bluuebunny/crossref_metadata_2025 huggingface dataset:
Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells
Authors: Zerrahn Jens , Volkmann Ariane , Coles Mark C. , Held Werner , Lemonnier Francois A. , Raulet David H.
Published: July 2002

The identity of cells that mediate positive selection of CD8+T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated β2-microglobulin mutant mice or mice with mutations in both the Kband Dbgenes were repopulated with fetal liver cells from class I+TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8+T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8+T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8+T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)–TCR+CD8+T cells was observed in class I+into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.
Soluble member(s) of the mesothelin/megakaryocyte potentiating factor family are detectable in sera from patients with ovarian carcinoma
Authors: Scholler Nathalie , Fu Ning , Yang Yi , Ye Zhengmao , Goodman Gary E. , Hellström Karl Erik , Hellström Ingegerd
Published: July 2002

mAb OV569 was made by immunizing mice with ovarian carcinoma cells. It binds to cells from ovarian carcinomas and, to a lesser extent, to cells from certain other carcinomas whereas the binding to normal tissues is low to nondetectable. It also binds to soluble molecule(s) in culture supernatants from antigen-positive carcinomas. OV569 recognizes a protein(s) of 42–45 kDa with the same N-terminal amino acid sequence as the membrane-bound portion of mesothelin and megakaryocyte potentiating factor (MPF). Binding assays with fusion proteins comprising either the N-terminal part of mesothelin/MPF (D1Ig), reported to be easily cleaved off, or a noncleavable, membrane-associated part (D2Ig) showed that OV569 only binds to D2hIg. A new member of the mesothelin/MPF family was discovered, which has an 82-bp insert in the membrane-associated part, leading to a frameshift of 212 bp, and whose predicted molecular structure indicates that it is soluble. To test patient sera for soluble tumor antigen, antigen was isolated from cell-free tumor culture supernatants via immunoadsorption with OV569 and used to generate murine mAbs to an epitope different from the one to which OV569 binds, after which mAbs to two different epitopes were used to develop a “sandwich ELISA.” Using this assay, the level of circulating antigen was elevated significantly in 23 of 30 sera from patients with ovarian carcinoma, as compared with 0 of 68 sera from healthy controls, 0 of 3 sera from patients with nonneoplastic diseases, and 25 of 75 sera from patients with other tumors. Soluble molecules of the mesothelin/MPF family may provide useful new marker(s) for diagnosis of ovarian carcinoma and/or monitoring its response to therapy.
Specific binding of proinsulin C-peptide to human cell membranes
Authors: Rigler R. , Pramanik A. , Jonasson P. , Kratz G. , Jansson O. T. , Nygren P.-Å. , Ståhl S. , Ekberg K. , Johansson B.-L. , Uhlén S. , Uhlén M. , Jörnvall H. , Wahren J.
Published: July 2002

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand–membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with
K
ass
> 3.3 × 10
9
M
−1
. Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 × 10
−4
s
−1
. The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative
d
-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.
Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes
Authors: Griffin Lisa D. , Mellon Synthia H.
Published: July 2002

The neurosteroid 3α-hydroxysteroid-5α-pregnan-20-one (allopregnanolone) acts as a positive allosteric modulator of γ-aminobutyric acid at γ-aminobutyric acid type A receptors and hence is a powerful anxiolytic, anticonvulsant, and anesthetic agent. Allopregnanolone is synthesized from progesterone by reduction to 5α-dihydroprogesterone, mediated by 5α-reductase, and by reduction to allopregnanolone, mediated by 3α-hydroxysteroid dehydrogenase (3α-HSD). Previous reports suggested that some selective serotonin reuptake inhibitors (SSRIs) could alter concentrations of allopregnanolone in human cerebral spinal fluid and in rat brain sections. We determined whether SSRIs directly altered the activities of either 5α-reductase or 3α-HSD, using an
in vitro
system containing purified recombinant proteins. Although rats appear to express a single 3α-HSD isoform, the human brain contains several isoforms of this enzyme, including a new isoform we cloned from human fetal brains. Our results indicate that the SSRIs fluoxetine, sertraline, and paroxetine decrease the
K
m
of the conversion of 5α-dihydroprogesterone to allopregnanolone by human 3α-HSD type III 10- to 30-fold. Only sertraline inhibited the reverse oxidative reaction. SSRIs also affected conversions of androgens to 3α- and 3α, 17β-reduced or -oxidized androgens mediated by 3α-HSD type II
Brain
. Another antidepressant, imipramine, was without any effect on allopregnanolone or androstanediol production. The region-specific expression of 3α-HSD type II
Brain
and 3α-HSD type III mRNAs suggest that SSRIs will affect neurosteroid production in a region-specific manner. Our results may thus help explain the rapid alleviation of the anxiety and dysphoria associated with late luteal phase dysphoria disorder and major unipolar depression by these SSRIs.
Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival
Authors: Ogilvy Sarah , Metcalf Donald , Print Cristin G. , Bath Mary L. , Harris Alan W. , Adams Jerry M.
Published: July 2002

