability to elicit CD8+ CTLs depends on a discrete 200–amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8+ T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8+ CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4+ T cell–deficient individuals.
Virus-Induced Maturation and Activation of Autoreactive Memory B Cells
Authors: Reed Amy J. , Riley Michael P. , Caton Andrew J.
Published: July 2002

We have examined B cell populations that participate in distinct phases of the immune response to the influenza virus A/PR/8/34 hemagglutinin (HA) for their susceptibility to negative selection in mice that express the HA as a neo–self-antigen (HA104 mice). We demonstrated previously that specificity for the neo–self-HA causes a population of immunoglobulin G antibody-secreting cells, which dominate the primary response to virus immunization in BALB/c mice, to be negatively selected in HA104 mice. We find here that in contrast to these primary response B cells, HA-specific memory response B cells developed equivalently in HA104 and nontransgenic (BALB/c) mice. Indeed, there was no indication that HA-specific B cells were negatively selected during memory formation in influenza virus–immunized HA104 mice, even though the neo–self-HA can be recognized by memory B cells. Furthermore, HA-specific autoantibodies were induced in the absence of virus immunization by mating HA104 mice with mice transgenic for a CD4+ HA-specific T cell receptor. These findings indicate that specificity for a self-antigen does not prevent the maturation of autoreactive B cells in the germinal center pathway. Rather, the availability of CD4+ T cell help may play a crucial role in regulating autoantibody responses to the HA in HA104 mice.
Human Cd1b and Cd1c Isoforms Survey Different Intracellular Compartments for the Presentation of Microbial Lipid Antigens
Authors: Briken Volker , Jackman Robin M. , Watts Gerald F.M. , Rogers Rick A. , Porcelli Steven A.
Published: July 2002

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.
Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock
Authors: Tsytsykova Alla V. , Goldfeld Anne E.
Published: July 2002

Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock.
Tec Family Kinases Modulate Thresholds for Thymocyte Development and Selection
Authors: Schaeffer Edward M. , Broussard Christine , Debnath Jayanta , Anderson Stacie , McVicar Daniel W. , Schwartzberg Pamela L.
Published: July 2002

Tec family kinases are implicated in T cell receptor (TCR) signaling, and combined mutation of inducible T cell kinase (Itk) and resting lymphocyte kinase (Rlk)/Txk in mice dramatically impairs mature T cell function. Nonetheless, mutation of these kinases still permits T cell development. While itk−/− mice exhibit mild reductions in T cells with decreased CD4/CD8 cell ratios, rlk−/−itk−/− mice have improved total T cell numbers yet maintain decreased CD4/CD8 ratios. Using TCR transgenics and an in vitro thymocyte deletion model, we demonstrate that mutation of Tec kinases causes graded defects in thymocyte selection, leading to a switch from negative to positive selection in rlk−/−itk−/− animals. The reduction in both positive and negative selection and decreased CD4/CD8 ratios correlates with decreased biochemical parameters of TCR signaling, specifically defects in capacitive Ca2+ influx and activation of the mitogen-activated kinases extracellular signal–regulated kinase 1 and 2. Thus, Tec kinases influence cell fate determination by modulating TCR signaling, leading to altered thresholds for thymocyte selection. These results provide support for a quantitative model for thymic development and provide evidence that defects in negative selection can substantially alter thymic cellularity.
Role of the High Affinity Immunoglobulin E Receptor in Bacterial Translocation and Intestinal Inflammation
Authors: Dombrowicz David , Nutten Sophie , Desreumaux Pierre , Neut Christel , Torpier Gérard , Peeters Marc , Colombel Jean-Frédéric , Capron Monique
Published: July 2002

A role for immunoglobulin E and its high affinity receptor (FcεRI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized FcεRI (hFcεRI), with a cell distribution similar to that in humans, to FcεRI-deficient animals. In hFcεRI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFcεRI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas FcεRI-deficient animals were protected from colitis, demonstrating that FcεRI can affect the onset of intestinal inflammation.