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human
bcl-2
gene in mice by using the promoter of the
vav
gene, which is active throughout this compartment but rarely outside it. The
vav-bcl-2
transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells
in vitro
remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.
Genetic epidemiology of single-nucleotide polymorphisms
Authors: Collins A. , Lonjou C. , Morton N. E.
Published: July 2002

On the causal hypothesis, most genetic determinants of disease are single-nucleotide polymorphisms (SNPs) that are likely to be selected as markers for positional cloning. On the proximity hypothesis, most disease determinants will not be included among markers but may be detected through linkage disequilibrium with other SNPs. In that event, allelic association among SNPs is an essential factor in positional cloning. Recent simulation based on monotonic population expansion suggests that useful association does not usually extend beyond 3 kb. This is contradicted by significant disequilibrium at much greater distances, with corresponding reduction in the number of SNPs required for a cost-effective genome scan. A plausible explanation is that cyclical expansions follow population bottlenecks that establish new disequilibria. Data on more than 1,000 locus pairs indicate that most disequilibria trace to the Neolithic, with no apparent difference between haplotypes that are random or selected through a major disease gene. Short duration may be characteristic of alleles contributing to disease susceptibility and haplotypes characteristic of particular ethnic groups. Alleles that are highly polymorphic in all ethnic groups may be older, neutral, or advantageous, in weak disequilibrium with nearby markers, and therefore less useful for positional cloning of disease genes. Significant disequilibrium at large distance makes the number of suitably chosen SNPs required for genome screening as small as 30,000, or 1 per 100 kb, with greater density (including less common SNPs) reserved for candidate regions.
Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation
Authors: Kobayashi Koichi , Hatano Masahiko , Otaki Masayuki , Ogasawara Takeshi , Tokuhisa Takeshi
Published: July 2002

The inhibitor of apoptosis (IAP) proteins form a highly conserved gene family that prevents cell death in response to a variety of stimuli. Herein we describe a newly defined murine IAP, designatedTiap,that proved to be a murine homologue of human survivin based on sequence comparison. TIAP has one baculovirus IAP repeat and lacks a C-terminal RING finger motif. TIAP interacted with the processed form of caspase 3 and inhibited caspase-induced cell death. Histological examinations revealed that TIAP is expressed in growing tissues such as thymus, testis, and intestine of adult mice and many tissues of embryos. Inin vitrostudies, TIAP was induced in splenic T cells activated with anti-CD3 antibody or Con A, and the expression of TIAP was up-regulated in synchronized NIH 3T3 cells at S to G2/M phase of the cell cycle. We propose that during cell proliferation, cellular protective activity may be augmented with inducible IAPs such as TIAP.
Mutation ofPten/Mmac1in mice causes neoplasia in multiple organ systems
Authors: Podsypanina Katrina , Ellenson Lora Hedrick , Nemes Adriana , Gu Jianguo , Tamura Masahito , Yamada Kenneth M. , Cordon-Cardo Carlos , Catoretti Giorgio , Fisher Peter E. , Parsons Ramon
Published: July 2002

Pten/Mmac1+/− heterozygous mice exhibited neoplasms in multiple organs including the endometrium, liver, prostate, gastrointestinal tract, thyroid, and thymus. Loss of the wild-type allele was detected in neoplasms of the thymus and liver. Surprisingly, tumors of the gastrointestinal epithelium developed in association with gut lymphoid tissue. Tumors of the endometrium, thyroid, prostate, and liver were not associated with lymphoid tissue and appeared to be highly mitotic. In addition, these mice have nonneoplastic hyperplasia of lymph nodes that was caused by an inherited defect in apoptosis detected in B cells and macrophages. Examination of peripheral lymphoid tissue including lymphoid aggregates associated with polyps revealed that the normal organization of B and T cells was disrupted in heterozygous animals. Taken together, these data suggest that PTEN is a regulator of apoptosis and proliferation that behaves as a “landscaper” tumor suppressor in the gut and a “gatekeeper” tumor suppressor in other organs.
Allelic association between marker loci
Authors: Lonjou C. , Collins A. , Morton N. E.
Published: July 2002

Allelic association has proven useful to refine the location of
major genes prior to positional cloning, but it is of uncertain value
for genome scans in complex inheritance. We have extended kinship
theory to give information content for linkage and allelic association.
Application to pairs of closely linked markers as a surrogate for
marker × oligogene pairs indicates that association is largely
determined by regional founders, with little effect of subsequent
demography. Sub-Saharan Africa has the least allelic association,
consistent with settlement of other regions by small numbers of
founders. Recent speculation about substantial advantages of isolates
over large populations, of constant size over expansion, and of
F
1
hybrids over incrosses is not supported by theory or
data. On the contrary, fewer affected cases, less opportunity for
replication, and more stochastic variation tend to make isolates less
informative for allelic association, as they are for linkage.
Induction of
ARF
tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1
Authors: Inoue Kazushi , Roussel Martine F. , Sherr Charles J.
Published: July 2002

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the
ARF
tumor suppressor gene. DMP1 binds to a single canonical recognition site in the
ARF
promoter to activate gene expression, and in turn, p19
ARF
synthesis causes p53-dependent cell cycle arrest. Unlike genes such as
Myc
, adenovirus
E1A
, and
E2F-1,
which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.
Shift in speed selectivity of visual cortical neurons: A neural basis of perceived motion contrast
Authors: Li Chao-Yi , Lei Jing-Jiang , Yao Hai-Shan
Published: July 2002

The perceived speed of motion in one part of the visual field is influenced by the speed of motion in its surrounding fields. Little is known about the cellular mechanisms causing this phenomenon. Recordings from mammalian visual cortex revealed that speed preference of the cortical cells could be changed by displaying a contrast speed in the field surrounding the cell’s classical receptive field. The neuron’s selectivity shifted to prefer faster speed if the contextual surround motion was set at a relatively lower speed, and vice versa. These specific center–surround interactions may underlie the perceptual enhancement of speed contrast between adjacent fields.
A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: Application to interaction screening
Authors: Shioda Toshi , Andriole Stephanie , Yahata Tetsuro , Isselbacher Kurt J.
Published: July 2002

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein–Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the
OriP
Epstein–Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an
OriP
-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/
Ori-P
system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.
Gene content phylogeny of herpesviruses
Authors: Montague Michael G. , Hutchison Clyde A.
Published: July 2002

Clusters of orthologous groups [COGs; Tatusov, R. L., Koonin, E. V. & Lipman, D. J. (1997)
Science
278, 631–637] were identified for a set of 13 completely sequenced herpesviruses. Each COG represented a family of gene products conserved across several herpes genomes. These families were defined without using an arbitrary threshold criterion based on sequence similarity. The COG technique was modified so that variable stringency in COG construction was possible. High stringencies identify a core set of highly conserved genes. Varying COG stringency reveals differences in the degree of conservation between functional classes of genes. The COG data were used to construct whole-genome phylogenetic trees based on gene content. These trees agree well with trees based on other methods and are robust when tested by bootstrap analysis. The COG data also were used to construct a reciprocal tree that clustered genes with similar phylogenetic profiles. This clustering may give clues to genes with related functions or with related histories of acquisition and loss during herpesvirus evolution.
LexA chimeras reveal the function of
Drosophila
Fos as a context-dependent transcriptional activator
Authors: Szüts David , Bienz Mariann
Published: July 2002

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic
Drosophila
embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing
Drosophila
Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a
Drosophila
Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.
Cell cycle inhibition by the anti-angiogenic agent TNP-470 is mediated by p53 and p21
WAF1/CIP1
Authors: Zhang Yi , Griffith Eric C. , Sage Julien , Jacks Tyler , Liu Jun O.
Published: July 2002

Angiogenesis has been demonstrated to be essential for tumor growth and metastasis, and inhibition of angiogenesis is emerging as a promising strategy for treating cancer. Among the most potent inhibitors of angiogenesis is the fumagillin family of natural products. An analog of fumagillin, known as TNP-470 or AGM-1470, has been undergoing clinical trials for treating a variety of cancers. TNP-470 has been shown to block endothelial cell cycle progression in the late G
1
phase. Although the direct molecular target for TNP-470 has been identified as the type 2 methionine aminopeptidase (MetAP2), how inhibition of this enzyme leads to cell cycle arrest has remained unclear. We report that treatment of endothelial and other drug-sensitive cell types leads to the activation of the p53 pathway, causing an accumulation of the G
1
cyclin-dependent kinase inhibitor p21
WAF1/CIP1
. The requirement of p53 and p21
WAF1/CIP1
for the cell cycle inhibition by TNP-470 is underscored by the observation that cells deficient in p53 and p21
WAF1/CIP1
are resistant to TNP-470. These results shed significant light on the mechanism of cell cycle inhibition by TNP-470 and suggest an alternative method of activating p53 in endothelial cells to halt angiogenesis and tumor progression.
The evolution of RNA viruses: A population genetics view
Authors: Moya Andrés , Elena Santiago F. , Bracho Alma , Miralles Rosario , Barrio Eladio
Published: July 2002

RNA viruses are excellent experimental models for studying
evolution under the theoretical framework of population genetics. For a
proper justification of this thesis we have introduced some properties
of RNA viruses that are relevant for studying evolution. On the other
hand, population genetics is a reductionistic theory of evolution. It
does not consider or make simplistic assumptions on the transformation
laws within and between genotypic and phenotypic spaces. However, such
laws are minimized in the case of RNA viruses because the phenotypic
space maps onto the genotypic space in a much more linear way than on
higher DNA-based organisms. Under experimental conditions, we have
tested the role of deleterious and beneficial mutations in the degree
of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus
of negative strand. We also have studied how effective population size,
initial genetic variability in populations, and environmental
heterogeneity shapes the impact of mutations in the evolution of
vesicular stomatitis virus. Finally, in an integrative attempt, we
discuss pros and cons of the quasispecies theory compared with classic
population genetics models for haploid organisms to explain the
evolution of RNA viruses.
Bacteria are different: Observations, interpretations, speculations, and opinions about the mechanisms of adaptive evolution in prokaryotes
Authors: Levin Bruce R. , Bergstrom Carl T.
Published: July 2002

To some extent, the genetic theory of adaptive evolution in
bacteria is a simple extension of that developed for sexually
reproducing eukaryotes. In other, fundamental ways, the process of
adaptive evolution in bacteria is quantitatively and qualitatively
different from that of organisms for which recombination is an integral
part of the reproduction process. In this speculative and opinionated
discussion, we explore these differences. In particular, we consider
(
i
) how, as a consequence of the low rates of
recombination, “ordinary” chromosomal gene evolution in bacteria
is different from that in organisms where recombination is frequent and
(
ii
) the fundamental role of the horizontal transmission
of genes and accessory genetic elements as sources of variation in
bacteria. We conclude with speculations about the evolution of
accessory elements and their role in the adaptive evolution
of bacteria.
Establishing a grassland signature in veins:18O in the leaf water of C3and C4grasses
Authors: Helliker Brent R. , Ehleringer James R.
Published: July 2002

We show that18O evaporative enrichment of bulk leaf water in grass species can be significantly more enriched than predicted by the Craig–Gordon model, with C4grasses considerably more enriched than C3grasses. Our results suggest that the unanticipated18O leaf water enrichment of grasses is attributable to the progressive evaporative enrichment along parallel veins (a function of both leaf length and interveinal distance), a pattern that does not occur in Dicotyledonous species. We propose that the differential18O enrichment of grasses will result in distinct C18O16O biospheric signals from grassland and forest ecosystems, allowing for further partitioning of terrestrial carbon fluxes.
Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast
Authors: Lyons Thomas J. , Gasch Audrey P. , Gaither L. Alex , Botstein David , Brown Patrick O. , Eide David J.
Published: July 2002

The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using
lacZ
reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.
Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1–MAGE-A1 from a nonimmunized phage-Fab library
Authors: Chames Patrick , Hufton Simon E. , Coulie Pierre G. , Uchanska-Ziegler Barbara , Hoogenboom Hennie R.
Published: July 2002

Antitumor antibodies with the same specificity as cytotoxic T lymphocytes that recognize antigenic peptides encoded by tumor-associated genes and presented by MHC class I molecules would be valuable tools to analyze the antigenicity or target tumor cellsin vivo. To obtain a human antibody directed against a peptide encoded by gene melanoma-associated antigen (MAGE)-A1 and presented by HLA-A1 molecules, we selected a large phage Fab antibody repertoire on a recombinant version of the complex HLA-A1–MAGE-A1 produced byin vitrorefolding. One of the selected phage antibodies shows binding to HLA-A1 complexed with the MAGE-A1 peptide, but does not show binding to HLA-A1 complexed with a peptide encoded by gene MAGE-A3 and differing from the MAGE-A1 peptide by only three residues. Phages carrying this recombinant antibody bind to HLA-A1+cells only afterin vitroloading with MAGE-A1 peptide. These results indicate that nonimmunized phage Fab libraries are a source of antibodies with a T cell antigen receptor-like specificity. The human anti-HLA-A1–MAGE-A1 antibody described here may prove very useful for monitoring the cell surface expression of these complexes, and eventually, as a targeting reagent for the specific immunotherapy of HLA-A1 patients bearing a MAGE-A1-positive tumor.
Internalization of CD26 by mannose 6-phosphate/insulin-like growth factor II receptor contributes to T cell activation
Authors: Ikushima Hideto , Munakata Yasuhiko , Ishii Tomonori , Iwata Satoshi , Terashima Masazumi , Tanaka Hirotoshi , Schlossman Stuart F. , Morimoto Chikao
Published: July 2002

CD26 is a T cell activation antigen known to bind adenosine deaminase and have dipeptidyl peptidase IV activity. Cross-linking of CD26 and CD3 with immobilized mAbs can deliver a costimulatory signal that contributes to T cell activation. Our earlier studies revealed that cross-linking of CD26 induces its internalization, the phosphorylation of a number of proteins involved in the signaling pathway, and subsequent T cell proliferation. Although these findings suggest the importance of internalization in the function of CD26, CD26 has only 6 aa residues in its cytoplasmic region with no known motif for endocytosis. In the present study, we have identified the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) as a binding protein for CD26 and that mannose 6-phosphate (M6P) residues in the carbohydrate moiety of CD26 are critical for this binding. Activation of peripheral blood T cells results in the mannose 6 phosphorylation of CD26. In addition, the cross-linking of CD26 with an anti-CD26 antibody induces not only capping and internalization of CD26 but also colocalization of CD26 with M6P/IGFIIR. Finally, both internalization of CD26 and the T cell proliferative response induced by CD26-mediated costimulation were inhibited by the addition of M6P, but not by glucose 6-phosphate or mannose 1-phosphate. These results indicate that internalization of CD26 after cross-linking is mediated in part by M6P/IGFIIR and that the interaction between mannose 6-phosphorylated CD26 and M6P/IGFIIR may play an important role in CD26-mediated T cell costimulatory signaling.
A framework for interpreting the leucine-rich repeats of the
Listeria
internalins
Authors: Marino Michael , Braun Laurence , Cossart Pascale , Ghosh Partho
Published: July 2002

The surface protein InlB of the bacterial pathogen
Listeria
monocytogenes
is required for inducing phagocytosis in various
nonphagocytic mammalian cell types
in vitro
. InlB
causes tyrosine phosphorylation of host cell adaptor proteins,
activation of phosphoinositide 3-kinase, and rearrangements of the
actin cytoskeleton. These events lead to phagocytic uptake of the
bacterium by the host cell. InlB belongs to the internalin family of
Listeria
proteins, which also includes InlA, another
surface protein involved in host cell invasion. The internalins are the
largest class of bacterial proteins containing leucine-rich repeats
(LRR), a motif associated with protein–protein interactions. The LRR
motif is found in a functionally diverse array of proteins, including
those involved in the plant immune system and in the mammalian innate
immune response. Structural and functional interpretations of the
sequences of internalin family members are presented in light of the
recently determined x-ray crystal structure of the InlB LRR domain.
Paxillin α and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation
Authors: Yano Hajime , Uchida Hiroshi , Iwasaki Teruo , Mukai Mutsuko , Akedo Hitoshi , Nakamura Kuniaki , Hashimoto Shigeru , Sabe Hisataka
Published: July 2002

Protein tyrosine phosphorylation accompanies and is essential for integrin signaling. We have shown that tyrosine phosphorylation of paxillin α and Crk-associated substrate (p130
Cas
) is a prominent event on integrin activation in normal murine mammary gland epithelial cells. Tyrosine phosphorylation of p130
Cas
has been demonstrated to facilitate cell migration. We show here that tyrosine phosphorylation of paxillin α acts to reduce haptotactic cell migrations as well as transcellular invasive activities in several different experimental cell systems, whereas tyrosine phosphorylation of p130
Cas
exerts opposing effects to those of paxillin α. Each of the phosphorylation-null mutants acts as a dominant negative for each phenotype. Moreover, we found that overexpression of paxillin α reduced the cell saturation density of normal murine mammary gland cells, whereas overexpression of p130
Cas
increased it. These effects also seemed to depend on tyrosine phosphorylation events. Cell growth rates and morphologies at growing phases were not significantly altered, nor were cells transformed. Addition of epidermal growth factor increased saturation density of the paxillin α-overexpressing cells, whereas no further increment was observed in p130
Cas
-overexpressing cells. We propose that tyrosine phosphorylation of paxillin α and p130
Cas
exerts opposing effects on several integrin-mediated cellular events, possibly through different signaling pathways.
Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M
3
subtype
Authors: Matsui Minoru , Motomura Daisuke , Karasawa Hiroshi , Fujikawa Toru , Jiang Jian , Komiya Yuriko , Takahashi Shin-ichi , Taketo Makoto M.
Published: July 2002

Muscarinic acetylcholine receptors consist of five distinct subtypes and have been important targets for drug development. In the periphery, muscarinic acetylcholine receptors mediate cholinergic signals to autonomic organs, but specific physiological functions of each subtype remain poorly elucidated. Here, we have constructed and analyzed mutant mice lacking the M
3
receptor and have demonstrated that this subtype plays key roles in salivary secretion, pupillary constriction, and bladder detrusor contractions. However, M
3
-mediated signals in digestive and reproductive organs are dispensable, likely because of redundant mechanisms through other muscarinic acetylcholine receptor subtypes or other mediators. In addition, we have found prominent urinary retention only in the male, which indicates a considerable sex difference in the micturition mechanism. Accordingly, this mutant mouse should provide a useful animal model for investigation of human diseases that are affected in the peripheral cholinergic functions.
Sexual conflict promotes speciation in insects
Authors: Arnqvist Göran , Edvardsson Martin , Friberg Urban , Nilsson Tina
Published: July 2002

Speciation rates among extant lineages of organisms vary extensively, but our understanding of the causes of this variation and, therefore, the processes of speciation is still remarkably incomplete. Both theoretical and empirical studies have indicated that sexual selection is important in speciation, but earlier discussions have focused almost exclusively on the potential role of female mate choice. Recent findings of postmating reproductive conflicts of interest between the sexes suggest a quite different route to speciation. Such conflicts may lead to perpetual antagonistic coevolution between males and females and may thus generate rapid evolutionary divergence of traits involved in reproduction. Here, we assess this hypothesis by contrasting pairs of related groups of insect species differing in the opportunity for postmating sexual conflict. Groups where females mate with many males exhibited speciation rates four times as high as in related groups where females mate only once. Our results not only highlight the general importance of postmating sexual selection in speciation, but also support the recent suggestion that sexual conflict is a key engine of speciation.
Calnuc, an EF-Hand Ca
2+
binding protein, specifically interacts with the C-terminal α5-helix of Gαi3
Authors: Lin Ping , Fischer Thierry , Weiss Thomas , Farquhar Marilyn Gist
Published: July 2002

Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and to be the major Golgi Ca
2+
binding protein. In this study we verified the existence of the cytosolic pool of calnuc and investigated its interaction with Gαi3. Cytosolic calnuc was released by mild digitonin permeabilization. In pulse–chase experiments, the two pools of calnuc had different mobilities, suggesting different posttranslational modifications. That calnuc interacts with Gαi3
in vivo
was verified by the finding that Gαi3 could be crosslinked intracellularly to calnuc and co-immunoprecipitated from NIH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) Gαi3. Binding was Ca
2+
and Mg
2+
-dependent. Calnuc and Gαi3-GFP codistributed primarily in the Golgi region. By yeast two-hybrid analysis, the binding site on Gαi3 for calnuc was mapped to the C-terminal region because removal of the last 12 amino acids (but not 11) abolished the interaction. Peptide competition indicated that calnuc, with its coiled-coil domain constituted by the two EF-hands, binds to Gαi3's C-terminal α5-helix. These results demonstrate that calnuc may play an important role in G protein- and Ca
2+
-regulated signal transduction events.
Detection of emphysema in rat lungs by using magnetic resonance measurements of
3
He diffusion
Authors: Chen X. Josette , Hedlund Laurence W. , Möller Harald E. , Chawla Mark S. , Maronpot Robert R. , Johnson G. Allan
Published: July 2002

Emphysema is a pulmonary disease characterized by alveolar wall
destruction, resulting in enlargement of gas exchange spaces without
fibrosis. This condition is a part of chronic obstructive pulmonary
disease (COPD), which causes 3.5% of deaths worldwide [Anonymous
(1990)
World Health Stat. Q.
Special
,
1–51] and contributes greatly to the global burden of disease
[Murray, C. J. & Lopez, A. D. (1996)
Science
274, 740–743]. Alveolar regeneration has been shown in animal models
and could have potential for clinical treatment of early-stage
emphysema. However, current techniques for detection of emphysema are
not sensitive at the initial stages. Early-stage human panacinar
emphysema is modeled in elastase-treated animals. Here, we provide an
in vivo
imaging method for differentiating normal and
emphysematous rat lungs by measuring the apparent diffusion coefficient
(ADC) of hyperpolarized
3
He by using magnetic resonance
imaging. These data show that the ADC is significantly larger in
elastase-treated rats, indicating alveolar expansion. Whereas these
rats were clinically asymptomatic, conventional histology confirmed
presence of injury. Our results indicate that measurement of the
hyperpolarized
3
He ADC can be a valuable research tool and
has potential application in the clinical setting.
The antiangiogenic agent TNP-470 requires p53 and p21
CIP/WAF
for endothelial cell growth arrest
Authors: Yeh Jing-Ruey J. , Mohan Royce , Crews Craig M.
Published: July 2002

Targeting the endothelial cell cycle as an antiangiogenic strategy
has been difficult given the ubiquitous expression of critical cell
cycle regulators. Here, we show that the antiangiogenic drug TNP-470
displays striking cell-type specificity insofar as it induces the
expression of p21
CIP/WAF
, a cyclin-dependent kinase
inhibitor, in endothelial cells but not in embryonic or adult
fibroblasts. Moreover, primary endothelial cells isolated from
p53
−/−
and p21
CIP/WAF−/−
mice are
resistant to the cytostatic activity of TNP-470. We also demonstrate
that p21
CIP/WAF−/−
mice are resistant to the
antiangiogenic activity of TNP-470 in the basic fibroblast growth
factor corneal micropocket angiogenesis assay. We conclude that TNP-470
induces p53 activation through a unique mechanism in endothelial cells
leading to p21
CIP/WAF
expression and subsequent growth
arrest.
Invariant size–frequency distributions along a latitudinal gradient in marine bivalves
Authors: Roy Kaustuv , Jablonski David , Martien Karen K.
Published: July 2002

In the most extensive analysis of body size in marine invertebrates
to date, we show that the size–frequency distributions of northeastern
Pacific bivalves at the provincial level are surprisingly invariant in
modal and median size as well as size range, despite a 4-fold change in
species richness from the tropics to the Arctic. The modal sizes and
shapes of these size–frequency distributions are consistent with the
predictions of an energetic model previously applied to terrestrial
mammals and birds. However, analyses of the Miocene–Recent history of
body sizes within 82 molluscan genera show little support for the
expectation that the modal size is an evolutionary attractor over
geological time.
Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function
Authors: Postel Edith H. , Abramczyk Bozena M. , Levit Mikhail N. , Kyin Saw
Published: July 2002

NM23/NDP kinases play an important role in development and cancer
but their biological function is unknown, despite an intriguing
collection of biochemical properties including nucleoside-diphosphate
kinase (NDP kinase), DNA binding and transcription, a mutator function,
and cleavage of unusually structured DNA by means of a covalent
enzyme–DNA complex. To assess the role of the nuclease in human
NM23-H2, we sought to identify the amino acid responsible for covalent
catalysis. By sequencing a DNA-linked peptide and by site-directed
mutagenesis, we identified lysine-12, a phylogenetically conserved
residue, as the amino acid forming the covalent complex with DNA. In
particular, the ɛ-amino group acts as the critical nucleophile,
because substitution with glutamine but not arginine completely
abrogated covalent adduct formation and DNA cleavage, whereas the
DNA-binding properties remained intact. These findings and chemical
modification data suggest that phosphodiester-bond cleavage occurs by a
DNA glycosylase/lyase-like mechanism known as the signature of base
excision DNA repair nucleases. Involvement of NM23/NDP kinase in a
DNA repair pathway would be consistent with its role in normal and
tumor cell development. Additionally, lysine-12, which is known in the
x-ray crystallographic structure to lie in the catalytic pocket
involved in the NDP kinase phosphorylation reaction, was found
essential also for the NDP kinase activity of NM23-H2, suggesting that
the two catalytic activities of NM23-H2 are fundamentally connected.
HO endonuclease-induced recombination in yeast meiosis resembles Spo11-induced events
Authors: Malkova Anna , Klein Franz , Leung Wai-Ying , Haber James E.
Published: July 2002

In meiosis, gene conversions are accompanied by higher levels of
crossing over than in mitotic cells. To determine whether the special
properties of meiotic recombination can be attributed to the way in
which Spo11p creates double-strand breaks (DSBs) at special hot spots
in
Saccharomyces cerevisiae
, we expressed the
site-specific HO endonuclease in meiotic cells. We could therefore
compare HO-induced recombination in a well-defined region both in
mitosis and meiosis, as well as compare Spo11p- and HO-induced meiotic
events. HO-induced gene conversions in meiosis were accompanied by
crossovers at the same high level (52%) as Spo11p-induced events.
Moreover, HO-induced crossovers were reduced 3-fold by a
msh4
Δ mutation that similarly affects Spo11p-promoted
events. In a
spo11
Δ diploid, where the only DSB is
made by HO, crossing over was significantly higher (27%) than in
mitotic cells (≤7%). This single meiotic DSB failed to induce the
formation of a synaptonemal complex. We also show that HO-induced gene
conversion tract lengths are shorter in meiotic than in mitotic cells.
We conclude that a hallmark of meiotic recombination, the production of
crossovers, is independent of the nature of Spo11p-generated DSBs at
special hotspots, but some functions of Spo11p are required in
trans
to achieve maximum crossing over.
Role of serum amyloid P component in bacterial infection: Protection of the host or protection of the pathogen
Authors: Noursadeghi Mahdad , Bickerstaff Maria C. M. , Gallimore J. Ruth , Herbert Jeff , Cohen Jonathan , Pepys Mark B.
Published: July 2002

Serum amyloid P component (SAP) binds toStreptococcus pyogenes, and we show here that it also binds toNeisseria meningitidis, including a lipopolysaccharide (LPS)-negative mutant, and to rough variants ofEscherichia coli. Surprisingly, this binding had a powerful antiopsonic effect bothin vitroandin vivo, reducing phagocytosis and killing of bacteria. Furthermore, SAP knockout mice survived lethal infection withS. pyogenesand roughE. coliJ5, organisms to which SAP binds. The susceptibility of SAP−/−mice was fully restored by injection of isolated human SAP. However, SAP−/−mice were more susceptible than wild-type animals to lethal infection withE. coliO111:B4, a smooth strain to which SAP does not bind, suggesting that SAP also has some host defense function. Although SAP binds to LPSin vitro, SAP−/−mice were only marginally more susceptible to lethal LPS challenge, and injection of large amounts of human SAP into wild-type mice did not affect sensitivity to LPS, indicating that SAP is not a significant modulator of LPS toxicityin vivo. In contrast, the binding of SAP to pathogenic bacteria enabled them to evade neutrophil phagocytosis and display enhanced virulence. Abrogation of this molecular camouflage is thus potentially a novel therapeutic approach, and we show here that administration to wild-type mice of (R)-1-[6-(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid, a drug that inhibits SAP binding, significantly prolonged survival during lethal infection withE. coliJ5.
Induction of topoisomerase I cleavage complexes by 1-β-
d
-arabinofuranosylcytosine (ara-C)
in vitro
and in ara-C-treated cells
Authors: Pourquier Philippe , Takebayashi Yuji , Urasaki Yoshimasa , Gioffre Christopher , Kohlhagen Glenda , Pommier Yves
Published: July 2002

1-β-
d
-Arabinofuranosylcytosine (Ara-C) is a nucleoside analog commonly used in the treatment of leukemias. Ara-C inhibits DNA polymerases and can be incorporated into DNA. Its mechanism of cytotoxicity is not fully understood. Using oligonucleotides and purified human topoisomerase I (top1), we found a 4- to 6-fold enhancement of top1 cleavage complexes when ara-C was incorporated at the +1 position (immediately 3′) relative to a unique top1 cleavage site. This enhancement was primarily due to a reversible inhibition of top1-mediated DNA religation. Because ara-C incorporation is known to alter base stacking and sugar puckering at the misincorporation site and at the neighboring base pairs, the observed inhibition of religation at the ara-C site suggests the importance of the alignment of the 5′-hydroxyl end for religation with the phosphate group of the top1 phosphotyrosine bond. This study also demonstrates that ara-C treatment and DNA incorporation trap top1 cleavage complexes in human leukemia cells. Finally, we report that camptothecin-resistant mouse P388/CPT45 cells with no detectable top1 are crossresistant to ara-C, which suggests that top1 poisoning is a potential mechanism for ara-C cytotoxicity.